RNA干扰抑制大鼠T淋巴细胞CD28基因表达的实验研究  

Depressive effect of siRNA technique on the expression of CD28 costimulatory molecule on lymphocytes

作　　者：薛利军[1] 尹路[1] 陈春球[1] 张贵阳[1] 万方军[1] 林谋斌[1] 倪俊声[1] 金志明[1] 施敏敏[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院外科器官移植中心,上海200025

出　　处：《外科理论与实践》2009年第2期193-197,共5页Journal of Surgery Concepts & Practice

基　　金：国家自然科学基金资助项目(30671972)

摘　　要：目的:探讨RNA干扰(RNAi)后对大鼠T淋巴细胞共刺激分子CD28基因的表达及其对细胞功能的影响。方法:设计针对目标基因的小干扰RNA(siRNA),转染大鼠T淋巴细胞。以FCAS检测CD28水平,MTT检测转染后T淋巴细胞对异体淋巴细胞增殖能力的影响,Realtime-PCR及ELISA法检测细胞因子水平。结果:siRNA转染大鼠T淋巴细胞后可抑制CD28的表达,T细胞增殖能力、IL-2、IFN-γ水平明显低于空白对照组(P<0.05)。结论:siRNA可特异性抑制大鼠T淋巴细胞共刺激分子CD28的表达,降低大鼠T细胞的增殖能力,抑制IL-2、IFN-γ细胞因子的基因水平表达和分泌水平,从而产生免疫耐受效应,为器官移植免疫研究提供实验依据。Objectives To study the RNA interference （RNAi） effect induced by small interfering RNA （siRNA） targeting CD28 mRNA on rat peripheral blood lymphoeytes. Methods According to the CD28 gene sequence, siRNAs were designed and synthesized for transfecting into freshly isolated rat T lymphocytes with the nueleofeetor technique. Cells were collected at 6,12,24 and 48 hrs after transfeetion, and the surface expression of CD28 was detected by flow cytometry, the cell viability of transfected lymphocytes was determined by methyl thiazolyt tetrazolium （MTr） assay and trypan blue dye exclusion assay. The mRNA expression levels of IL-2, IL-10, TNF-α and IFN-γ production were examined by Reahime-PCR, and IL-2, IL-10, TNF-α, IFN-γ eytokine were determined by enzyme-linked immunosorbent assay （ELISA）. Results The CD28 expression and the viable transfeeted lymphocytes were significantly reduced in the siRNA groups 24 hrs after transfection （P〈0.05）. The expression levels of IL-2 and IFN-γ were significantly down -regulated as compared with those of the control group, while a comparative up-regnlation of IL-10 was observed after transfection of siRNAs. Conclusions The silencing effect on CD28 mRNA induced by the siRNA technique may contribute to costimulate blockade and immune tolerance. The siRNA technique may be useful for further studies on graft-versus-host disease after allogeneic organ transplantation.

关 键 词：免疫耐受 T淋巴细胞 RNA干扰 基因表达 

分 类 号：R392.4[医药卫生—免疫学]