【作者】 王艳芬；

【导师】 田德安；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 硕士

【摘要】 目的:探讨针对大鼠RhoA基因的RNA干扰技术对HSC-T6 RhoA、Ⅰ型胶原(CollagenⅠ,ColⅠ)、Ⅲ型前胶原(Procollagen typeⅢ,PCⅢ)、透明质酸(Hyaluronic acid ,HA)及层粘连蛋白(Laminin,LN)的影响。方法:将阴性质粒(HK-A质粒)及大鼠RhoA基因特异性siRNA表达质粒(Rat1质粒、Rat2质粒)转染至大鼠肝星状细胞HSC-T6。将细胞分组如下:A组(正常培养)、B组(转染HK-A质粒)、C组(转染Rat1质粒)、D组(转染Rat2质粒)。用RT-PCR技术检测细胞RhoA、ColⅠ、PCⅢmRNA的表达。用ELISA方法检测培养上清中HA及LN的含量。结果:Rat1质粒所介导的RNAi技术能有效抑制大鼠肝星状细胞HSC-T6 RhoA、PCⅢ、ColⅠmRNA表达量及培养上清中HA及LN生成量。与A组相比,C组RhoA、PCⅢmRNA表达量下降(p﹤0.05),ColⅠmRNA、HA及LN表达量也显著下降(p﹤0.01),五者抑制率分别为43.77%、43.68%、71.00%、59.53%、93.08%。而B组、D组与A组相比,表达量均无统计学差异(p﹥0.05)。结论:以RhoA为干预点,Rat1质粒所介导的RNAi技术能有效抑制大鼠肝星状细胞HSC-T6细胞外基质的生成,为体内的进一步研究提供了实验基础。更多还原

【Abstract】 OBJECTIVE: To investigate the effect of RNA interference targeting RhoA gene on the expression of RhoA, Collagen typeⅠ(ColⅠ), Prollagen typeⅢ(PCⅢ), hyaluronic acid(HA)and laminin(LN)in rat hepatic stellate cell HSC-T6.METHODS: Transfect negative control plasmid vectors (named HK-A) and rat RhoA gene specific siRNA expressing plasmid vectors(named Rat1 and Rat2)into rat hepatic stellate cell HSC-T6 respectively. Divide the HSC-T6 cells into four groups as following: group A (cultured normally)、group B (transfected with HK-A)、group C (transfected with Rat1)、group D (transfected with Rat2). The mRNA expression of RhoA, ColⅠand PCIII in HSC-T6 was detected by reverse-transcription polymerase chain reaction(RT-PCR)respectively in each group. The content of HA and LN in culture serum was measured by specific enzyme-linked immunosorbent assay (ELISA).RESULTS: Compared with group A, the expression of RhoA, ColⅠ, PCIII, HA and LN were decreased in group C (p﹤0.05 for RhoA and PCIII, the inhibition ratios were 43.77% and 43.68%. p﹤0.01 for ColⅠ, HA and LN , the inhibition ratios were 71.00%, 59.53% and 93.08% respectively). Otherwise, compared with group A, there was no significant difference in group B or group D for the five indexs (p﹥0.05).CONCLUSION: RNA interference mediated by Rat1 plasmids targeting RhoA can depress the expression of ColⅠ, PCⅢ, HA and LN in rat hepatic stellate cell HSC-T6, which has provided experimental foundation for further research in vivo.更多还原