FGD6基因对肝干细胞分化的调控作用

发布时间：2018-05-20 11:43

本文选题：干细胞 + RNA干扰　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的本研究拟探讨FGD6(faciogenital dysplasia)基因对肝干细胞分化的调控作用。方法 1.选取FGD6基因的RNA干扰靶序列,将目的片段克隆到穿梭载体p SES-HUS上,再与骨架质粒p Adeasy-1共转化到感受态细胞中,然后接种到卡那霉素平板中,挑选出阳性克隆。将构建的质粒测序。2.将测序证实正确的质粒使用Ad Easy系统构建腺病毒载体,包装并扩增重组腺病毒载体p SES-FGD6-si RNA。3.使用细胞免疫荧光法检测FGD6蛋白在肝干细胞HP14.5细胞中的表达水平及表达定位。4.用腺病毒载体p SES-FGD6-si RNA感染HP14.5细胞,使用实时荧光定量PCR检测FGD6、甲胎蛋白(AFP)及白蛋白(Alb)的mRNA水平,Western blot检测FGD6、AFP及Alb的蛋白表达水平。每组细胞均设置p SES-Ad-RFP腺病毒空载体感染进行对照。5.本研究所有数据使用均数±标准差(x±s)表示,并采用单因素方差分析的方法进行数据统计分析。结果 1.成功构建腺病毒载体p SES-FGD6-si RNA,并完成病毒的扩增及纯化。2.细胞免疫荧光检测结果显示FGD6蛋白在肝干细胞HP14.5细胞中呈高表达,主要定位在细胞核中。3.用腺病毒载体p SES-FGD6-si RNA感染HP14.5细胞,成功下调FGD6基因在HP14.5细胞中的表达,同时,AFP的mRNA及蛋白的表达降低,而Alb的mRNA及蛋白的表达升高(P0.01)。结论抑制FGD6基因在肝干细胞HP14.5细胞中的表达,可使HP14.5细胞向肝细胞方向分化。因此,FGD6基因对肝干细胞的分化可能起着重要的调控作用。
[Abstract]:Objective to investigate the effect of FGD6(faciogenital dysplasia gene on the differentiation of hepatic stem cells. Method 1. The RNA interference target sequence of FGD6 gene was selected, the target fragment was cloned into shuttle vector p SES-HUS, and then transformed into receptive cells with skeleton plasmid p Adeasy-1, and then inoculated into kanamycin plate to select positive clones. The constructed plasmid was sequenced. The adenovirus vector was constructed by using Ad Easy system and the recombinant adenovirus vector p SES-FGD6-si RNA.3was amplified by sequencing. The expression and localization of FGD6 protein in HP14.5 cells of liver stem cells were detected by immunofluorescence assay. HP14.5 cells were infected with adenovirus vector p SES-FGD6-si RNA. The mRNA levels of FGD6, AFP and Alb) were detected by real-time fluorescence quantitative PCR. The protein expression levels of FGD6 and Alb were detected by Western blot. Each group of cells were infected with empty vector of p SES-Ad-RFP adenovirus for control. 5. In this study, the data are expressed by the mean 卤standard deviation (x 卤s), and the statistical analysis of the data is carried out by using the method of univariate analysis of variance (ANOVA). Result 1. Adenovirus vector p SES-FGD6-si RNA was successfully constructed, and the amplification and purification of the virus. 2. 2. The results of cellular immunofluorescence assay showed that FGD6 protein was highly expressed in HP14.5 cells of liver stem cells, mainly located in the nucleus. HP14.5 cells were infected with adenovirus vector p SES-FGD6-si RNA, and the expression of FGD6 gene in HP14.5 cells was down-regulated, while the expression of mRNA and protein was decreased, while the expression of mRNA and protein of Alb was increased (P0.01). Conclusion inhibiting the expression of FGD6 gene in HP14.5 cells of liver stem cells can induce the differentiation of HP14.5 cells into hepatocytes. Therefore, FGD6 gene may play an important role in regulating the differentiation of hepatic stem cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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