背景:RT-PCR技术是将RNA的反转录和cDNA的聚合酶链反应相结合的技术.常用于检测细胞组织中基因表达水平、细胞中RNA病毒的含量和直接克隆特定基因的cDNA序列等.目的:运用RT-PCR技术检测Stealth siRNA对转化生长因子β1的抑制作用.方法:实验分为空白对照组、空转染组、stealth-48组、stealth-166组和stealth-594组.针对BALB/c小鼠转化生长因子β1基因组,选择不同位点设计3套siRNA基因序列,转染体外培养的小鼠肺成纤维细胞,RT-PCR法检测其对转化生长因子β1和下游结缔组织生长因子表达的影响.结果与结论:①RT-PCR结果显示3种Stealth siRNA对转化生长因子β1表达在不同时间有不同程度的抑制作用,以Stealth-166效果更为明显;抑制效果与转染时间长短相关,48 h后就可检测出明显抑制,72 h达到最高峰,96 h后开始减弱;②结果说明,RT-PCR技术可用于特异性Stealth siRNA抑制转化生长因子β1在小鼠肺成纤维细胞的表达效果的检测.%BACKGROUND: As a combination of reverse transcription (RT) and polymerase chain reaction (PCR), RT-PCR has been used to detect gene expression levels in cells and tissues, RNA virus contents in cells and specific gene cloned cDNA sequences.OBJECTIVE: To detect the inhibitory effcet of Stealth siRNAs on the expression of transforming growth factor β1 (TGF-β1).METHODS: There were blank control, empty vector transfection, stealth-48, stealth-166, and stealth-594 groups. Three stealth siRNAs aimed at different sequences in TGF-β1 mRNA were made, and were then transfected into BALB/c mouse lung fibroblasts in vitro. The expressions of TGF-β1 and connective tissue growth factor were detected by RT-PCR.RESULTS AND CONCLUSION: In different time periods, the TGF-β1 expression was differentially depressed by three stealth siRNAs, especially stealth-166. The inhibitory effects varied with time, which could be detective at 48 hours,reached the peak at 72 hours and then began to attenuate at 96 hours. Our findings show that the inhibitory effect of stealth siRNAs on the TGF-β1 expression in mouse lung fibroblasts can be detected by RT-PCR.
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