小干扰RNA对小鼠淋巴细胞cbl-b基因表达的抑制作用  

Inhibitory effects of small interfering RNA on the expression of cbl-b gene in mouse lymphocytes

作　　者：史振铎[1] 陈卫华[1] 陆英[2] 杨波[1] 温晓飞[1] 李学峰[3] 李良[1] 胡昕海[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]同济大学附属东方医院中心实验室,上海200120 [3]滨州医学院附属医院生殖医学中心

出　　处：《中华实验外科杂志》2011年第7期1172-1174,共3页Chinese Journal of Experimental Surgery

摘　　要：目的设计合成及筛选自身免疫关键基因——chl—b（Casitas B-cell lineage lymphoma-b,cbl—b）基因小分子干扰RNA（siRNA）。方法应用RNA设计软件，模拟cbl—b小鼠cbl—bmRNA二级结构，设计并合成针对cbl—bmRNA的4对21核苷酸（nt）siRNA，96孔板转染小鼠淋巴细胞，以空白及转染非特异siRNA（与cbl—bmRNA无同源性的21ntsiRNA）作为对照，应用蛋白免疫印迹（Western blot）方法检测小鼠淋巴细胞cbl—b蛋白表达。结果cbl—b基因siRNA工作浓度为100nmol／L时转染小鼠淋巴细胞转染率最高，可达（87．48±1．94）％，平均荧光强度最强，可达33．09±1．77。与对照相比，转染cbl—b siRNA的小鼠淋巴细胞蛋白表达明显下调，以siRNA-4最显著，抑制率达85％，转染非特异性siRNA的小鼠淋巴细胞cbl—b蛋白表达水平无明显变化。结论得到cbl—b siRNA转染小鼠淋巴细胞最佳转染条件，成功筛选能高效抑制cbl—b蛋白表达的siRNA-4，其有效抑制时间约为48h，有望通过沉默cbl—b基因直接活化淋巴细胞，增强机体主动免疫杀伤肿瘤细胞。Objective To design, synthesize screen small interfering RNA （siRNA） targeting to Casitas B-cell lineage lymphoma-b （cbl-b）. Methods Four pairs of 21 nucleotide siRNAs directed to Cbl-b mRNA were designed and synthesized by utilizing RNA design software to simulate secondary structure of cbl-b mRNA in mice. These siRNAs were respectively transfected into lymphocytes in 96 shadows mask by oligofectamine package, and untreated and unspecific siRNA-transfected lymphocytes served as controls. The expression of cbl-b protein was detected by Western blotting. Results When the work concentration of siRNA was 100 nmol/L, transfection efficiency of lymphocytes was highest, up to （ 87.48 ± 1.94） % and the mean fluorescence intensity was strongest, up to 33.09 ±1.77. Compared with bland controls, the expression of cbl-b protein level was markedly down-regulated in siRNA-transfected lymphocytes. The inhibitory rate of the siRNA of the target-4 was highest, up to 85%. The expression of cbl-b protein in unspecific siRNA-transfected lymphocytes had no significant changes. Conclusion siRNA-4, which can highly effectively inhibit protein expression of cbl-b gene, was screened successfully, and its inhibition effect can maintain near 48 h. It is hopeful that the cbl-b siRNA will activate lymphocytes directly by cbl-b gene silencing, and kill tumor by activate immunization.

关 键 词：cbl—b 小干扰RNA 淋巴细胞 

分 类 号：R285.5[医药卫生—中药学]