特异性小干扰RNA沉默Cbl—b基因增强小鼠脾脏T淋巴细胞的免疫活性  

Enhanced immune activity of mouse spleen T lymphocytes by small specific interfering RNA silen-cing expression of casitas B cell lymphoma-b gene

作　　者：周术奎[1] 史振铎 陈卫华[1] 王顺平[1] 李良[1] 王跃闽[1] 

机构地区：[1]同济大学附属东方医院泌尿外科,上海200120 [2]徐州中心医院

出　　处：《中华实验外科杂志》2013年第6期1207-1209,共3页Chinese Journal of Experimental Surgery

基　　金：上海科委基金资助项目（PKJ2008-Y43）

摘　　要：目的观察特异性小干扰RNA（siRNA）沉默Cbl—b基因对于小鼠脾脏T淋巴细胞免疫活性影响。方法尼龙毛柱法分离T淋巴细胞，加入抗CD3抗体（1mg／L）在体外模拟T淋巴细胞活化的第一信号，脂质体转染12、24、48h后，流式细胞仪检测早、中、晚期活化标志分子CD69、CD25、CD71的表达变化；转染48h后细胞计数试剂盒（CCK-8）试剂盒检测T淋巴细胞增殖程度。结果利用特异性Cbl．bsiRNA能有效沉默T淋巴细胞Cbl-b基因，转染12h后，转染组、对照组、空白组CD69分子表达阳性率分别为（46．78±4．89）％、（21．36±2．06）％、（25．09±2．27）％（P〈0．01）；转染24h后，转染组、对照组、空白组CD25分子表达阳性率分别为（19．27±1．84）％、（8．18±0．71）％、（9．25±0．65）％（P〈0．01）；转染48h后，转染组、对照组、空白组CD71分子表达阳性率分别为（43．26±3．89）％、（23．99±1．97）％、（28．09±2．15）％（P〈0．01）；转染48h后，CCK-8试剂盒检测转染组、对照组、空白组吸光度值分别为0．763±0．063、0．356±0．039、0．383±0．015（P〈0．01）。结论利用特异性小干扰沉默Cbl—b基因能够增强小鼠脾脏T淋巴细胞免疫活性。Objective To investigate the effect of specific small interfering RNA （siRNA） silen- cing the expression of casitas B cell lymphoma-b （Cbl-b） gene on the immune activity of the mouse spleen T lymphocytes. Methods T lymphocytes were isolated by Nylon Wool Fiber Columns. By using anti-CD3 antibodies （ 1 mg/L） as the first signal of T lymphocyte activation in vitro, and at 12, 24 and 48 h after transfecting by liposome, the expression of CD69, CD25 and CD71 was detected and analyzed by using flu- orescence conjugated monoclonal antibodies and flowcytometry. At 48 h after transfection, the proliferation of the T lymphocyte was measured with CCK-8 kit. Results Specific Cbl-b siRNA could effectively silence the expression of Cbl-b gene. At 12 h after transfection, CD69 molecular expression levels in transfection group, negative control group and blank group were （46. 78±4. 89）%, （21.36±2. 06）%, and （25.09±2. 27）% respectively （P 〈0. 01 ）. At 24 h after transfection, CD25 molecular expression levels in trans- fection group, negative control group and blank group were （19. 2±1.84）% , （8.18±0. 71 ）% , and （9.25±0. 65） % respectively （ P 〈 0. 01 ）. At 48 h after transfection, CD71 molecular expression levels in transfection group, negative control group and blank group were （43.26±3.89 ）%, （23.99 ±1.97）%, and （28. 09 ±2. 15）% respectively （P 〈0. 01 ）. At 48 h after transfection, A values detected by CCK-8 kit in transfection group, control group, and blank group were 0. 763± 0. 063, 0. 356±0. 039, and 0. 383±0. 015 respectively （ P 〈 0. 01 ）. Conclusion Specific siRNA silencing Cbl-b gene can markedly promote the immune activity of mouse T lymphocytes.

关 键 词：CBL-B 小分子干扰RNA 基因沉默 T淋巴细胞 

分 类 号：R3[医药卫生—基础医学]