肝癌细胞通过Fas/FasL途径触发T淋巴细胞凋亡的双重阻断研究

目的通过阻断Fas/FasL介导的T细胞凋亡,建立快速制备大量激活的肿瘤特异性细胞毒性T淋巴细胞(CTL)的方法。方法选择FasL表达阳性的肝癌标本,分离肝癌细胞和肿瘤浸润淋巴细胞(TIL),然后在体外将两者混合,并在CD28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,与对照组比较用流式细胞仪和DNA ladder Test同时检测阻断凋亡作用,通过3H掺入法和51Cr释放法了解T细胞增殖力及杀伤活性。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47．82％±0．13％,对照组静息性T淋巴细胞组为3．76%±0．25％,阻断组为8．22％±0．26％(P<0．01),DNA ladder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。51Cr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<0．01)。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<0．01)。结论体外实验可获得大量激活T细胞,并可杀伤肿瘤细胞。

Experimental study on double blocking of T lymphocytes apoptosis induced by Fas/FasL

Abstract Objective To block the apoptosis of T lymphocytes induced by Fas/FasL in order to establish a method of the large-scale preparation of large amounts of tumor-specific cytoxic T-lymphocytes (CTL). Methods Liver cancer cells and tumor infiltrating lymphocytes (TIL) were isolated from FasL positive fresh specimens, and co-cultured. Specific CTL were activated and prepared in the presence of the co-stimulation of monoclonal antibody CD28. Then the blocking and activation of apoptosis of T lymphocytes was activated by soluble Fas receptor, which was detected by cytometry and DNA ladder test simultaneously. The apoptosis-blocking effect was compared with the control group. Furthermore, the changes of T cell proliferation and killing activity were detected by the method of ³H thymidine incorporation and ⁵¹Cr release test. Results There was a significant increase in apoptosis rate in unblocking group compared with blocking group and quiescent group, with the unblocking group of 47.82%±0.13%, quiescent group of 3.76%±0.25%, and the blocking group of 8.22%±0.26% respectively (P<0.01). T cell-ladder appeared in unblocking group by DNA ladder test. Both the killing ability and proliferation rate of T cells were significantly increased after blocking. There was significant difference among blocking group, unblocking group and quiescent group (P<0.01). Conclusion With this method we obtained large amounts of tumor-specific T lymphocytes, which was able to kill liver cancer cells effectively. This project was supported by a grant from the National Natural Science Foundation of China (No. 03030302).