SiRNA干扰靶向抑制酪氨酸蛋白激酶Lck对哮喘小鼠T细胞功能的影响  

The effect of siRNA inhibited tyrosine protein kinase Lck on the function of T cells in asthmatic mice

作　　者：季巧英 方双燕 舒彩敏 杨琼芳 宋东莉[2] 郑永华[2] 

机构地区：[1]东阳市人民医院呼吸内科,浙江省东阳322100 [2]复旦大学附属中山医院实验研究中心

出　　处：《中国医师杂志》2011年第10期1323-1326,共4页Journal of Chinese Physician

基　　金：浙江省金华市科技计划项目（2010-3-030）

摘　　要：目的通过siRNA技术靶向抑制哮喘小鼠T细胞非受体酪氨酸蛋白激酶Lck的基因表达，研究Lck特异性siRNA对哮喘小鼠T细胞功能的影响。方法化学法合成小鼠T细胞Lck基因21—23bp的RNA片段，以INTERFERinTMsiRNA Transfection Reagent作为转染试剂，将合成的siRNA片段转染哮喘小鼠脾脏来源的T细胞，作用48h后再与哮喘小鼠骨髓来源的树突状细胞（DC）混合反应48h，收集细胞上清液，ELISA法检测细胞因子IL4、IL-13、IL-2、INF-1；Western Blot法检测T细胞Lck蛋白的含量，判断其表达是否被沉默。结果siRNA干扰组T细胞中几乎检测不到Lck蛋白的表达，且其细胞上清液中IL-4、IL-13的水平（10．19±1．66、12．34±0．79）较非siRNA干扰组（28．06±2．88、27．87±1．61）及对照组（22．07±2．51、20．47±2．37）明显下降，差异有统计学意义（P〈0．01）。结论特定的21—23bp的RNA片段能够以siRNA干扰的方式有效的抑制特定基因的表达，Lck特异性siRNA可以阻断哮喘小鼠T细胞的激活和分化，减少哮喘炎症因子的释放。Objective Using the technology of siRNA to inhibit gene expression of T cells＇ non - receptor tyrosine protein kinase Lck in asthmatic mice, and to study the effect of siRNA inhibited Lck to the function of T cells in asthmatic mice. Methods The 21 -23 bp RNA fragments of mouse T cell Lek were made by ehemosynthesis. INTERFERinTMsiRNA Transfection Reagent was used as transfeetion reagent to transfect the siRNA into the spleen T ceils of asthmatic mice for 48 hours. Then T cells were mixed with bone marrow dendritic cells （DC） of asthmatic mice for another 48 hours. Cell culture suspension was collected and the level of IL-4, IL-13, IL-2, INF-γ were detected with respondent ELISA kits; Western Blot was used to identify if the expression of Lek was blocked. Results The expression of Lck in T cells almost could not be detected in siRNA interference group. The levels of IL-4 and IL-13 in siRNA interference group（ 10. 19 ± 1.66,12. 34 ±0. 79） were lower than no-siRNA interference （28.06 ± 2. 88,27.87 ± 1.61 ） and control group （ 22. 07± 2. 51,20.47 ± 2. 37 ） , and the difference was statistical significant （ P 〈 0. 01 ）. Conclusions Special siRNA could block the expression of special gene, and Lck specific siRNA could block the activation and differentiation of T cells and reduce the secretion of inflammatory eytokines in asthmatic mice.

关 键 词：RNA 小分子干扰 淋巴细胞特异蛋白质酪氨酸激酶p56(Lck) 哮喘/免疫学 细胞因子类/代谢 

分 类 号：R562.25[医药卫生—呼吸系统]