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目的 探讨CD80、CD86和CD137L基因联合表达增强宿主细胞毒性T淋巴细胞(CTL)杀伤活性的机制.方法 BAL B/c小鼠随机分为5组,A组接种H22-Wt细胞,B组接种H22-neo细胞,C组接种H22-CD80/CD86+细胞,D组接种H22-CD137L+细胞,E组接种H22-CD80/CD86/CD137L+细胞.分别于第14、35、56和84天,每组每次随机选取2只小鼠处死取材.原位末端标记法(TUNEL)和DNA ladder法检测脾淋巴细胞凋亡.电泳迁移率法(EMSA)检测T细胞核因子KB(NF-KB)活性.结果 TUNEL检测结果显示,接种后第14天,A、B组脾淋巴细胞即出现大量凋亡,D组凋亡虽然较A、B组明显减少,但仍远高于C、E组.随接种时间推移,C组脾淋巴细胞凋亡逐渐增多,而E组这一趋势不明显,至第84天,C组和E组的脾淋巴细胞凋亡指数分别为30.8±9.2和14.4±4.5.DNA ladder检测结果显示,接种后第14、35、56和84天,C组和E组均检测出典型阳性结果,其中以C组第35、56和84天凋亡最明显.EMSA检测结果显示,A、B组T细胞NF-KB活性很低,D组显著高于A、B组,C、E组则显著高于A、B和D组.随着接种时间推移,E组NF-KB活性一直维持较高水平,而C组呈现逐渐下降趋势,至第84天,C组和E组的T细胞NF-KB活性分别为14.01±1.04和41.16±5.78.结论 H22-CD80/CD86/CD137L+变异株接种荷瘤鼠后,CD28信号和CD137信号的协同作用可显著增强活化T细胞NF-KB活性,这可能是CD80、CD86和CD137L基因联合表达显著增强宿主CTL杀伤活性的机制之一.

Objective To study the mechanism of enhancement of the CTL activity in mice co-expressing of CD80, CD86 and CD137L genes. Methods The mice were randomly divided into five group, named A, B, C, D and E. The group A and B were control group (CG). H22-BAL B/c HCC mouse model was established by subcutaneous injection with hepatoceUular carcinoma cells of cell line H22-Wt (group A), H22-neo (group B), H22-CD80/CD86+ (group C), H22-CD137L+ (group D) and H22-CD80/CD86/CD137L+ (group E), respectively. On the 14th, 35th, 56th and 84th day after the first inoculation of tumor cells, TUNEL staining and DNA ladder examination were used to detect apoptosis of splenic T lymphocytes in all groups at each post-inoculation time point. Electroplioretie mobility-shift assay (EMSA) method was used to detect the activity of nuclear factor KB (NF-KB) in splenic T lymphocytes in each group at each time point post-inoculation. Results Apoptosis was found in a great number of T lymphecytes in CG on the 14th day, much more than that in group C and E. The number of apoptotic T cells in group C had a significant difference compared with that in the group E from 14th to 84th day (P=0.003). DNA ladder analysis showed typical positive results in group C and E. The significant apoptosia fragments were found in group C on 21st, 35th and 84th days. NF-KB activity ofT cells in groups C and E was remarkably higher than that of groups CG and D, with higher in group D than that of CG (P=0.002), and with no significant difference between group C and E on 14th day. The activity in group E was stable and remarkably higher than that of group C on 56th and 84th days after the first inoculation. Conclusion H22-CD80/CD86/CD137L+ induces higher NF-KB activity of the host T ceils by synergistic action of CD28 and CD137, which may be one of the mechanisms of enhancement of the host CTL activity induced by co-expression of CDS0, CD86 and CD137L genes.