通过Tet-on系统研究Hes1基因在调控肝干细胞胆向分化中的作用

发布时间：2018-07-02 20:28

  本文选题：Hes1基因 + 肝干细胞系　； 参考：《宁夏医科大学》2011年硕士论文

【摘要】：目的Hes1基因在肝干细胞胆向分化中发挥重要作用,但其具体的作用机制尚不清楚。本研究利用Tet-on真核表达系统,构建了连接目的基因Hes1的载体,在肝干细胞系LEPCs(Liver Epithelia Progenitor Cells,也称为肝原始细胞系)中,筛选出可调控性表达Hes1基因的单克隆来源的细胞,通过检测在表达不同水平Hes1基因的LEPCs细胞中特异性肝细胞及胆管上皮细胞的标志性分子,评价Hes1基因在LEPCs分化中的作用。 方法通过构建实验组pTRE2hyg-Hesl和对照组pTRE2hyg-EGFP-Hesl两种可调控性载体,结合pTet-on真核表达系统,分别转染LEPCs并筛选得到稳定表达Hes1基因的细胞系,通过加入不同终浓度(0、0.1、1、5、10、50、100、500μg/ml)强力霉素(Doxycycline,DOX)诱导Hes1基因表达。采用PCR、RT-PCR、Real-Time PCR、免疫细胞化学、Western blot等方法检测并验证Hes1基因在LEPCs中表达的可调控性;采用RT-PCR检测当外在调控性Hes1表达增高时LEPCs表达肝细胞(Gss)及胆管细胞(Krt19、Krt7及Foxa1)的标志性分子,探讨Hes1基因在LEPCs胆向分化中的作用。 结果成功构建真核表达载体pTRE2hyg-Hesl和pTRE2hyg-EGFP-Hesl;RT-PCR和Real-time PCR检测结果均显示,随着DOX浓度的增加,Hes1在LEPCs细胞中的表达量也逐渐增加,当DOX浓度为10μg/ml时Hes1的表达可达到最高效率,是未加DOX时的11.21倍,第5天Hes1的表达处于高峰,高峰至少持续到第7天;Western blot检测显示HES1蛋白水平的表达也随着DOX浓度的增高而有所增加,比未经处理的LEPCs的HES1表达量高,同样免疫细胞化学染色也显示转染了pTet-on和pTRE2hyg-Hes1的98%以上的细胞呈阳性反应,着色主要见于细胞核和细胞质。当外源性Hes1在LEPCs细胞中表达量上调后,肝细胞的分子标志物Gss有所下降,胆管细胞分子标志物Krt19等表达出现逐渐增加的趋势。 结论可调控性表达载体pTRE2hyg-Hesl或pTRE2hyg-EGFP-Hesl与pTet-on共转染入LEPCs并经筛选后的所得的细胞系,通过加入不同浓度的DOX ,可调控性地诱导Hes1基因的表达,并与Hes1基因的表达存在一定的剂量依赖性。本实验表明当Hes1基因表达增高时,将参与调控肝干细胞LEPCs向胆管细胞方向分化;提示Notch信号通路参与调控肝干细胞分化命运的选择,可能与肝干细胞的分化、增殖,以及肝脏或胆管肿瘤的发生发展有一定密切的联系。本课题为进一步研究和了解Hes1基因的在肝干细胞胆向分化的调控机制提供了一定的理论依据。
[Abstract]:Objective Hes1 gene plays an important role in the gallbladder differentiation of hepatic stem cells, but the mechanism of Hes1 gene is still unclear. In this study, Tet-on eukaryotic expression system was used to construct the vector of Hes1 gene. In LEPCs (liver stem cell line LEPCs, also known as liver primitive cell line), cells derived from monoclonal expression of Hes1 gene were screened. The role of Hes1 gene in the differentiation of LEPCs was evaluated by detecting the specific markers of hepatocytes and bile duct epithelial cells expressing different levels of Hes1 gene. Methods two kinds of regulatory vectors pTRE2hyg-Hesl and control group pTRE2hyg-EGFP-Hesl were constructed, and pTet-on eukaryotic expression system was used to transfect LEPCs and screen stable Hes1 cell lines. The expression of Hes1 gene was induced by adding Doxycycline doxorubicin (Doxycycline doxycycline DOX) at different final concentrations (0 0. 1 g/ml). The expression of Hes1 gene in LEPCs was detected by PCR RT-PCR-1 Real-Time PCRand immunocytochemistry, and the expression of Hes1 gene in LEPCs was detected by RT-PCR, and the iconic molecules of hepatocytes (GSS) and bile duct cells (Krt19pKrt7 and Foxa1) were detected by RT-PCR when the expression of Hes1 was increased. To investigate the role of Hes1 gene in the differentiation of LEPCs into gallbladder. Results Eukaryotic expression vectors pTRE2hyg-Hesl and pTRE2hyg-EGFP-Hesltr RT-PCR and Real-time PCR showed that the expression of Hes1 in LEPCs cells increased with the increase of DOX concentration, and the expression of Hes1 could reach the highest efficiency when dox concentration was 10 渭 g/ml. The expression of Hes1 was at its peak on the 5th day, and the expression of HES1 protein increased with the increase of DOX concentration, which was higher than that of untreated LEPCs. Immunocytochemical staining also showed that more than 98% of the cells transfected with pTet-on and pTRE2hyg-Hes1 were positive, and the staining was mainly found in the nucleus and cytoplasm. When the expression of exogenous Hes1 was up-regulated in LEPCs cells, the expression of GSS, a molecular marker of hepatocytes, decreased, and the expression of Krt19, a molecular marker of bile duct cells, gradually increased. Conclusion the regulated expression vector pTRE2hyg-Hesl or pTRE2hyg-EGFP-Hesl co-transfected with pTet-on into LEPCs and screened cell lines can induce the expression of Hes1 gene in a dose-dependent manner by adding different concentrations of DOX. This study suggests that when Hes1 gene expression is increased, LEPCs will be involved in the differentiation of hepatic stem cells into bile duct cells, which suggests that Notch signaling pathway is involved in the selection of differentiation fate of hepatic stem cells, which may be related to the differentiation and proliferation of hepatic stem cells. And the occurrence and development of liver or bile duct tumor have certain close relation. This study provides a theoretical basis for further study and understanding of the regulation mechanism of Hes1 gene in hepatic stem cell gallbladder differentiation.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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