通过FasL通路阻抑肿瘤免疫逃逸的研究  

Study on Blocking the Tumor Immune Escape by Fas Ligand Pathway

作　　者：胡中波[1] 邹萍[1] 李爱香[1] 王良利[1] 刘凌波[1] 

机构地区：[1]华中科技大学同济医学院血液病研究所,武汉430022

出　　处：《中国实验血液学杂志》2003年第6期616-621,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目 编号 3 9970 70 8

摘　　要：许多白血病和实体瘤细胞通过高表达Fas配体 (FasL)而发生肿瘤的免疫逃逸。为了观察腺病毒载体向肿瘤细胞导入小鼠可溶性Fas(sFas)基因后能否阻抑肿瘤细胞通过FasL发生肿瘤免疫逃逸作用 ,采用AdEasy腺病毒载体系统 ,利用大肠杆菌内质粒间同源重组的方法分别构建携带小鼠sFas基因及增强型绿色荧光蛋白 (EGFP)的重组腺病毒载体 ,扩增纯化后利用空斑试验测定滴度 ,Westernblot检测蛋白的表达。然后 ,分别感染肿瘤细胞 EL4细胞 ,用氚胸腺嘧啶核苷 ( 3 H thymidine,3 H TdR)掺入法检测其诱导靶细胞YAC 1的凋亡率。结果表明 ,获得了重组成功的复制缺陷的腺病毒载体AdsFas和AdEGFP ,密度梯度离心纯化后其滴度达到 10 11pfu/ml,Westernblot分析证实前者能够高效表达出sFas蛋白。转染肿瘤细胞EL4细胞之后与YAC 1细胞混合培养 ,导入sFas组的YAC 1凋亡率在效靶比 (E∶T)为 3∶1,10∶1和 30∶1时分别为 6 %、7%和 9% ,与对照组 (分别为 2 8%、37%和4 5 % )相比明显下降 ,统计学有显著性差异 (P <0 .0 1) ;而导入EGFP组YAC 1凋亡率 (分别为 30 %、36 %和 4 8% )与对照组相似 ,没有统计学差异 (P >0 .0 5 )。结论 :腺病毒介导sFas的导入能够明显抑制肿瘤细胞EL4诱导Fas+细胞 YAC 1的凋亡 。The expression of Fas ligand (FasL) on the membrane of many kinds of leukemia or solid tumor cells played an important role in the immune escape of tumor cells. This study was aimed to know if the soluble Fas (sFas), expressed by adenovirus, could block the immune escape of tumor cells by FasL pathway. The two recombinant adenoviral vectors, AdsFas with murine soluble Fas gene and AdEGFP with enhanced GFP protein gene, were constructed by homologous recombination between two plasmids in Escherichia coli with the AdEasy adenovirus vector system. The viruses were propagated and purified by two times ultracentrifugation. Their titres were detected by plaque assays. The expressed protein was evaluated by Western blot analysis. Then the tumor EL4 cells were infected with AdsFas and AdEGFP respectively. The apoptosis ratio of the target cells YAC 1 cells induced by EL4 cells was respectively detected by 3H thymidine ( 3H TdR) labeling. The results showed that the recombinant adenoviral vectors AdsFas and AdEGFP were successfully obtained. The titres of viruses purified by two times ultracentrifugation were up to 10 11 pfu/ml by plaque assays. The sFas protein was highly expressed in the target cells by Western blot analysis. After the EL4 cells were transfected with the adenoviruses AdsFas,the apoptosis rate of YAC 1 cells in the sFas transfection group (respectively 6%?7% and 9% when the effector∶target (E∶T) was 3∶1?10∶1 and 30∶1) was obviously lower than that in the control group(respectively 28%?37% and 45%), P<0.01. But when the EL4 cells were transfected with AdEGFP, the apoptosis rate of YAC 1 cells(respctively 30%?36% and 48%)was similar to the control group, P>0 05. In conclusion, the transfer of sFas by adenovirus could inhibit the apoptosis of Fas + cells YAC 1 cells induced by tumor EL4 cells. It showed that the transduction of sFas could block the effect of the immune escape of EL4 cells through FasL in vitro. These results thus provide a new direction to find a way to

关 键 词：FAS配体 重组腺病毒 肿瘤 免疫逃逸 白血病 FASL 基因 免疫系统 

分 类 号：R730[医药卫生—肿瘤] R733.7[医药卫生—临床医学]