高效小鼠CD40siRNA基因片段的筛选及对淋巴细胞CD40基因表达的影响  

Effect of highly effective selection of mouse CD40 small interference RNA and on the expression of CD40 in mouse lymphocytes

作　　者：严金川[1] 杨萍[1] 龚杰[1] 王标[1] 徐良洁[1] 

机构地区：[1]江苏大学附属医院心血管内科,江苏镇江212001

出　　处：《江苏大学学报（医学版）》2010年第6期476-480,共5页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省自然科学基金资助项目(BK2008239);江苏省高校自然科学基金资助项目(08KJB320001)

摘　　要：目的:应用RNA干扰技术筛选高效小鼠CD40siRNA基因序列片段。方法:体外化学合成小鼠CD40基因为靶标的siRNA3对(siCD40-1、siCD40-2、siCD40-3),通过脂质体瞬时转染小鼠脾淋巴细胞(实验组),并分别以转染无关siRNA片段及未转染细胞作为对照(对照组)。分别采用流式细胞术、实时荧光定量PCR检测转染24 h细胞表面CD40抗原、CD40 mRNA表达,蛋白质印迹检测转染48 h蛋白表达的变化。结果:与对照组相比,实验组CD40抗原表达、CD40 mRNA及蛋白表达明显下降(P<0.01);3对实验组siRNA能明显抑制CD40基因表达,抑制率分别为69%、78%和41%,差异有统计学意义(P<0.05)。而各对照组之间比较差异无统计学意义(P>0.05)。结论:siRNA可抑制小鼠淋巴细胞表达CD40,并具有良好的特异性,其中siRNA-2为最高效干扰片段。Objective： To select highly effective mouse CD40 small interfering RNA（siRNA）by the technology of interfering RNA.Methods： Three specific chemically synthesized siRNAs targeting on CD40 of mouse were designed and were transfected into lymphocytes with Lipofectamine RNAiMAX via reverse transfection,while nonspecific siRNA and untreated cell served as the control separately,The expression of CD40 and CD40 mRNA in lymphocytes were measured by flow cytometry and real-time fluorogenic quantitative PCR（RFQ-PCR） respectively after 24 h of transfection.The expression of CD40 protein levels were assessed by Western blot after 48 h of transfection.Results： Compared with the experimental and controls,siRNA group showed significantly down-regulation of CD40 mRNA and protein in lymphocytes.The total inhibitory rate of CD40 expression for siCD40-1,siCD40-2,siCD40-3 were 69%,78% and 41% respectively.There was no significant difference found in controls.Conclusion： siRNA of CD40 could specifically down-regulate the expression of CD40 mRNA and protein in mouse lymphocytes.siRNA-2 has the highly effective inhibition.

关 键 词：小分子RNA干扰 淋巴细胞 CD40 反向转染 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]