本文選題：Hes1基因 + 肝干細(xì)胞系��； 參考：《寧夏醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的Hes1基因在肝干細(xì)胞膽向分化中發(fā)揮重要作用,但其具體的作用機(jī)制尚不清楚。本研究利用Tet-on真核表達(dá)系統(tǒng),構(gòu)建了連接目的基因Hes1的載體,在肝干細(xì)胞系LEPCs(Liver Epithelia Progenitor Cells,也稱為肝原始細(xì)胞系)中,篩選出可調(diào)控性表達(dá)Hes1基因的單克隆來源的細(xì)胞,通過檢測在表達(dá)不同水平Hes1基因的LEPCs細(xì)胞中特異性肝細(xì)胞及膽管上皮細(xì)胞的標(biāo)志性分子,評價Hes1基因在LEPCs分化中的作用。 方法通過構(gòu)建實(shí)驗(yàn)組pTRE2hyg-Hesl和對照組pTRE2hyg-EGFP-Hesl兩種可調(diào)控性載體,結(jié)合pTet-on真核表達(dá)系統(tǒng),分別轉(zhuǎn)染LEPCs并篩選得到穩(wěn)定表達(dá)Hes1基因的細(xì)胞系,通過加入不同終濃度(0、0.1、1、5、10、50、100、500μg/ml)強(qiáng)力霉素(Doxycycline,DOX)誘導(dǎo)Hes1基因表達(dá)。采用PCR、RT-PCR、Real-Time PCR、免疫細(xì)胞化學(xué)、Western blot等方法檢測并驗(yàn)證Hes1基因在LEPCs中表達(dá)的可調(diào)控性;采用RT-PCR檢測當(dāng)外在調(diào)控性Hes1表達(dá)增高時LEPCs表達(dá)肝細(xì)胞(Gss)及膽管細(xì)胞(Krt19、Krt7及Foxa1)的標(biāo)志性分子,探討Hes1基因在LEPCs膽向分化中的作用。 結(jié)果成功構(gòu)建真核表達(dá)載體pTRE2hyg-Hesl和pTRE2hyg-EGFP-Hesl;RT-PCR和Real-time PCR檢測結(jié)果均顯示,隨著DOX濃度的增加,Hes1在LEPCs細(xì)胞中的表達(dá)量也逐漸增加,當(dāng)DOX濃度為10μg/ml時Hes1的表達(dá)可達(dá)到最高效率,是未加DOX時的11.21倍,第5天Hes1的表達(dá)處于高峰,高峰至少持續(xù)到第7天;Western blot檢測顯示HES1蛋白水平的表達(dá)也隨著DOX濃度的增高而有所增加,比未經(jīng)處理的LEPCs的HES1表達(dá)量高,同樣免疫細(xì)胞化學(xué)染色也顯示轉(zhuǎn)染了pTet-on和pTRE2hyg-Hes1的98%以上的細(xì)胞呈陽性反應(yīng),著色主要見于細(xì)胞核和細(xì)胞質(zhì)。當(dāng)外源性Hes1在LEPCs細(xì)胞中表達(dá)量上調(diào)后,肝細(xì)胞的分子標(biāo)志物Gss有所下降,膽管細(xì)胞分子標(biāo)志物Krt19等表達(dá)出現(xiàn)逐漸增加的趨勢。 結(jié)論可調(diào)控性表達(dá)載體pTRE2hyg-Hesl或pTRE2hyg-EGFP-Hesl與pTet-on共轉(zhuǎn)染入LEPCs并經(jīng)篩選后的所得的細(xì)胞系,通過加入不同濃度的DOX ,可調(diào)控性地誘導(dǎo)Hes1基因的表達(dá),并與Hes1基因的表達(dá)存在一定的劑量依賴性。本實(shí)驗(yàn)表明當(dāng)Hes1基因表達(dá)增高時,將參與調(diào)控肝干細(xì)胞LEPCs向膽管細(xì)胞方向分化;提示Notch信號通路參與調(diào)控肝干細(xì)胞分化命運(yùn)的選擇,可能與肝干細(xì)胞的分化、增殖,以及肝臟或膽管腫瘤的發(fā)生發(fā)展有一定密切的聯(lián)系。本課題為進(jìn)一步研究和了解Hes1基因的在肝干細(xì)胞膽向分化的調(diào)控機(jī)制提供了一定的理論依據(jù)。
[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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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