可溶性Fas阻断肝癌细胞通过FasFasL途径触发T淋巴细胞的凋亡

Soluble Fas blocks the apoptosis of T lymphocytes induced by Fas/FasL of hepatic cancer cells

目的 通过阻断Fas介导T细胞凋亡 ,建立快速大量激活制备肿瘤特异性细胞毒性T淋巴细胞 (CTL)的方法。方法 分离肝癌细胞和肿瘤浸润淋巴细胞 (TIL)。选择FasL表达阳性的肝癌标本 ,在体外将两者混合 ,并在CD2 8单抗共刺激下 ,激活制备特异性CTL。应用可溶性Fas受体阻断肝癌细胞通过Fas FasL途径触发激活T细胞凋亡 ,与对照组比较观察阻断凋亡作用 ,通过3H掺入法和51 Cr释放法了解T细胞增殖杀伤活性。结果 流式细胞术仪检测未阻断组较阻断组和未阻断对照组凋亡率明显升高 ,未阻断组凋亡率达 4 7 82 %± 0 13% ,静息性T淋巴细胞组为 3 76 %± 0 2 5 % ,阻断组为 8 2 2 %± 0 2 6 % (P <0 0 1) ,DNAladder显示未阻断组T淋巴细胞出现明显梯状条带 ,阻断组为阴性。51 Cr释放法表明阻断后T淋巴细胞杀伤活性增加 ,较未阻断组及未阻断对照组差异有显著性 (P<0 0 1)。3H掺入法检测证实可溶性Fas受体阻断激活T细胞凋亡后 ,细胞增殖明显升高 ,较未阻断组差异有显著性 (P <0 0 1)。结论 体外实验可获得大量激活T细胞 ,并可杀伤肿瘤细胞 ,

Objectives The purpose of this study is to block the apoptosis of T lymphocytes induced by Fas, to establish a method of quick preparation of abundant carcinoma specific CTL Methods Liver carcinoma cells and TIL were isolated from FasL positive fresh specimens, and mixed Specific CTL was activated and prepared in the presence of the co stimulation of monoclonal antibody CD28 After that, the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder, simultaneously The effect was compared with the control to measure the apoptosis blocking effect Moreover, the changes of T cell proliferation and killing activity were determined by the method of 3H thymidine incorporation and 51 Cr release, respectively Results The results showed that there was a significant increase in apoptosis rate in unblocked group compared with blocked group and stable lymphocyte group, with the unblocked group of 47 82%±0 13%, stable lymphocyte group of 3 76%±0 25%, and the blocked group of 8 22%±0 26% ( P <0 01) Additionally, DNA ladder found T cell ladder appeared in unblocked group The killing ability of T cells significantly increased after blocking The high proliferation rate of T cells after the blocking of apoptosis was agitated by soluble Fas receptor There was significant difference among blocked group, unblocked group and stable lymphocyte group ( P <0 01) Conclusion With this method massive T lymphocytes were obtained, which is able to kill hepatic carcinoma cells