CAJ | 학술논문

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 - 23 bp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48 h,收集细胞上清液,ELISA法检测细胞因子IL-4 、IL-13、IL-2、INF-γ; Western Blot法检测T细胞kk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 -23 bp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.
목적 통과siRNA기술파향억제효천소서T세포비수체락안산단백격매Lck적기인표체,연구Lck특이성siRNA대효천소서T세포공능적영향.방법 화학법합성소서T세포Lck기인21 - 23 bp적RNA편단,이INTERFERinTMsiRNA Transfection Reagent작위전염시제,장합성적siRNA편단전염효천소서비장래원적T세포,작용48h후재여효천소서골수래원적수돌상세포(DC)혼합반응48 h,수집세포상청액,ELISA법검측세포인자IL-4 、IL-13、IL-2、INF-γ; Western Blot법검측T세포kk단백적함량,판단기표체시부피침묵.결과 siRNA간우조T세포중궤호검측불도Lck단백적표체,차기세포상청액중IL-4、IL-13적수평(10.19±1.66、12.34±0.79)교비siRNA간우조(28.06±2.88、27.87±1.61)급대조조(22.07±2.51、20.47±2.37)명현하강,차이유통계학의의(P＜0.01).결론 특정적21 -23 bp적RNA편단능구이siRNA간우적방식유효적억제특정기인적표체,Lck특이성siRNA가이조단효천소서T세포적격활화분화,감소효천염증인자적석방.
Objective Using the technology of siRNA to inhibit gene expression of T cells'nonreceptor 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 Lck were made by chemosynthesis.INTERFERinTMsiRNA Transfection Reagent was used as transfection reagent to transfect the siRNA into the spleen T cells 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 Lck 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 ± 2.5 1,20.47 ± 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 cytokines in asthmatic mice.