Ko ngā matū whakarerekētanga ārai mate he āhuatanga matua o te taiao moroiti pukupuku (TME), engari haunga ētahi tūponotanga, kāore i te tino mōhiotia ō rātou tuakiri. I konei, i tātarihia e mātou ngā pukupuku me ngā pūtau-T mai i ngā pukupuku me ngā ascites o ngā tūroro he carcinoma serous teitei (HGSC) hei whakaatu i te metabolome o ēnei wāhanga TME rerekē. He nui ngā rerekētanga matū whakarerekētanga o ngā ascites me ngā pūtau pukupuku. Ki te whakatauritea ki ngā ascites, he nui te 1-methylnicotinamide (MNA) o ngā pūtau-T e uru ana ki te pukupuku. Ahakoa he teitei ake te taumata o te MNA i roto i ngā pūtau-T, ko te whakaaturanga o te nicotinamide N-methyltransferase (he whākōkī e whakatere ana i te whakawhiti o ngā rōpū methyl mai i te S-adenosylmethionine ki te nicotinamide) he iti noa iho ki ngā fibroblasts me ngā pūtau pukupuku. I runga i te mahi, ka akiaki te MNA i ngā pūtau-T ki te huna i te cytokine whakatairanga pukupuku tumor necrosis factor alpha. Nō reira, ka whai wāhi te MNA i ahu mai i te TME ki te whakahaere i te ārai mate o ngā pūtau-T, ā, he whāinga ārai mate pea mō te maimoatanga o te mate pukupuku tangata.
Ka taea e ngā matū whai hua i ahu mai i te pukupuku te whai pānga nui ki te aukati i te ārai mate pukupuku, ā, kei te piki haere te maha o ngā taunakitanga e whakaatu ana ka taea hoki e rātou te mahi hei kaha matua mō te whanaketanga o te mate (1). Haunga te pānga Warburg, kua tīmata ngā mahi hou ki te whakaahua i te āhua o te pākia o ngā pūtau pukupuku me tōna whanaungatanga ki te āhua ārai mate o te taiao moroiti pukupuku (TME). Kua whakaatuhia e ngā rangahau mō ngā tauira kiore me ngā pūtau-T tangata ka taea e te pākia o te glutamine (2), te pākia o te waikura (3) me te pākia o te huka (4) te mahi motuhake i runga i ngā rōpū pūtau ārai mate. He maha ngā matū whai hua i roto i ēnei ara e aukati ana i te mahi ārai mate pukupuku o ngā pūtau-T. Kua whakamātauhia ko te aukati i te coenzyme tetrahydrobiopterin (BH4) ka taea te kino i te tipu o ngā pūtau-T, ā, ko te pikinga o te BH4 i roto i te tinana ka taea te whakarei ake i te urupare ārai mate pukupuku e whakahaerehia ana e te CD4 me te CD8. Hei tāpiri, ka taea te whakaora i te pānga ārai mate o te kynurenine mā te whakahaere i te BH4 (5). I roto i te glioblastoma mutant isocitrate dehydrogenase (IDH), ko te whakaputanga o te enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) ka aukati i te whakahohenga, te tipu me te mahi cytolysis pūtau-T (6). Nō nā tata nei, kua whakaaturia ko te methylglyoxal, he hua taha o te glycolysis, ka puta mai i ngā pūtau aukati o te takenga mai o te myeloid, ā, ka taea e te whakawhiti pūtau-T o te methylglyoxal te aukati i te mahi a te pūtau-T effector. I roto i te maimoatanga, ka taea e te whakakorenga o te methylglyoxal te wikitoria i te mahi a ngā pūtau aukati i ahu mai i te myeloid (MDSC) me te whakarei ake i te maimoatanga aukati tirotiro i roto i ngā tauira kiore (7). E whakanuia ana e ēnei rangahau te mahi matua o ngā metabolites i ahu mai i te TME ki te whakahaere i te mahi me te mahi a te pūtau-T.
Kua whānuitia te pūrongo mō te ngoikore o te pūtau-T i roto i te mate pukupuku ovarian (8). Ko tētahi wāhanga o tēnei ko ngā āhuatanga pākia o te hypoxia me te toto totoka rerekē (9), e hua ake ai te hurihanga o te glucose me te tryptophan ki ngā hua taha pērā i te waikawa lactic me te kynurenine. Ko te nui rawa o te lactate extracellular ka whakaiti i te hanga o te interferon-γ (IFN-γ) ā, ka akiaki i te rerekētanga o ngā rōpū iti myelosuppressive (10, 11). Ko te kai o te tryptophan ka aukati tika i te tipu o te pūtau-T me te aukati i te tohu a te kaiwhiwhi pūtau-T (12-14). Ahakoa ēnei kitenga, he nui ngā mahi e pā ana ki te pākia o te pūnaha ārai mate i mahia i roto i te ahurea pūtau-T in vitro mā te whakamahi i ngā pāpāho kua arotauhia, i whakawhāitihia rānei ki ngā tauira kiore homologous in vivo, kāore tetahi o ēnei e whakaata katoa ana i te rerekētanga o ngā mate pukupuku tangata me te taiao macro me te micro Physiological.
Ko tētahi āhuatanga noa o te mate pukupuku ovarian ko te horapa o te peritoneal me te putanga mai o ngā ascites. Ko te kohikohinga o te wai pūtau i roto i ngā ascites e pā ana ki te mate whakamua me te matapae kino (15). E ai ki ngā pūrongo, he hypoxic tēnei wāhanga ahurei, he nui ngā taumata o te vascular endothelial growth factor (VEGF) me te indoleamine 2,3-dioxygenase (IDO), ā, ka uruhia e ngā pūtau T regulatory me ngā pūtau myeloid inhibitory (15-18). Tērā pea he rerekē te taiao metabolic o ngā ascites i te taiao o te pukupuku tonu, nō reira kāore i te mārama te hōtaka anō o ngā pūtau T i roto i te wāhi peritoneal. Hei tāpiri, ko ngā rerekētanga matua me te rerekētanga i waenga i ngā ascites me ngā metabolites kei roto i te taiao pukupuku ka ārai pea i te urunga o ngā pūtau ārai mate me tā rātou mahi ki ngā pukupuku, ā, me rangahau anō.
Hei whakaoti i ēnei raruraru, i hangaia e mātou he tikanga wehewehe pūtau tairongo me te chromatography wai tandem mass spectrometry (LC-MS/MS) hei ako i ngā momo pūtau rerekē (tae atu ki ngā pūtau CD4 + me CD8 + T) tae atu ki roto me waenga i ngā pukupuku. Ka horahia e ōna matūkai ngā pūtau i roto i te ascites me te taiao pukupuku kotahi o te tūroro. Ka whakamahia e mātou tēnei tikanga me te cytometry rere-ahu teitei me te raupapatanga RNA pūtau-kotahi (scRNA-seq) hei whakarato i tētahi whakaahua tino mārama o te āhua pākia o ēnei taupori matua. I whakaatuhia e tēnei tikanga he pikinga nui o te taumata o te 1-methylnicotinamide (MNA) i roto i ngā pūtau T pukupuku, ā, i whakaatuhia e ngā whakamātautau in vitro kāore i te mōhiotia i mua te pānga immunomodulatory o te MNA ki te mahi pūtau T. I te nuinga o te wā, ka whakakitea e tēnei tikanga ngā taunekeneke pākia i waenga i ngā pukupuku me ngā pūtau ārai mate, ā, ka whakaratohia he māramatanga ahurei ki ngā matūkai whakahaere ārai mate, e whai hua ana pea mō te maimoatanga o te mate pukupuku ovarian e ahu mai ana i te pūtau T. Ngā whai wāhitanga maimoatanga immunotherapy.
I whakamahia e mātou te cytometry rere-rahi-rahi hei ine i te tangohanga huka [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) me te mahi mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) he tohu noa e wehewehe ana i ngā pūtau ārai mate me ngā taupori pūtau pukupuku (Ripanga S2 me te Pikitia S1A). I ​​whakaatuhia e tēnei tātaritanga, ki te whakaritea ki ngā pūtau T, he nui ake ngā taumata tangohanga huka o ngā ascites me ngā pūtau pukupuku, engari he iti ake ngā rerekētanga o te mahi mitochondrial. Ko te tangohanga huka toharite o ngā pūtau pukupuku [CD45-EpCAM (EpCAM)+] e toru ki te whā ngā wā i ngā pūtau T, ā, ko te tangohanga huka toharite o ngā pūtau CD4 + T he 1.2 ngā wā i ngā pūtau CD8 + T, e tohu ana he rerekē ngā whakaritenga pākia o ngā lymphocytes whakauru pukupuku (TIL) ahakoa i roto i te TME kotahi (Pikitia 1A). He rerekē, he rite te mahi mitochondrial i roto i ngā pūtau pukupuku ki te mahi a ngā pūtau CD4 + T, ā, he teitei ake te mahi mitochondrial o ngā momo pūtau e rua i te mahi a ngā pūtau CD8 + T (Pikitia 1B). I te nuinga o te wā, e whakaatu ana ēnei hua i te taumata pākia. He teitei ake te mahi pākia o ngā pūtau pukupuku i te mahi a ngā pūtau CD4 + T, ā, he teitei ake te mahi pākia o ngā pūtau CD4 + T i te mahi a ngā pūtau CD8 + T. Ahakoa ēnei pānga puta noa i ngā momo pūtau, kāore he rerekētanga pumau i roto i te āhua pākia o ngā pūtau CD4 + me CD8 + T, i ō rātou ōwehenga whanaunga rānei i roto i te ascites ki te whakaritea ki ngā pukupuku (Pikitia 1C). He rerekē, i roto i te wāhanga pūtau CD45, i piki ake te ōwehenga o ngā pūtau EpCAM+ i roto i te pukupuku ki te whakaritea ki ngā ascites (Pikitia 1D). I kite anō mātou i tētahi rerekētanga pākia mārama i waenga i ngā wāhanga pūtau EpCAM+ me EpCAM-. He nui ake te tango huka me te mahi mitochondrial o ngā pūtau EpCAM+ (pukupuku) i ngā pūtau EpCAM-, he nui ake i te mahi pākia o ngā fibroblasts i roto i ngā pūtau pukupuku i roto i te TME (Pikitia 1, E me te F).
(A me B) Te kaha o te hihi o te rā (MFI) o te tango huka (2-NBDG) (A) me te mahi mitochondrial o ngā pūtau CD4 + T (MitoTracker whero pouri) (B) Ngā kauwhata māngai (maui) me ngā raraunga kua tātaitia (Matau), ngā pūtau CD8 + T me ngā pūtau EpCAM + CD45-pukupuku mai i te ascites me te pukupuku. (C) Te ōwehenga o ngā pūtau CD4 + me CD8 + (o ngā pūtau CD3 + T) i roto i te ascites me te pukupuku. (D) Te ōwehenga o ngā pūtau EpCAM + pukupuku i roto i te ascites me te pukupuku (CD45−). (E me F) Te tango huka EpCAM + CD45-pukupuku me te EpCAM-CD45-matrix (2-NBDG) (E) me te mahi mitochondrial (MitoTracker whero pouri) (F) ngā kauwhata māngai (maui) me ngā raraunga kua tātaitia (Matau) Ngā ascites me ngā pūtau pukupuku. (G) Ngā kauwhata māngai o te whakaaturanga CD25, CD137 me te PD1 mā te flow cytometry. (H me I) Te whakaaturanga o te CD25, CD137 me te PD1 i runga i ngā pūtau CD4 + T (H) me ngā pūtau CD8 + T (I). (J me K) Ngā momo āhua o te mahara matua (Tcm), te kaiwhakaputa (Teff) me te mahara kaiwhakaputa (Tem) i runga i te whakaaturanga o CCR7 me CD45RO. Ngā whakaahua māngai (maui) me ngā raraunga ripanga (matau) o ngā pūtau CD4 + T (J) me ngā pūtau CD8 + T (K) i roto i ngā ascites me ngā pukupuku. Ko ngā uara P i whakatauhia e te whakamātautau-t takirua (*P<0.05, **P<0.01 me ***P<0.001). Ko te rārangi e tohu ana i ngā tūroro ōrite (n = 6). FMO, fluorescence tango kotahi; MFI, te kaha fluorescence waenga.
I whakaatuhia e te tātaritanga anō ētahi atu rerekētanga nui i waenga i te āhua o te pūtau-T kua tino whakatauhia. Ko te mahara whakahohe (Pikitia 1, G ki I) me te mahara whaihua (Pikitia 1, J me K) i roto i ngā pukupuku he nui ake te auau i te ascites (te ōwehenga o ngā pūtau-T CD3 +). Waihoki, ko te tātari i te phenotype mā te whakaaturanga o ngā tohu whakahohe (CD25 me CD137) me ngā tohu whakahekenga [pūmua mate pūtau kua whakaritea 1 (PD1)] i whakaatu ahakoa he rerekē ngā āhuatanga pākia o ēnei taupori (Pikitia S1, B ki E), engari kāore he rerekētanga pākia nui i kitea i waenga i ngā huinga iti naive, effector, mahara rānei (Pikitia S1, F ki I). I whakaūhia ēnei hua mā te whakamahi i ngā tikanga ako mīhini hei tohu aunoa i ngā phenotype pūtau (21), i whakaatu anō i te aroaro o te maha o ngā pūtau hinu wheua (CD45 + / CD3- / CD4 + / CD45RO +) i roto i ngā ascites o te tūroro (Pikitia S2A). I roto i ngā momo pūtau katoa i tautuhia, ko tēnei taupori pūtau myeloid te mea i whakaatu i te nui rawa o te tango huka me te mahi mitochondrial (Pikitia S2, B ki G). E whakaatu ana ēnei hua i ngā rerekētanga kaha o te pākia o te pūtau i waenga i ngā momo pūtau maha i kitea i roto i ngā ascites me ngā pukupuku i roto i ngā tūroro HGSC.
Ko te wero matua ki te mārama ki ngā āhuatanga metabonomic o te TIL ko te hiahia ki te wehe i ngā tauira pūtau-T he parakore, he kounga me te rahinga rawaka mai i ngā pukupuku. Kua whakaatuhia e ngā rangahau tata nei ko ngā tikanga whakarōpū me te whakarei ake i ngā pītiti i runga i te cytometry rere ka arahi pea ki ngā huringa o ngā kōtaha metabolite pūtau (22-24). Hei whakakore i tēnei raruraru, i arotauhia e mātou te tikanga whakarei ake i ngā pītiti hei wehe me te wehe i te TIL mai i te mate pukupuku ovarian tangata i tangohia mā te pokanga i mua i te tātaritanga mā te LC-MS/MS (tirohia ngā Rauemi me ngā Tikanga; Pikitia 2A). Hei aromatawai i te pānga whānui o tēnei kawa ki ngā huringa metabolite, i whakaritea e mātou ngā kōtaha metabolite o ngā pūtau-T i whakahohehia e ngā kaituku hauora i muri i te taahiraa wehenga pītiti i runga ake nei ki ngā pūtau kāore i wehea ngā pītiti engari i noho tonu ki runga i te hukapapa. I kitea e tēnei tātaritanga whakahaere kounga he hononga nui i waenga i ēnei āhuatanga e rua (r = 0.77), ā, he teitei te whakahokinga hangarau o te rōpū o te 86 metabolites (Pikitia 2B). Nō reira, ka taea e ēnei tikanga te whakahaere i te tātaritanga tika o ngā matūkai i roto i ngā pūtau e whakarei ake ana i te momo pūtau, ā, koinei te tūāpapa tuatahi mō te tautuhi i ngā matūkai motuhake i roto i te HGSC, ā, ka taea ai e te tangata te mārama ake ki te motuhaketanga o ngā pūtau.
(A) Kauwhata hoahoa o te whakarei ake i ngā pirepire aukume. I mua i te tātaritanga mā te LC-MS/MS, ka toru ngā huringa whakarei ake i ngā pirepire aukume, ka noho rānei ki runga i te hukapapa. (B) Te pānga o te momo whakarei ake ki te nui o ngā matū whaihua. Ko te toharite o ngā inenga e toru mō ia momo whakarei ake ± SE. Ko te rārangi hina e tohu ana i te whanaungatanga 1:1. Ko te hononga ā-roto (ICC) o ngā inenga kua whakahuatia e whakaaturia ana i te tapanga tuaka. NAD, nicotinamide adenine dinucleotide. (C) Kauwhata hoahoa o te rere mahi o te tātari matū whaihua tūroro. Ka kohia ngā ascites, ngā pukupuku rānei mai i ngā tūroro, ā, ka tiakina. I tātarihia he wāhanga iti o ia tauira mā te cytometry rere, ko ngā tauira e toe ana i toru ngā huringa whakarei ake mō ngā pūtau CD4+, CD8+ me CD45-. I tātarihia ēnei wāhanga pūtau mā te whakamahi i te LC-MS/MS. (D) Mahere wera o te nui o te matū whaihua paerewa. Ko te dendrogram e tohu ana i te whakarōpūtanga a Ward o ngā tawhiti Euclidean i waenga i ngā tauira. (E) Te tātaritanga wāhanga matua (PCA) o te mahere tauira matū matū, e whakaatu ana i ngā tāruarua e toru o ia tauira, ka honoa ngā tauira mai i te tūroro kotahi mā te rārangi. (F) Ko te PCA o te kōtaha matū matū o te tauira e pā ana ki te tūroro (arā, mā te whakamahi i te taurite ā-wāhanga); ka whakawhāitihia te momo tauira e te anga kōpiko. PC1, wāhanga matua 1; PC2, wāhanga matua 2.
Muri iho, i whakamahia e mātou tēnei tikanga whakarei ake hei tātari i te 99 ngā matū whaihua i roto i ngā wāhanga pūtau CD4+, CD8+ me te CD45 i roto i ngā ascites tuatahi me ngā pukupuku o ngā tūroro HGSC e ono (Pikitia 2C, Pikitia S3A me te Ripanga S3 me te S4). Ko te taupori e hiahiatia ana e 2% ki te 70% o te tauira nui taketake o ngā pūtau ora, ā, he rerekē rawa te ōwehenga o ngā pūtau i waenga i ngā tūroro. I muri i te wehewehe i ngā pītiti, ko te wāhanga whakarei ake e hiahiatia ana (CD4+, CD8+, CD45- rānei) e neke atu i te 85% o ngā pūtau ora katoa i roto i te tauira i te toharite. Mā tēnei tikanga whakarei ake ka taea e mātou te tātari i ngā taupori pūtau mai i te pākia o te kiko pukupuku tangata, he mea kāore e taea te mahi mai i ngā tauira nui. Mā te whakamahi i tēnei tikanga, i whakatauhia e mātou ko te l-kynurenine me te adenosine, ēnei matū whaihua ārai mate e rua kua tautuhia pai, i piki ake i roto i ngā pūtau T pukupuku, i ngā pūtau pukupuku rānei (Pikitia S3, B me te C). Nō reira, e whakaatu ana ēnei hua i te pono me te kaha o tā mātou hangarau wehenga pūtau me te hangarau ine papatipu ki te kimi i ngā matū whaihua nui ki te koiora i roto i ngā kiko tūroro.
I whakaatuhia hoki e tā mātou tātaritanga he wehenga kaha o ngā momo pūtau i roto, i waenga hoki i ngā tūroro (Pikitia 2D me te Pikitia S4A). Ina koa, ki te whakatauritea ki ētahi atu tūroro, i whakaatuhia e te tūroro 70 ngā āhuatanga rerekē o te pākia (Pikitia 2E me te Pikitia S4B), e tohu ana tera pea he rerekētanga nui o te pākia i waenga i ngā tūroro. He mea tika kia mōhiotia, ki te whakatauritea ki ētahi atu tūroro (1.2 ki te 2 rita; Ripanga S1), he iti ake te nui o ngā ascites i kohia i roto i te tūroro 70 (80 ml). Ko te whakahaere i te rerekētanga i waenga i ngā tūroro i te wā o te tātaritanga wāhanga matua (hei tauira, mā te whakamahi i te tātaritanga tauutuutu wāhanga) e whakaatu ana i ngā huringa ōrite i waenga i ngā momo pūtau, ā, ko ngā momo pūtau me/ranei te taiao moroiti kua whakakotahihia kia rite ki te kōtaha metabolite (Pikitia 2F). I whakanuia e te tātaritanga o ngā metabolites takitahi ēnei pānga, ā, i whakaatuhia ngā rerekētanga nui i waenga i ngā momo pūtau me te taiao moroiti. He mea tika kia mōhiotia ko te rerekētanga tino nui i kitea ko te MNA, e whakanuia ana i roto i ngā pūtau CD45- me ngā pūtau CD4+ me CD8+ e uru ana ki te pukupuku (Pikitia 3A). Mō ngā pūtau CD4+, ko tēnei pānga te mea tino kitea, ā, ko te MNA i roto i ngā pūtau CD8+ te āhua nei he tino pāngia hoki e te taiao. Heoi, kāore tēnei i te mea nui, nā te mea e toru anake o ngā tūroro e ono ka taea te aromatawai mō ngā kaute CD8+ pukupuku. Haunga te MNA, i roto i ngā momo pūtau rerekē i roto i ngā ascites me ngā pukupuku, ko ētahi atu matū whaihua kāore i te tino kitea i roto i te TIL he rerekē hoki te nui (Pikitia S3 me te S4). Nō reira, e whakaatu ana ēnei raraunga i tētahi huinga matū whaihua ārai mate mō ētahi atu rangahau.
(A) Te ihirangi MNA kua whakatauritea i roto i ngā pūtau CD4+, CD8+ me CD45- mai i ngā ascites me te pukupuku. E whakaatu ana te kauwhata pouaka i te waenga (raina), te whānuitanga interquartile (anga anga) me te whānuitanga raraunga, tae atu ki te 1.5 ngā wā o te whānuitanga interquartile (anga anga). E ai ki te whakaahuatanga i roto i ngā Rauemi me ngā Tikanga Tūroro, whakamahia te uara limma o te tūroro hei whakatau i te uara P (*P<0.05 me **P<0.01). (B) Te kauwhata kauwhata o te pākia o te MNA (60). Ngā matūkai: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl-2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Ngā whākōkī (kakariki): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyl transferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE o te scRNA-seq o te ascites (hina) me te pukupuku (whero; n = 3 ngā tūroro). (D) Te whakaaturanga NNMT i roto i ngā taupori pūtau rerekē i tautuhia mā te whakamahi i te scRNA-seq. (E) Te whakaaturanga o NNMT me AOX1 i roto i te SK-OV-3, te tākihi embryonic tangata (HEK) 293T, ngā pūtau T me ngā pūtau T i rongoatia ki te MNA. Ka whakaaturia te whakaaturanga kua whakapikohia e pā ana ki te SK-OV-3. Ka whakaaturia te tauira whakaaturanga me te SEM (n = 6 ngā kaituku hauora). Ko ngā uara Ct neke atu i te 35 e kiia ana kāore e kitea (UD). (F) Te whakaaturanga o SLC22A1 me SLC22A2 i roto i te SK-OV-3, HEK293T, ngā pūtau T me ngā pūtau T i rongoatia ki te 8mM MNA. Ka whakaaturia te whakaaturanga kua whakapikohia e pā ana ki te SK-OV-3. Ka whakaaturia te tauira whakaaturanga me te SEM (n = 6 ngā kaituku hauora). Ko ngā uara Ct neke atu i te 35 e kiia ana kāore e kitea (UD). (G) Te ihirangi MNA pūtau i roto i ngā pūtau T kaituku hauora kua whakahohehia i muri i te 72 hāora o te whakatipuranga ki te MNA. Ka whakaaturia te tauira whakaaturanga me te SEM (n = 4 ngā kaituku hauora).
Ka puta te MNA mā te whakawhiti i te rōpū methyl mai i te S-adenosyl-1-methionine (SAM) ki te nicotinamide (NA) mā te nicotinamide N-methyltransferase (NNMT; Pikitia 3B). He nui rawa te whakaaturanga o te NNMT i roto i ngā momo mate pukupuku tangata, ā, e pā ana ki te tipu, te whakaekenga me te metastasis (25-27). Hei mārama ake i te pūtake o te MNA i roto i ngā pūtau T i roto i te TME, i whakamahia e mātou te scRNA-seq hei whakaahua i te whakaaturanga o te NNMT puta noa i ngā momo pūtau i roto i ngā ascites me ngā pukupuku o ngā tūroro HGSC e toru (Ripanga S5). I whakaatuhia e te tātaritanga o te 6,500 pūtau tata ki te 6,500 i roto i ngā taiao ascites me te pukupuku, ko te whakaaturanga NNMT i herea ki ngā taupori pūtau fibroblast me te pukupuku e kiia ana (Pikitia 3, C me te D). He mea tika kia mōhio kāore he whakaaturanga NNMT mārama i roto i tētahi taupori e whakaatu ana i te PTPRC (CD45 +) (Pikitia 3D me te Pikitia S5A), e tohu ana kua whakaurua te MNA i kitea i roto i te whānuitanga metabolite ki roto i ngā pūtau T. Ko te whakapuakanga o te aldehyde oxidase 1 (AOX1) ka huri i te MNA ki te 1-methyl-2-pyridone-5-carboxamide (2-PYR) 1-methyl-4-pyridone-5-carboxamide (4-PYR) 1-methyl-4-pyridone-5-carboxamide (4-PYR) rānei; Pikitia 3B) ka herea hoki ki te taupori o ngā fibroblast e whakaatu ana i te COL1A1 (Pikitia S5A), e tohu tahi ana kāore i te pūtau-T te kaha o te pākia o te MNA tikanga. I manatokohia te tauira whakapuakanga o ēnei ira e pā ana ki te MNA mā te whakamahi i tētahi huinga raraunga pūtau motuhake tuarua mai i ngā ascites mai i ngā tūroro HGSC (Pikitia S5B; n = 6) (16). Hei tāpiri, ko te tātaritanga tauhohenga mekameka polymerase quantitative (qPCR) o ngā pūtau-T kaituku hauora i rongoatia ki te MNA i whakaatu i te whakataurite ki ngā pūtau pukupuku ovarian SK-OV-3 mana whakahaere, tata kore te NNMT, te AOX1 rānei i whakapuakanga (Pikitia 3E). Ko ēnei hua ohorere e tohu ana ka huna pea te MNA mai i ngā fibroblast, i ngā pukupuku rānei ki roto i ngā pūtau-T tata i roto i te TME.
Ahakoa kei roto i ngā kaitono te whānau o ngā kaikawe cation organic 1 ki te 3 (OCT1, OCT2 me OCT3) i whakakōrerohia e te whānau kaikawe wairewa 22 (SLC22) (SLC22A1, SLC22A2 me SLC22A3), kāore anō kia tautuhia ngā kaikawe pea o te MNA (28). I whakaatuhia e te QPCR o te mRNA mai i ngā pūtau T kaituku hauora he iti ngā taumata whakaaturanga o SLC22A1 engari he iti ngā taumata o SLC22A2 e kitea ana, ā, i whakaūtia kua pūrongohia i mua i roto i ngā tuhinga (Pikitia 3F) (29). He rerekē, i whakaatuhia e te raina pūtau pukupuku ovarian SK-OV-3 he taumata teitei o ngā kaikawe e rua (Pikitia 3F).
Hei whakamātautau i te āheinga o ngā pūtau-T ki te mimiti i ngā MNA ke, i whakatōngia ngā pūtau-T kaitāko hauora mō ngā hāora 72 i te aroaro o ngā kukū rerekē o te MNA. I te korenga o te MNA o waho, kāore i taea te kite i te ihirangi pūtau o te MNA (Pikitia 3G). Heoi, ko ngā pūtau-T whakahohe i rongoatia ki te MNA o waho i whakaatu i te pikinga o te ihirangi MNA i roto i ngā pūtau, tae atu ki te 6 mM MNA (Pikitia 3G). E tohu ana tēnei hua ahakoa te iti o te taumata o te whakaaturanga kaikawe me te korenga o te whākōkī matua e kawenga ana mō te pākia o te MNA o roto, ka taea tonu e te TIL te tango i te MNA.
Ko te whānuitanga o ngā matū matū i roto i ngā pūtau-T o ngā tūroro me ngā whakamātautau mimiti MNA in vitro ka whakanui ake i te tūponotanga ka tukuna e ngā fibroblasts e pā ana ki te mate pukupuku (CAF) te MNA, ā, ka taea e ngā pūtau pukupuku te whakahaere i te āhua me te mahi a te TIL. Hei whakatau i te pānga o te MNA ki ngā pūtau-T, i whakahohehia ngā pūtau-T kaituku hauora i roto i te vitro i te aroaro, i te ngaro rānei o te MNA, ā, i aromatawaihia tō rātou tipu me te hanga cytokine. I muri i ngā rā e 7 o te tāpiri i te MNA i te horopeta teitei rawa, i whakaitihia te tau ruarua o te taupori, i te mea i mau tonu te kaha i ngā horopeta katoa (Pikitia 4A). Hei tāpiri, ko te maimoatanga o te MNA exogenous i hua ake ai te pikinga o te ōwehenga o ngā pūtau-T CD4 + me CD8 + e whakaatu ana i te tumor necrosis factor-α (TNFα; Pikitia 4B). He rerekē, ko te hanga IFN-γ i roto i te pūtau-T i tino heke i roto i ngā pūtau-T CD4 +, engari kāore i roto i ngā pūtau-T CD8 +, ā, kāore he huringa nui i roto i te interleukin 2 (IL-2; Pikitia 4, C me D). Nō reira, i whakaatuhia e te whakamātautau immunosorbent linked-enzyme (ELISA) o ngā wai i roto i ngā ipu mai i ēnei ahurea pūtau-T i rongoatia ki te MNA he pikinga nui o te TNFα, he hekenga o te IFN-γ, ā, kāore he huringa o te IL-2 (Pikitia 4, E ki te G). . Ko te hekenga o te IFN-γ e tohu ana tera pea ka whai wāhi te MNA ki te aukati i te mahi ārai-pukupuku o ngā pūtau-T. Hei whakatauira i te pānga o te MNA ki te cytotoxicity e whakahaerehia ana e te pūtau-T, ka whakaputaina ngā pūtau chimeric antigen receptor T (FRα-CAR-T) e aro ana ki te folate receptor α me ngā pūtau CAR-T (GFP) e whakahaerehia ana e ngā pūtau pūmua fluorescent matomato (GFP) -CAR-T) e ngā pūtau mononuclear toto peripheral donor hauora (PBMC). I whakatipuria ngā pūtau CAR-T mō te 24 hāora i te aroaro o te MNA, kātahi ka whakatipuria tahitia me ngā pūtau pukupuku ovarian SK-OV-3 tangata e whakaatu ana i te folate receptor α i te ōwehenga effector ki te ūnga o te 10:1. Nā te maimoatanga MNA i heke nui ai te mahi whakamate o ngā pūtau FRα-CAR-T, he rite tonu ki ngā pūtau FRα-CAR-T i rongoatia ki te adenosine (Pikitia 4H).
(A) Te tapeke o ngā pūtau ora me te takirua o te taupori (PD) mai i te ahurea i te rā 7. Ko te kauwhata pae e tohu ana i te toharite + SEM o ngā kaituku hauora e ono. E tohu ana i ngā raraunga mai i te iti rawa n = 3 ngā whakamātautau motuhake. (B ki D) I whakamahia a CD3/CD28 me IL-2 hei whakahohe i ngā pūtau T i ō rātou kukū MNA mō ngā rā e 7. I mua i te tātari, i whakaohokia ngā pūtau ki te PMA/ionomycin me GolgiStop mō ngā haora e 4. Te whakaaturanga TNFα (B) i roto i ngā pūtau T. Tauira ahua (maui) me ngā raraunga ripanga (matau) o te whakaaturanga TNFα i roto i ngā pūtau ora. Te whakaaturanga IFN-γ (C) me te IL-2 (D) i roto i ngā pūtau T. I inehia te whakaaturanga o ngā cytokine mā te cytometry rere. Ko te kauwhata pae e tohu ana i te toharite (n = 6 ngā kaituku hauora) + SEM. Whakamahia te tātaritanga kotahi-ara o te rerekētanga me ngā inenga anō (*P<0.05 me **P<0.01) hei whakatau i te uara P. E tohu ana i ngā raraunga mai i te iti rawa n = 3 ngā whakamātautau motuhake. (E ki G) I whakamahia te CD3/CD28 me te IL-2 hei whakahohe i ngā pūtau-T i ō rātou kukū MNA mō ngā rā e 7. I kohia te reo i mua atu i te 4 hāora o te whakaohooho PMA/ionomycin, ā, i muri mai. I inehia ngā kukū o te TNFα (E), IFN-γ (F) me te IL-2 (G) mā te ELISA. Ko te kauwhata pae e tohu ana i te toharite (n = 5 ngā kaituku hauora) + SEM. I whakatauhia te uara P mā te whakamahi i te tātaritanga rerekētanga kotahi-ara me ngā inenga anō (*P<0.05). Ko te rārangi iraira e tohu ana i te rohe kitenga o te kitenga. (H) Whakamātautau pakaru pūtau. I whakatikatikaina ngā pūtau FRα-CAR-T, GFP-CAR-T rānei ki te adenosine (250μM) MNA rānei (10 mM) mō ngā hāora 24, i waiho rānei kia kore e rongoatia (Ctrl). I inehia te ōrau whakamate o ngā pūtau SK-OV-3. I whakatauhia te uara P mā te whakamātautau t Welch (*P<0.5 me **P<0.01).
Hei whiwhi i tētahi māramatanga tikanga mō te whakahaere i te whakapuakanga TNFα e whakawhirinaki ana ki te MNA, i arotakehia ngā huringa o te TNFα mRNA o ngā pūtau T i rongoatia ki te MNA (Pikitia 5A). Ko ngā pūtau T kaitāko hauora i rongoatia ki te MNA i whakaatu i te pikinga rua o ngā taumata whakawhiti TNFα, e tohu ana kei te whakawhirinaki te MNA ki te whakahaere whakawhiti TNFα. Hei tirotiro i tēnei tikanga whakahaere pea, e rua ngā take whakawhiti e mōhiotia ana e whakahaere ana i te TNFα, arā, ko te take karihi pūtau T i whakahohetia (NFAT) me te pūmua motuhake 1 (Sp1), i arotakehia hei whakautu ki te herenga MNA ki te kaiwhakatairanga TNFα tata (30). Kei roto i te kaiwhakatairanga TNFα e 6 ngā pae herenga NFAT kua tautuhia me ngā pae herenga Sp1 e 2, e taupatupatu ana i te pae kotahi [-55 ngā takirua turanga (bp) mai i te 5'cap] (30). I whakaatuhia e te Chromatin immunoprecipitation (ChIP) i te wā i rongoatia ai ki te MNA, ka toru ngā pikinga o te herenga o Sp1 ki te kaiwhakatairanga TNFα. I piki ake hoki te whakaurunga o te NFAT, ā, i tata ki te hiranga (Pikitia 5B). E whakaatu ana ēnei raraunga ka whakahaere te MNA i te whakapuakanga o te TNFα mā te whakawhiti Sp1, ā, he iti iho te whakapuakanga o te NFAT.
(A) Ki te whakatauritea ki ngā pūtau-T i whakatipuria me te kore MNA, ko te panonitanga o te whakaaturanga TNFα i roto i ngā pūtau-T i rongoatia ki te MNA. Ka whakaaturia te tauira whakaaturanga me te SEM (n = 5 ngā kaituku hauora). E tohu ana i ngā raraunga mai i te iti rawa n = 3 ngā whakamātautau motuhake. (B) Ko te kaiwhakatairanga TNFα o ngā pūtau-T i rongoatia me te 8 mM MNA, kāore rānei i muri i te NFAT me te Sp1 i whakakotahihia ki te (Ctrl) me te whakaoho PMA/ionomycin mō ngā haora 4. I whakamahia te Immunoglobulin G (IgG) me te H3 hei mana whakahaere kino me te pai mō te immunoprecipitation. I whakaatuhia e te inenga o te ChIP ko te here o Sp1 me te NFAT ki te kaiwhakatairanga TNFα i roto i ngā pūtau i rongoatia ki te MNA i piki ake i ngā wā maha ki te whakataurite ki te mana whakahaere. E tohu ana i ngā raraunga mai i te iti rawa n = 3 ngā whakamātautau motuhake. Ko te uara P i whakatauhia e ngā whakamātautau-t maha (*** P <0.01). (C) Ki te whakatauritea ki ngā ascites o te HGSC, i whakaatuhia e ngā pūtau-T (kore-cytotoxic) te pikinga o te whakaaturanga o te TNF i roto i te pukupuku. Ko ngā tae e tohu ana i ngā tūroro rerekē. Kua tangohia matapōkeretia ngā pūtau kua whakaaturia ki te 300, ā, kua nekenekehia kia iti ai te nui o te toto (** Padj = 0.0076). (D) Tauira MNA kua whakaarohia mō te mate pukupuku ovarian. Ka hangaia te MNA i roto i ngā pūtau pukupuku me ngā fibroblast i roto i te TME, ā, ka tangohia e ngā pūtau T. Ka whakanuia e te MNA te here o Sp1 ki te kaiwhakatairanga TNFα, ka piki ake te whakaputanga TNFα me te hanga cytokine TNFα. Ka heke hoki te IFN-γ i roto i te MNA. Ko te aukati i te mahi a te pūtau T ka heke te kaha patu me te tere ake o te tipu o te pukupuku.
E ai ki ngā pūrongo, he pānga ā-mua, ā-muri hoki o te TNFα ki te ārai i te pukupuku me te ārai i te pukupuku, engari he tūranga rongonui tōna ki te whakatairanga i te tipu me te horapa o te mate pukupuku ovarian (31-33). E ai ki ngā pūrongo, he teitei ake te kukū o te TNFα i roto i ngā ascites me ngā kiko pukupuku i roto i ngā tūroro he mate pukupuku ovarian i ērā i roto i ngā kiko pai (34-36). Mō te tikanga, ka taea e te TNFα te whakahaere i te whakahohenga, te mahi me te tipu o ngā toto toto ma, me te whakarerekē i te momo o ngā pūtau mate pukupuku (37, 38). E rite ana ki ēnei kitenga, i whakaatuhia e te tātaritanga rerekētanga o te whakaaturanga ira he nui ake te pikinga o te TNF i roto i ngā pūtau T i roto i ngā kiko pukupuku ki te whakataurite ki te ascites (Pikitia 5C). Ko te pikinga o te whakaaturanga TNF i kitea noa i roto i ngā taupori pūtau T me te momo kore-cytotoxic (Pikitia S5A). Hei whakarāpopototanga, e tautoko ana ēnei raraunga i te tirohanga he rua ngā pānga ārai mate me te whakatairanga pukupuku o te MNA i roto i te HGSC.
Ko te tapanga hihi-ā-rangi i runga i te cytometry rere kua noho hei tikanga matua mō te ako i te pākia o te TIL. Kua whakaatuhia e ēnei rangahau, ki te whakatauritea ki ngā lymphocytes toto ā-taha, ki ngā pūtau T rānei mai i ngā whekau lymphoid tuarua, he nui ake te hiahia o te TIL kiore me te tangata ki te tango i te huka (4, 39) me te ngaronga āta o te mahi mitochondrial (19, 40). Ahakoa kua kitea e mātou ngā hua ōrite i roto i tēnei rangahau, ko te whanaketanga matua ko te whakataurite i te pākia o ngā pūtau pukupuku me te TIL mai i te kiko pukupuku kotahi i tangohia. E rite ana ki ētahi o ēnei pūrongo o mua, he nui ake te tango huka o ngā pūtau pukupuku (CD45-EpCAM +) mai i ngā ascites me ngā pukupuku i ngā pūtau T CD8 + me CD4 +, e tautoko ana i te taea te whakataurite i te tango huka teitei o ngā pūtau pukupuku ki ngā pūtau T. Ko te ariā o te whakataetae pūtau T. TME. Heoi, he teitei ake te mahi mitochondrial o ngā pūtau pukupuku i ngā pūtau T CD8 +, engari he rite te mahi mitochondrial ki ngā pūtau T CD4 +. Ka whakakaha ake ēnei hua i te kaupapa hou ko te pākia o te oxidative he mea nui mō ngā pūtau pukupuku (41, 42). E kī ana hoki rātou he ngāwari ake pea ngā pūtau CD8 + T ki te ngoikore o te waikura i ngā pūtau CD4 + T, he whakamahi rānei ngā pūtau CD4 + T i ngā pūtake waro ehara i te huka hei pupuri i te mahi mitochondrial (43, 44). Me mōhio kāore mātou i kite i tētahi rerekētanga i roto i te tango huka, i te mahi mitochondrial rānei i waenga i ngā CD4 + T effectors, te mahara T effector me ngā pūtau mahara T pokapū i roto i te ascites. Waihoki, ko te āhua rerekētanga o ngā pūtau CD8 + T i roto i ngā pukupuku kāore he pānga ki ngā huringa o te tango huka, e whakaatu ana i te rerekētanga nui i waenga i ngā pūtau T i whakatipuria i roto i te vitro me te TIL tangata i roto i te vivo (22). I whakaūhia hoki ēnei kitenga e te whakamahinga o te tohatoha taupori pūtau aunoa kore whakaaro, i whakaatu anō i te mea he nui ake te tango huka me te mahi mitochondrial o ngā pūtau pukupuku engari he taupori pūtau hohe Metabolic. Tērā pea ko tēnei taupori te taupori iti pea o ngā pūtau pehi myeloid, ngā pūtau dendritic plasmacytoid rānei i tautuhia i roto i te tātaritanga scRNA-seq. Ahakoa kua pūrongohia ēnei e rua i roto i ngā pukupuku whatukuhu tangata [45], me mahi tonu ēnei. Ko ētahi atu mahi ko te whakaahua i tēnei taupori myeloid.
Ahakoa ka taea e ngā tikanga e hangai ana ki te rere o te cytometry te whakamārama i ngā rerekētanga whānui o te huka me te pākia o te waikura i waenga i ngā momo pūtau, kāore anō kia whakatauhia ngā matū tika i hangaia e te huka, e ētahi atu puna waro rānei mō te pākia o te mitochondrial i roto i te TME. Ko te tohu i te aroaro, te ngaro rānei o ngā matū ki tētahi wāhanga TIL kua whakaritea me purenga te taupori pūtau mai i te kiko kua tangohia. Nō reira, ko tā mātou tikanga whakarei pūtau me te ine papatipu ka taea te whakarato māramatanga ki ngā matū e whakareihia ana i roto i ngā pūtau T me ngā taupori pūtau pukupuku i roto i ngā tauira tūroro e ōrite ana. Ahakoa he painga tēnei tikanga i runga i te whakarōpūtanga pūtau kua whakahohehia e te fluorescence, ka pāngia pea ētahi whare pukapuka matū nā te pumau ā-roto me/te tere hurihuri rānei (22). Heoi anō, i taea e tā mātou tikanga te tautuhi i ngā matū e rua e whakaiti ana i te kaha o te pūnaha ārai mate, te adenosine me te kynurenine, nā te mea he rerekē rawa atu i waenga i ngā momo tauira.
Mā tā mātou tātaritanga metabonomic o ngā pukupuku me ngā momo TIL ka nui ake ngā māramatanga ki te tūranga o ngā metabolites i roto i te ovarian TME. Tuatahi, mā te whakamahi i te flow cytometry, i whakatauhia e mātou kāore he rerekētanga o te mahi mitochondrial i waenga i ngā pukupuku me ngā pūtau CD4 + T. Heoi, i whakaatuhia e te tātaritanga LC-MS/MS ngā huringa nui o te nui o ngā metabolites i roto i ēnei taupori, e tohu ana me āta whakamārama ngā whakatau mō te metabolism TIL me tōna mahi metabolic whānui. Tuarua, ko te MNA te metabolite me te rerekētanga nui rawa atu i waenga i ngā pūtau CD45 me ngā pūtau T i roto i te ascites, ehara i te pukupuku. Nō reira, tera pea he rerekē ngā pānga o te wehewehenga me te taunga pukupuku ki te metabolism TIL, e whakaatu ana i te rerekētanga pea i roto i tētahi taiao moroiti. Tuatoru, ko te whakaaturanga o te whākōkī whakaputa MNA NNMT he iti noa iho te CAF, arā, ko ngā pūtau pukupuku, engari ka kitea ngā taumata MNA ka kitea i roto i ngā pūtau T i ahu mai i te pukupuku. Ko te nui rawa o te whakaaturanga o te NNMT i roto i te ovarian CAF he pānga whakatairanga matepukupuku e mōhiotia ana, nā te whakatairanga i te metabolism CAF, te whakaekenga pukupuku me te metastasis (27). Ahakoa he taurite te taumata whānui o te TIL, he hononga tata te whakaaturanga o te NNMT i roto i te CAF ki te momo mesenchymal Cancer Genome Atlas (TCGA), e hono ana ki te matapae kino (27, 46, 47). Hei whakamutunga, ko te whakaaturanga o te whākōkī AOX1 e kawenga ana mō te whakahekenga MNA e herea ana ki te taupori CAF, e tohu ana kāore i te kaha ngā pūtau-T ki te tukatuka i te MNA. E tautoko ana ēnei hua i te whakaaro ahakoa me mahi anō hei manatoko i tēnei kitenga, ko ngā taumata teitei o te MNA i roto i ngā pūtau-T ka tohu pea i te noho o tētahi taiao moroiti CAF e pehi ana i te pūnaha ārai mate.
I te mea he iti te taumata whakaaturanga o ngā kaikawe MNA me ngā taumata kore e kitea o ngā pūmua matua e whai wāhi ana ki te pākia o te MNA, he mea ohorere te aroaro o te MNA i roto i ngā pūtau-T. Kāore i taea te kite i te NNMT me te AOX1 mā te tātaritanga scRNA-seq me te qPCR kua arotahihia o ngā rōpū motuhake e rua. E whakaatu ana ēnei hua kāore te MNA i hangaia e ngā pūtau-T, engari i mimitihia mai i te TME e karapoti ana. E whakaatu ana ngā whakamātautau in vitro he maha ngā pūtau-T e kohikohi ana i te MNA o waho.
Kua whakaatuhia e ā mātou rangahau in vitro ko te MNA o waho e whakaoho ana i te whakaaturanga o te TNFα i roto i ngā pūtau T, ā, e whakarei ake ana i te here o Sp1 ki te kaiwhakatairanga TNFα. Ahakoa he mahi ārai-puku me te ārai-puku a te TNFα, i roto i te mate pukupuku ovarian, ka taea e te TNFα te whakatairanga i te tipu o te mate pukupuku ovarian (31-33). Ko te whakakorenga o te TNFα i roto i te ahurea pūtau pukupuku ovarian, te whakakorenga rānei o te tohu TNFα i roto i ngā tauira kiore ka taea te whakapai ake i te hanga cytokine mumura e whakahaerehia ana e te TNFα me te aukati i te tipu o te pukupuku (32, 35). Nō reira, i roto i tēnei take, ka taea e te MNA i ahu mai i te TME te mahi hei metabolite pro-inflammatory mā roto i te tikanga e whakawhirinaki ana ki te TNFα mā roto i te koropiko autocrine, ā, ka whakatairanga i te putanga mai me te horapa o te mate pukupuku ovarian (31). I runga i tēnei pea, kei te rangahauhia te aukati TNFα hei kaihoko rongoā pea mō te mate pukupuku ovarian (37, 48, 49). Hei tāpiri, ka whakararu te MNA i te cytotoxicity o ngā pūtau CAR-T ki ngā pūtau pukupuku ovarian, e whakarato ana i ētahi atu taunakitanga mō te pehitanga ārai mate e whakahaerehia ana e te MNA. I te katoa, e whakaatu ana ēnei hua i tētahi tauira e whakaputa ana ngā pūtau pukupuku me ngā pūtau CAF i te MNA ki roto i te TME o waho o te pūtau. Mā te (i) whakaohooho tipu o te mate pukupuku ovarian i puta mai i te TNF me te (ii) aukati i te mahi paitini pūtau-T i puta mai i te MNA, tera pea he pānga pukupuku takirua tēnei (Pikitia 5D).
Hei whakamutunga, mā te whakamahi i te huinga o te whakarei ake tere o te pūtau, te raupapatanga pūtau-kotahi me te whakatauira pākia, i whakakitea e tēnei rangahau ngā rerekētanga nui o te immunometabolomic i waenga i ngā pukupuku me ngā pūtau ascites i roto i ngā tūroro HGSC. I whakaatuhia e tēnei tātaritanga whānui he rerekētanga kei roto i te tango huka me te mahi mitochondrial i waenga i ngā pūtau-T, ā, i tautuhia te MNA hei metabolite whakahaere ārai mate motuhake kore-pūtau. He pānga ēnei raraunga ki te pānga o te TME ki te pākia o te pūtau-T i roto i ngā mate pukupuku tangata. Ahakoa kua pūrongohia te whakataetae tika mō ngā matūkai i waenga i ngā pūtau-T me ngā pūtau mate pukupuku, ka taea hoki e ngā metabolite te mahi hei kaiwhakahaere ā-korekore hei whakatairanga i te whanaketanga o te pukupuku me te pehi pea i ngā urupare ārai mate taketake. Ko te whakaahuatanga atu o te mahi a ēnei metabolite whakahaere ka whakatuwhera pea i ngā rautaki rerekē hei whakarei ake i te urupare ārai mate anti-pukupuku.
I kohia ngā tauira tūroro me ngā raraunga haumanu mā te putunga kiko pukupuku pukupuku BC kua whakamanahia e te Whatunga Putunga Tissue o Kanata. E ai ki te kawa i whakaaetia e te Komiti Matatika Rangahau Mate Pukupuku BC me te Whare Wānanga o British Columbia (H07-00463), i whiwhi whakaaetanga tuhituhi whai whakaaro, i whakakorea rānei ō rātou whakaaetanga e ngā tauira me ngā raraunga haumanu katoa a ngā tūroro. Kei te rongoa ngā tauira ki te BioBank kua whakamanahia (BRC-00290). Kei roto i ngā Ripanga S1 me S5 ngā āhuatanga taipitopito o te tūroro. Mō te tiaki-kāore, ka whakamahia he maripi hei wawahi ā-mīhini i te tauira pukupuku o te tūroro, kātahi ka panaia mā roto i te tātari 100-micron kia whiwhi ai i tētahi whakatārewatanga pūtau kotahi. I poipoia te ascites o te tūroro i te 1500 rpm mō te 10 meneti i te 4°C hei whakapūpū i ngā pūtau me te tango i te waikura. Ko ngā pūtau i tangohia mai i te puku me te ascites i tiakina ki roto i te 50% serum AB tangata i whakakorea ki te wera (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) me te 10% dimethyl sulfoxide. I whakarewaina ēnei whakatārewatanga pūtau kotahi i tiakina, ā, i whakamahia mō te whakatau i te metabolomics me te metabolite e whakaahuatia ana i raro nei.
Ko te rauemi katoa he 0.22 μm kua tātarihia 50:50 kua tāpirihia ki te RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) kua tāpirihia ki te 10% serum tangata AB kua whakakorea-wera (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x otinga Penicillin Streptomycin (PenStrep) (Thermo Fisher Scientific) me te 50 μMB-mercaptoethanol. Kua tāpirihia te AimV (Invitrogen) ki te 20 mM Hepes (Thermo Fisher Scientific) me te 2 mM l-glutamine (Thermo Fisher Scientific). Ko te arai tae cytometer rere he 0.22μm saline phosphate buffered kua tātarihia (PBS; Invitrogen) kua tāpirihia ki te 3% serum tangata AB kua whakakorea-wera (Sigma). Ko te arai whakarei pūtau he mea tito mai i te 0.22μm PBS kua tātarihia, ā, he mea tāpiri atu ki te 0.5% serum AB tangata kua whakakorea-werahia (Sigma-Aldrich).
I roto i te reo katoa 37°C, i pania ngā pūtau ki te 10 nM MT DR me te 100 μM 2-NBDG mō te 30 meneti. Muri iho, i pania ngā pūtau ki te tae ora eF506 i te 4°C mō te 15 meneti. Whakatārewatia anō ngā pūtau ki roto i te FC Block (eBioscience) me te Brilliant Stain Buffer (BD Biosciences), whakaranua ki roto i te pūtāhui paninga cytometry rere (e ai ki ngā tohutohu a te kaihanga), ā, whakatōkia mō te 10 meneti i te pāmahana rūma. Whakatārewatia ngā pūtau ki tētahi huinga paturopi (Ripanga S2) ki roto i te pūtāhui paninga cytometry rere i te 4°C mō te 20 meneti. Whakatārewatia anō ngā pūtau ki roto i te pūtāhui paninga cytometry rere (Cytek Aurora; whirihoranga 3L-16V-14B-8R) i mua i te tātari. Whakamahia a SpectroFlo me FlowJo V10 hei tātari i ngā raraunga tatau pūtau, ā, whakamahia a GraphPad Prism 8 hei hanga i ngā raraunga. I whakaōritehia te kaha o te hihi-mārama waenga (MFI) o te 2-NBDG me te MT DR mā te takiuru, kātahi ka whakamahia he whakamātautau-t takirua mō te tātari tatauranga hei whakauru i ngā tūroro ōrite. Tangohia ngā taupori katoa he iti iho i te 40 ngā takahanga mai i te tātari; whakauruhia he uara MFI o te 1 mō ngā uara kino i mua i te whakahaere i te tātari tatauranga me te whakaaturanga raraunga.
Hei tāpiri atu ki te rautaki ārai ā-ringa o te papa tukanga i runga ake nei, i whakamahia e mātou te tuhipoka katoa e te rākau here āhua (FAUST) (21) hei tuku aunoa i ngā pūtau ki te taupori i muri i te tango i ngā pūtau mate i roto i te FlowJo. Ka whakahaerehia ā-ringa e mātou te putanga hei whakakotahi i ngā taupori e ahua hē ana te tohatoha (te whakakotahi i te PD1+ me ngā pūtau pukupuku PD1) me ngā taupori e puritia ana. Kei roto i ia tauira he toharite neke atu i te 2% o ngā pūtau, mō te katoa o ngā taupori 11.
I whakamahia te whakapūtanga kiato o te Ficoll hei wehe i te PBMC mai i ngā hua wehenga leukocyte (STEMCELL Technologies). I wehea ngā pūtau CD8 + T mai i te PBMC mā te whakamahi i te CD8 MicroBeads (Miltenyi) ā, i whakawhanuitia ki roto i te reo katoa mā te whakamahi i te TransAct (Miltenyi) mō ngā wiki e rua e ai ki ngā tohutohu a te kaihanga. I tukuna ngā pūtau kia tū mō ngā rā e rima i roto i te reo katoa kei roto ko te IL-7 (10 ng/ml; PeproTech), kātahi ka whakaohokia anō ki te TransAct. I te rā 7, e ai ki ngā tohutohu a te kaihanga, i whakamahia ngā MicroBeads CD45 tangata (Miltenyi) hei whakarei ake i ngā pūtau i roto i ngā rauna e toru karapīpiti. I wehea ngā pūtau mō te tātaritanga cytometry rere (e ai ki te whakaahuatanga i runga ake nei), ā, kotahi miriona ngā pūtau i wehea kia toru ngā wā mō te tātaritanga LC-MS/MS. I tukatukahia ngā tauira mā te LC-MS/MS e ai ki te whakaahuatanga i raro nei. I whakatau tata mātou i te uara metabolite ngaro me te tau katote o te 1,000. Ka whakatauritea ia tauira e te tau katote katoa (TIC), ka tahurihia mā te taupū, ā, ka whakatauritea aunoatia ki MetaboAnalystR i mua i te tātari.
I whakarewaina te whakatārewatanga pūtau kotahi o ia tūroro, ā, i tātarihia mā te tātari 40 μm ki roto i te reo katoa (e ai ki te whakaahuatanga i runga ake nei). E ai ki te tikanga a te kaihanga, e toru ngā huringa whiriwhiri pai mā te wehenga pirepire aukume mā te whakamahi i ngā MicroBeads (Miltenyi) i whakamahia hei whakarei ake i ngā tauira mō ngā pūtau CD8+, CD4+ me CD45- (i runga i te hukapapa). I roto i te poto, ka whakatārewatia anō ngā pūtau ki roto i te arai whakarei pūtau (e ai ki te whakaahuatanga i runga ake nei) ā, ka tatauhia. I whakatōkia ngā pūtau ki ngā pirepire CD8 tangata, ngā pirepire CD4 tangata, ngā pirepire CD45 tangata rānei (Miltenyi) i te 4°C mō te 15 meneti, kātahi ka horoia ki te arai whakarei pūtau. Ka tukuna te tauira mā te pou LS (Miltenyi), ā, ka kohia ngā wāhanga pai me ngā wāhanga kino. Hei whakaiti i te roa me te whakanui ake i te taahiraa whakaora pūtau, ka whakamahia te wāhanga CD8 mō te huringa tuarua o te whakarei CD4+, ā, ka whakamahia te wāhanga CD4 mō te whakarei CD45 e whai ake nei. Puritia te otinga ki runga i te hukapapa puta noa i te tukanga wehenga.
Hei whakarite i ngā tauira mō te tātaritanga matū matū, i horoia ngā pūtau kotahi ki te wai tote makariri, ā, i tāpirihia he 1 ml o te 80% methanol ki ia tauira, kātahi ka hurihia, ka tio ki roto i te hauota wai. I tukuna ngā tauira ki ngā huringa tio-rewa e toru, ā, i whakapūtāhia i te 14,000 rpm mō te 15 meneti i te 4°C. Ka mimitia te wai kei roto ngā matū ... kore matū matū matū kore matū matū kore matū matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū kore matū
Tangohia ngā matū matū e ai ki te whakaahuatanga i runga ake nei. Whakawhitihia te wai i roto i te pounamu chromatography wai mahi teitei mō te rangahau metabolomics. Whakamahia he tikanga maimoatanga matapōkere hei rongoā i ia tauira me te maha rite o ngā pūtau hei ārai i ngā pānga puranga. I whakahaerehia e mātou he aromatawai kounga o ngā matū matū matū ā-ao i whakaputaina i mua i runga i te AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). I whakahaerehia te tātari chromatographic me te whakaurunga rohe tihi mā te whakamahi i te pūmanawa MultiQuant putanga 2.1 (Applied Biosystems SCIEX).
I whakamahia he tatau katote o te 1000 hei whakatau tata i te uara metabolite ngaro, ā, i whakamahia te TIC o ia tauira hei tatau i te horahanga tihi kua whakatauritea o ia metabolite kua kitea hei whakatika i ngā huringa i puta mai i te tātari taputapu mai i te tukatuka tauira. I muri i te whakatauritea o te TIC, ka whakamahia te MetaboAnalystR(51) (tawhā taunoa) mō te tahuritanga logarithmic me te tauine raina paerewa aunoa. I whakamahia e mātou te PCA me te kete vegan R hei mahi tātari tūhura i ngā rerekētanga metabolome i waenga i ngā momo tauira, ā, i whakamahia te tātari tauutuutu wāhanga hei tātari i ngā tūroro. Whakamahia te tikanga Ward hei hanga i tētahi dendrogram mahere wera hei whakarōpū i te tawhiti Euclidean i waenga i ngā tauira. I whakamahia e mātou te limma (52) i runga i te nui o te metabolite paerewa hei tautuhi i ngā metabolite rerekē te nui puta noa i te momo pūtau katoa me te taiao moroiti. Hei whakangawari i te whakamārama, ka whakamahia e mātou te tawhā toharite rōpū hei tohu i te tauira, ā, ka whakaarohia ngā momo pūtau i roto i te taiao moroiti hei ia rōpū (n = 6 ngā rōpū); mō te whakamātautau hiranga, i mahia e mātou e toru ngā inenga tāruarua mō ia metabolite Hei karo i te tāruarua teka, i whakaurua te tūroro hei arai i roto i te hoahoa limma. Hei tirotiro i ngā rerekētanga o ngā metabolites i waenga i ngā tūroro rerekē, i whakatikatikaina e mātou te tauira limma tae atu ki ngā tūroro i runga i tētahi huarahi pumau. Ka ripoatahia e mātou te hiranga o te rerekētanga kua tohua i mua i waenga i te momo pūtau me te taiao moroiti o Padj <0.05 (whakatikatika Benjamini-Hochberg).
I muri i te whakarei ake i te kaha mā te whakamahi i te Miltenyi Dead Cell Removal Kit (>80% te ora), i whakahaerehia te raupapatanga transcriptome pūtau-kotahi ki runga i ngā tauira ascites tio ora katoa me ngā tauira pukupuku mā te whakamahi i te kawa whakaaturanga ira 10x 5′. E rima ngā take me ngā pukupuku me ngā ascites e ōrite ana i tātarihia, ahakoa ko te iti o te ora mai i te tauira pukupuku kotahi i aukati i tōna whakaurunga. Hei whakatutuki i ngā kōwhiringa maha o ngā tūroro, i whakakotahihia e mātou ngā tauira o ia tūroro ki ngā ara o te kaiwhakahaere chromium 10x, ā, i tātarihia motuhaketia ngā ascites me ngā wāhi pukupuku. I muri i te raupapatanga [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; [he toharite o te 73,488 me te 41,378 ngā pānui mō ia pūtau mō te pukupuku me te ascites]], i whakamahia e mātou a CellSNP me Vireo (53) (i runga i te CellSNP hei Ko te SNP tangata noa (VCF) i whakaratohia e GRCh38 kua tohaina he tuakiri kaituku. Ka whakamahia e mātou a SNPRelate hei whakatau i te tuakiri tata (IBS) o te tūnga iraira o te tūroro (IBS), me te kore e whakauru i ngā pūtau me ngā pūtau kāore i tohaina hei takirua me ngā kaituku ōrite i waenga i ngā ascites me ngā tauira pukupuku (54). I runga i te kaupapa o tēnei mahi, i puritia e mātou e toru ngā take me te nui o te whakaaturanga pūtau i roto i te pukupuku me te ascites mō te tātaritanga o raro. Whai muri i te mahi i te taahiraa tātari papatipu i roto i te takai BioConductor scater (55) me te scran (56), i puta mai he 6975 pūtau (2792 me te 4183 pūtau mai i te pukupuku me te ascites, ia) mō te tātaritanga. Ka whakamahia e mātou te whakarōpū Louvain a igraph (57) o te whatunga tata tata tiritahi (SNN) i runga i te tawhiti Jaccard ki ngā pūtau whakarōpū mā te whakaaturanga. I tātaitia ā-ringa ngā tautau ki ngā momo pūtau pea i runga i te whakaaturanga ira tohu, ā, i tirohia ki te t-SNE. Ko ngā pūtau T cytotoxic ka tautuhia e te whakaaturanga o te CD8A me te GZMA, engari kāore ngā tautau iti he iti te whakaaturanga pūmua ribosomal. I uru atu mātou ki ngā raraunga kua whakaputaina a Izar et al. (16), tae atu ki tā rātou whakaurunga t-SNE, ka taea te whakahaere i te taupatupatu whakaaturanga i waenga i ngā tohu pūtau ārai mate me te whakaaturanga NNMT.
I wehea ngā PBMC mai i ngā hua wehenga leukocyte (STEMCELL Technologies) mā te whakamahi i te Ficoll gradient density centrifugation. I wehea ngā pūtau CD3 + mai i te PBMC mā te whakamahi i ngā pītiti CD3 (Miltenyi). I te aroaro, i te ngaro rānei o te MNA, i whakahohehia ngā pūtau CD3+ me te CD3 here-pereti (5μg/ml), te CD28 wairewa (3μg/ml) me te IL-2 (300 U/ml; Proleukin). I te rā whakamutunga o te whakawhānui, i aromatawaihia te oranga (Fixable Viability Dye eFluor450, eBioscience) me te tipu (123count eBeads, Thermo Fisher Scientific) mā te whakamahi i te flow cytometry. Aromatawaihia te mahi a te kaiwhakaputa mā te whakaoho i ngā pūtau ki te PMA (20 ng/ml) me te ionomycin (1μg/ml) me te GolgiStop mō ngā hāora e 4, ā, aroturukihia te CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) me te TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Whakaohokia ngā pūtau qPCR me te ChIP ki te PMA (20 ng/ml) me te ionomycin (1μg/ml) mō ngā hāora e 4. I kohia te wai whakapūkara ELISA i mua atu, ā, i muri mai i te whakaoho ki te PMA (20 ng/ml) me te ionomycin (1 μg/ml) mō ngā hāora e 4.
Whāia te tikanga a te kaihanga hei wehe i te RNA mā te whakamahi i te RNeasy Plus Mini Kit (QIAGEN). Whakamahia te QIAshredder (QIAGEN) hei whakakotahi i te tauira. Whakamahia te kete RNA ki te cDNA kaha-teitei (Thermo Fisher Scientific) hei hanga i te DNA tautoko (cDNA). Whakamahia te TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) hei ine i te whakaaturanga ira (e ai ki te tikanga a te kaihanga) me ēnei whakamātautau: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] me Hs01010726_m1 (SLC22A2). I whakahaerehia ngā tauira i runga i te pūnaha PCR wā-tūturu a StepOnePlus (Applied Biosystems) (Applied Biosystems) i roto i te pereti tauhohenga whatu tere 96-puna MicroAmp (Applied Biosystems) me te kiriata whatu MicroAmp. Ko te uara Ct e neke atu ana i te 35 ka kiia kei runga ake i te paepae kitenga, ā, ka tohua he kore e taea te kite.
Mahia te ChIP e ai ki te whakaahuatanga o mua (58). I roto i te poto, i rongoatia ngā pūtau ki te formaldehyde (te kukū whakamutunga 1.42%), ā, i whaowhia ki te pāmahana rūma mō te 10 meneti. Whakamahia te pūwero pupuhi tāpiri (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl me te 0.1% NP-40) ki runga i te hukapapa mō te 10 meneti, kātahi ka whakatārewatia anō ki roto i te pūwero immunoprecipitation e ai ki te whakaahuatanga (58). Kātahi ka sonicated te tauira me ngā huringa e whai ake nei: 10 huringa (20 ngā pātere 1-hēkona) me te wā tūmau o te 40 hēkona. Whakawhaowhia te immunoglobulin G ChIP-grade (Hangarau Tohu Pūtau; 1μl), histone H3 (Hangarau Tohu Pūtau; 3μl), NFAT (Invitrogen; 3μl) me te SP1 (Hangarau Tohu Pūtau; 3μl) me te tauira i te 4°CC rūrūtia i te pō. Whakatōkia ngā poroporo pūmua A (Thermo Fisher Scientific) ki te tauira i te 4°C me te rūrū mārie mō te 1 hāora, kātahi ka whakamahia ngā poroporo chelex (Bio-Rad) hei whakarei ake i te DNA, ā, whakamahia te proteinase K (Thermo Fisher) hei nakunaku pūmua. I kitea te kaiwhakatairanga TNFα mā te PCR: whakamua, GGG TAT CCT TGA TGC TTG TGT; i te ritenga kē, GTG CCA ACA ACT GCC TTT ATA TG (hua 207-bp). I hangaia ngā whakaahua e Image Lab (Bio-Rad) ā, i tauahia mā te whakamahi i te pūmanawa ImageJ.
I kohia te wai whakapūkara o te ahurea pūtau e ai ki te whakaahuatanga i runga ake nei. I mahia te whakatau i runga anō i ngā tikanga a te kaihanga mō te kete TNFα ELISA tangata (Invitrogen), te kete IL-2 ELISA tangata (Invitrogen) me te kete IFN-γ ELISA tangata (Abcam). E ai ki te tikanga a te kaihanga, i whakaranua te wai whakapūkara ki te 1:100 hei kite i te TNFα me te IL-2, me te 1:3 hei kite i te IFN-γ. Whakamahia te EnVision 2104 Multilabel Reader (PerkinElmer) hei ine i te mimiti i te 450 nm.
I wehea ngā PBMC mai i ngā hua wehenga leukocyte (STEMCELL Technologies) mā te whakamahi i te Ficoll gradient density centrifugation. I wehea ngā pūtau CD3 + mai i te PBMC mā te whakamahi i ngā titi CD3 (Miltenyi). I te aroaro, i te ngaro rānei o te MNA, i whakahohehia ngā pūtau CD3+ ki te CD3 here-pereti (5μg/ml), te CD28 wairewa (3μg/ml) me te IL-2 (300 U/ml; Proleukin) mō ngā rā e 3. I muri i ngā rā e 3, i kohia ngā pūtau, ā, i horoia ki te 0.9% saline, ā, i tio wawetia te pirepire. I mahia te tatau pūtau mā te flow cytometry (Cytek Aurora; whirihoranga 3L-16V-14B-8R) mā te whakamahi i te 123count eBeads.
Tangohia ngā matū matū e ai ki te whakaahuatanga i runga ake nei. I hangaia anō te tango maroke ki te kukū o te 4000 pūtau ōrite/μl. Tātarihia te tauira mā te chromatography wāhanga-hurihuri (1290 Infinity II, Agilent Technologies, Santa Clara, CA) me te pou CORTECS T3 (2.1×150 mm, rahi matūriki 1.6-μm, rahi pore 120-Å; #186008500, Waters). Ine papatipu polar (6470, Agilent), e mahi ana te electrospray ionization i roto i te aratau pai. Ko te wāhanga pūkoro A he 0.1% waikawa formic (i roto i te H2O), ko te wāhanga pūkoro B he 90% acetonitrile, 0.1% waikawa formic. Ko te pikinga LC he 0 ki te 2 meneti mō te 100% A, 2 ki te 7.1 meneti mō te 99% B, me te 7.1 ki te 8 meneti mō te 99% B. Kātahi ka whakataurite anō i te pou ki te wāhanga neke A i te tere rere o te 0.6 ml/min mō te 3 meneti. . Ko te tere rere he 0.4ml/min, ā, ka whakamahanahia te rūma pou ki te 50°C. Whakamahia te paerewa matū parakore a MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) hei whakatū i te wā pupuri (RT) me te whakawhiti (RT = 0.882 meneti, whakawhiti 1 = 137→94.1, whakawhiti 2 = 137→92, Whakawhiti 3 = 137→78). Ina puta ngā whakawhiti e toru i te wā pupuri tika, ka whakamahia te whakawhiti 1 mō te ine hei whakarite i te motuhaketanga. I hangaia te pihi paerewa o te MNA (Toronto Research Chemical Company) mā te ono o ngā whakakōkī raupapa o te otinga rakau (1 mg/ml) hei whiwhi i ngā paerewa o te 0.1, 1.0, 10 me te 100 ng/ml me te 1.0 me te 10μg/ml wai. Ko te rohe kitenga he 1 ng/ml, ā, ko te urupare rārangi kei waenganui i te 10 ng/ml me te 10μg/ml. Ka whakamahia ia werohanga o ngā rita iti e rua o te tauira me te paerewa mō te tātari LC/MS, ā, ka whakahaerehia he tauira whakahaere kounga whakauru i ia waru werohanga hei whakarite i te pumau o te tūāpapa tātari. Ko ngā urupare MNA o ngā tauira pūtau katoa i rongoatia ki te MNA i roto i te awhe rārangi o te whakamātautau. I mahia te tātari raraunga mā te whakamahi i te pūmanawa tātari tauanga MassHunter (v9.0, Agilent).
I tangohia te hanganga αFR-CAR o te whakatupuranga tuarua mai i a Song et al. (59). I roto i te poto, kei roto i te hanganga ngā ihirangi e whai ake nei: te raupapatanga kaiārahi CD8a, te wāhanga taurangi mekameka kotahi motuhake αFR tangata, te rohe hononga CD8a me te rohe whakawhiti-membrane, te rohe pūtau CD27 me te rohe pūtau CD3z. I hangaia te raupapatanga CAR katoa e GenScript, kātahi ka tāruatia ki roto i te vector whakaaturanga lentiviral whakatupuranga tuarua i mua o te rīpene whakaaturanga GFP i whakamahia hei aromatawai i te whai huatanga o te whakawhiti.
Ka whakaputahia te Lentivirus mā te whakawhiti i ngā pūtau HEK293T [American Type Culture Collection (ATCC); ka whakatipuria i roto i te reo Eagle whakarerekētia a Dulbecco kei roto ko te 10% fetal bovine serum (FBS) me te 1% PenStrep, ā, ka whakamahia te CAR-GFP vector me te whakamahi i te lipofection amine (Sigma-Aldrich). I kohia te waikeri kei roto i te huaketo i muri i te 48 me te 72 hāora i muri i te whakawhiti, ka tātarihia, ka whakapūmau mā te ultracentrifugation. Penapenahia te waikeri huaketo kua whakapūmau ki te -80°C tae noa ki te whakawhiti.
Ka wehea ngā PBMC mai i ngā hua wehenga leukocyte kaituku hauora (STEMCELL Technologies) mā te whakamahi i te Ficoll gradient density centrifugation. Whakamahia ngā poitūkohu CD8 pai (Miltenyi) hei wehe i ngā pūtau CD8+ mai i te PBMC. Whakaohokia ngā pūtau T ki te TransAct (Miltenyi) me te reo TexMACS [Miltenyi; tāpirihia ki te 3% serum tangata kua whakakorea e te wera, 1% PenStrep me te IL-2 (300 U/ml)]. E rua tekau mā whā hāora i muri i te whakaoho, ka whakawhitia ngā pūtau T ki te lentivirus (10 μl supernatant huaketo kukū mō ia 106 pūtau). 1 ki te 3 rā i muri i te whakawhiti i runga i te Cytek Aurora (i runga i te FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), aromatawaihia te whakaaturanga GFP o ngā pūtau hei whakaatu i te whai huatanga whakawhiti o te iti rawa 30%.
I whakatōngia ngā pūtau CAR-T mō ngā hāora 24 i roto i te Immunocult (STEMCELL Technologies; i tāpirihia ki te 1% PenStrep) i raro i ngā āhuatanga e whai ake nei: kāore i rongoātia, i rongoātia ki te 250 μM adenosine, ki te 10 mM MNA rānei. I muri i te maimoatanga o mua, i horoia ngā pūtau CAR-T ki te PBS, ā, i honoa ki te 20,000 ngā pūtau SK-OV-3 [ATCC; i roto i te reo McCoy 5A (Sigma-Aldrich) i tāpirihia ki te 10% FBS me te 1% PenStrep i te 10: I whakanuia te ōwehenga pānga ki te ūnga o te 1 i roto i te toru o ngā tārua i roto i te reo Immunocult i tāpirihia. I whakamahia ngā pūtau SK-OV-3 me ngā pūtau SK-OV-3 i wetekina ki te saponin digitalis (0.5mg/ml; Sigma-Aldrich) hei mana whakahaere kino me te mana whakahaere pai. I muri i ngā hāora 24 o te whakatipu tahi, ka kohia te wai i roto i te ipu, ā, ka inehia te lactate dehydrogenase (LDH) e ai ki ngā tohutohu a te kaihanga (LDH Glo Cytotoxicity Assay Kit, Promega). I whakaranua te wai i roto i te ipu LDH i te 1:50 i roto i te LDH buffer. I inehia te ōrau o te patunga mā te whakamahi i te tātai e whai ake nei: ōrau o te patunga = ōrau o te whakatikatika / te tere patunga mōrahi x 100%, ko te ōrau o te whakatikatika = ngā pūtau-T whakatipu tahi anake, ā, ko te tere patunga mōrahi = te mana whakahaere pai-mana whakahaere kino.
E ai ki te tuhinga, ki ngā rauemi me ngā tikanga kua whakamāramahia, whakamahia te GraphPad Prism 8, Microsoft Excel, R v3.6.0 rānei mō te tātari tatauranga. Mena he maha ngā tauira ka kohia mai i te tūroro kotahi (pēnei i te ascites me te pukupuku), ka whakamahia e mātou he whakamātautau-t takirua, ka whakaurua rānei te tūroro hei pānga matapōkere i roto i tētahi tauira raina, tauira whānui rānei e tika ana. Mō te tātari metabolomics, ka mahia te whakamātautau hiranga i roto i ngā tāruarua e toru.
Mō ngā rauemi tāpiri mō tēnei tuhinga, tirohia http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
He tuhinga urunga-tuwhera tēnei e tohatohahia ana i raro i ngā tikanga o te Raihana Creative Commons Attribution-Non-Commercial, e āhei ai te whakamahinga, te tohatoha me te tārua i roto i tētahi momo pāpāho, mena kāore te whakamahinga whakamutunga mō te whai hua arumoni, ā, ko te whakaaro matua he tika te mahi taketake. Tohutoro.
Kia mōhio: Ko tā mātou anake he tuku mai i tō wāhitau īmēra kia mōhio ai te tangata e taunaki ana koe ki te whārangi e hiahia ana koe kia kite rātou i te īmēra, ā, ehara i te pāme. Kāore mātou e hopu i tētahi wāhitau īmēra.
Ka whakamahia tēnei pātai hei whakamātautau mēnā he manuhiri koe, hei ārai hoki i te tuku pāme aunoa.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (GBerardi Jones), De Bellardini (GBerardi Jones), De Bellardini (GBerardi Jones) Pinea T. Kirikiriroa (Phineas T.
Ka whai wāhi te MNA ki te pehi i te ārai mate o ngā pūtau-T, ā, he whāinga pea tēnei mō te rongoā i te mate pukupuku tangata.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (GBerardi Jones), De Bellardini (GBerardi Jones), De Bellardini (GBerardi Jones) Pinea T. Kirikiriroa (Phineas T.
Ka whai wāhi te MNA ki te pehi i te ārai mate o ngā pūtau-T, ā, he whāinga pea tēnei mō te rongoā i te mate pukupuku tangata.
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