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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。
OBJECTIVE: To establish a method for rapid preparation of a large number of activated tumor-specific cytotoxic T lymphocytes (CTLs) by blocking the Fas / FasL-mediated T cell apoptosis. Methods FasL positive hepatocellular carcinoma (HCC) specimens were selected and isolated from hepatocellular carcinoma cells and tumor infiltrating lymphocytes (TILs). Then the two were mixed in vitro and activated by CD28 monoclonal antibody. The soluble Fas receptor was used to block the apoptosis of hepatocellular carcinoma cells through the Fas / FasL pathway. Compared with the control group, apoptosis was detected by flow cytometry and DNA ladder test simultaneously. 3H-incorporation and 51Cr release Law to understand T cell proliferation and killing activity. Results The apoptosis rate of non-blocking group was significantly higher than that of non-blocking group and non-blocking group (47.82% ± 0.13%) in control group The percentage of T lymphocyte was 3.76% ± 0.25% in the T-lymphocyte group and 8.22% ± 0.26% in the blocking group (P <0.01) Strips, blocking group was negative. The results of 51Cr release showed that the cytotoxicity of T lymphocytes after blocking was significantly higher than that of non-blocking and non-blocking groups (P <0.01). 3H incorporation assay confirmed that soluble Fas receptor blocks the activation of T cell apoptosis, the cell proliferation was significantly higher than the non-blocked group difference was significant (P <0.01). Conclusion In vitro experiments can be a large number of activated T cells, and kill tumor cells.