胆管癌单胺氧他酶A对CD8＋T细胞抗肿瘤免疫功能的影响

Effect of monoamine oxidase A on anti - cancer immunity of CD8 ＋ T cells in cholangiocarcinoma

目的观察胆管癌细胞株单胺氧化酶A（MAOA）对CD8＋T细胞抗肿瘤免疫功能的影响。方法采用Tet—on3G诱导表达系统在人胆管癌细胞株中过表达MAOA；从正常人外周血中分离CD8＋T细胞，分别与过表达MAOA及低表达MAOA的胆管癌细胞混合培养48h，流式细胞仪检测CD8＋T细胞表达颗粒酶B、穿孔素、程序性死亡受体（PD）-1、细胞毒T淋巴细胞相关抗原（CTLA）4的水平，检测各组CD8＋T细胞的坏死和凋亡；酶联免疫吸附试验（ELISA）检测CD8＋T细胞分泌干扰素（IFN）-γ的水平；CD8＋T细胞分别与上述胆管癌细胞株混合培养48h进行肿瘤杀伤试验，检测各组肿瘤细胞释放乳酸脱氢酶（LDH）的水平。结果与过表达MAOA胆管癌细胞组比较，低表达MAOA胆管癌细胞组的CD8＋T细胞表达颗粒酶B、穿孔素减少（分别为44．2％比51．5％，P=0．000和51．8％比74．3％，P=0．000），表达PD-1及CTLA-4水平较高（分别为81．70％比21．30％，P=0．000和16．00％比8．49％，P=0．000），分泌IFN-1减少[（7562．0±927．5）ps／ml比（10107．0±1162．0）pg／ml，P=0．229]，CD8＋T细胞凋亡比例较高（26．43％比13．35％，P=0．000），肿瘤细胞释放LDH水平较低[（12．70±1．49）％比（22．88±2．93）％，P=0．036]，提示该组CD8＋T细胞肿瘤杀伤能力降低。结论胆管癌MAOA表达下调可抑制CD8＋T细胞抗肿瘤免疫功能，MAOA可望作为调控胆管癌肿瘤免疫的新靶点。

Objective To investigate the effect of monoamine oxidase A （MAOA） in cholangio- carcinoma on anti - tumor immunity of CD8 ＋ T cells. Methods We established stably inducible MAOA expression human cholangiocarcinoma cell lines by transfecting Tet - on 3G system. CD8 ＋ T cells were iso- lated from peripheral blood donated by healthy donors. After coculture with above celllines for 48 h, CD8 ＋ T cells were analyzed by flow cytometry to detect the expression of granzyme B, perforin, programmed death 1 （PD - 1 ）, cytotoxic T lymphocyte - associated antigen - 4 （ CTLA - 4）, as well as apoptosis rate. En- zyme linked immunosorbent assay （ELISA） was applied to assess the excretion of IFN - γ from CD8 ＋ T cells. Lactate dehydrogenase （LDH） test was performed to evaluate the cytotoxicity against the mixed cul- tured tumor cells by CD8 ＋ T cells. Results CD8 ＋ T cells in mock transfected groups expressed lower level of granzyme B （44. 2% vs. 51.5% , P =0. 000）, perforin （51.8% vs. 74. 3% , P =0. 000）, expressed higher level of PD - 1 and CTLA -4 （81.70% vs. 21.30% , P =0. 000;16. 00% vs. 8.49% ,P =0. 000 respectively）, secreted fewer IFN - γ （7 562.0 ± 927. 5 ） pg/ml vs. （ 10 107.0 ± 1 162.0） pg/ml, P = 0. 229） and had a higher apoptosis rate （26. 43% vs. 13.35% ,P =0. 000）, and the cytotoxieity CD8 ＋ T cells was also suppressed [ （12. 70 ± 1. 49 ） % vs. （22.88±2.93）%,P=0.036].Conclusion Down- regulation of MAOA in cholangiocarcinoma would impede anti - tumour immunity of CD8＋ T cells.