【作者】 金晔；

【导师】 曲强；

【作者基本信息】 中国协和医科大学 ， 外科学， 2008， 硕士

【摘要】 研究背景和目的:肝脏是体内进行物质代谢的重要器官,其中肝细胞约占肝脏组织的80%。肝细胞特异表达一系列编码血浆蛋白、凝血因子和多种肝脏解毒作用及维持糖类物质、脂肪和胆固醇代谢相关酶的功能基因。肝细胞特异表达基因的功能分析研究发现这些基因的调节区由结合不同家族的肝细胞转录因子的顺式作用元件组成。不同家族的肝脏富集转录因子形成蛋白复合物协同调控肝细胞特异基因表达的启动和维持。肝细胞核因子4α(HNF4α)在很多肝细胞功能基因的启动子或增强子上有结合活性,可以直接调控多达40%左右的肝细胞特异基因的表达。此外,HNF4α还可以通过调控多种其它家族肝细胞分化相关转录因子的表达间接调节肝细胞功能。因此,转录因子HNF4α很可能位于肝细胞相关转录因子调控网络的上游并对于肝细胞功能基因的表达起核心调控作用。目前,对于HNF4α的功能研究集中在胚胎肝脏发育和成熟肝细胞功能维持方面,但是对于肝细胞分化过程尤其是体外诱导分化过程中HNF4α的表达和尚未见报道。骨髓干细胞(bone marrow stem cells,BMSCs)可以分化为肝前体细胞和肝细胞,并具有取材容易,增殖能力强,体外培养、传代及扩增容易的特点,可以作为研究体外诱导分化肝细胞的可靠来源。但是骨髓干细胞诱导向成熟肝细胞分化的具体诱导机制目前尚不清楚。因此,本研究在骨髓间充质干细胞体外向肝细胞诱导分化过程中的培养细胞进行肝细胞标志基因及转录因子mRNA表达水平检测。在此基础上应用RNA干扰技术抑制转录因子HNF4α的转录水平,并深入研究大鼠骨髓间充质干细胞向肝细胞体外诱导分化过程中转录因子HNF4α调控的作用机制,深入了解细胞分化机制,并为临床促进肝细胞分化增殖提供理论基础。实验方法:1、提取大鼠骨髓细胞诱导其向肝细胞分化。通过形态学表现,ALB免疫荧光染色定位,半定量逆转录—聚合酶链反应(RT-PCR)方法检测肝细胞特征性标志ALB、AFP的mRNA的表达,生化方法检测白蛋白、尿素和ALT的在细胞培养液中的浓度以及PAS染色方法检测细胞糖原合成能力来验证大鼠骨髓间充质干细胞经过体外诱导分化培养后是否具有成熟肝细胞的功能。2、应用半定量RT-PCR方法比较肝细胞分化过程中肝脏富集转录因子肝细胞核因子4α(HNF4α),CCAAT增强子结合蛋白α和β(C/EBPα和C/EBPβ)在诱导分化组和对照组细胞中的mRNA表达水平和免疫荧光染色方法在荧光显微镜下观察HNF4α细胞中的表达定位,获得骨髓间充质肝细胞向肝细胞体外诱导分化过程中上述三种转录因子的表达特征。3、分别在大鼠骨髓干细胞的诱导分化过程中的三个时间点应用RNA干扰技术抑制转录因子HNF4α的mRNA表达。应用Realtime RT-PCR及HNF4α免疫荧光染色方法检测HNF4αmRNA和蛋白水平的表达,检验RNA干扰的抑制效果。同时,应用Realtime RT-PCR,细胞培养液生化检测,ALB免疫荧光染色和PAS染色方法检测HNF4α表达抑制对于诱导分化细胞的肝细胞特异基因ALB、AFP和HNF1α的mRNA表达水平,白蛋白表达水平和分泌能力,合成尿素和储存糖原能力的影响。研究结果:1、大鼠骨髓间充质干细胞经诱导分化培养后细胞具有成熟肝细胞的形态。在大鼠骨髓间充质干细胞向肝细胞诱导分化培养的7天、14天、18天和21天四个时间点,成熟肝细胞标志ALB和AFP mRNA的表达水平明显高于非诱导培养对照组细胞(p＜0.05)。应用ALB的特异性抗体进行的免疫荧光染色显示,骨髓间充质干细胞在分化14天后,细胞质中充满了大量点状,或颗粒状的绿色的荧光染色。此外,诱导分化细胞产生白蛋白、尿素和ALT以及合成及储存糖原的功能要明显强于对照组细胞。2、在骨髓间充质干细胞体外诱导分化培养细胞中,转录因子HNF4α、C/EBPα和C/EBPβmRNA均有表达,其中诱导分化组HNF4α和C/EBPα在细胞分化早期、C/EBPβ在分化晚期表达分别显著高于对照组(p＜0.05)。在HNF4αmRNA表达水平显著高于对照组的诱导分化14天时间点,免疫荧光染色显示诱导分化组细胞有HNF4α特异染色定位于分化细胞核内,而对照组细胞中并未观察到细胞核内有特异染色。3、在骨髓间充质干细胞向肝细胞诱导分化的7天、14天和21天三个时间点,对诱导分化细胞瞬时转染HNF4α的siRNA后,HNF4α的mRNA表达水平相比于正常诱导分化细胞降低了40%,而HNF4α免疫荧光染色显示转染siRNA的细胞核内HNF4α的特异染色明显减少,说明转染HNF4αsiRNA有效抑制了诱导分化细胞中HNF4αmRNA和蛋白水平的表达。在HNF4α表达下调的细胞中,成熟肝细胞标志AFP和HNF1α的mRNA表达水平、尿素产生及糖原合成储存能力显著低于同时间点正常诱导分化细胞。另一个肝细胞标志ALB mRNA和蛋白水平的表达则仅在诱导分化早期受HNF4α的调控。而在诱导分化进行至21天时,ALB的mRNA和蛋白水平的表达水平都与HNF4α表达下调无关。研究结论:1、大鼠骨髓间充质肝细胞在生长因子HGF,EGF,bFGF,地塞米松和胰岛素的诱导培养下可以分化为具有成熟肝细胞功能和形态的肝细胞样细胞,可以作为体外诱导骨髓间充质干细胞向肝细胞的可靠模型。2、肝细胞分化过程中转录因子HNF4α、C/EBPα和C/EBPβ呈特征性时序表达,表明骨髓间充质干细胞在向肝细胞分化时,肝细胞相关转录因子的表达与细胞分化的启动和维持密切相关。3、HNF4α对骨髓间充质干细胞来源的肝细胞的特异标志和大部分生理功能有直接和间接的调控作用,是体外诱导大鼠骨髓间充质干细胞向成熟肝细胞分化过程中的关键转录因子更多还原

【Abstract】 Background and ObjectiveThe liver,which is composed of eighty percent cells of hepatocytes,is an essential organ to maintein substance metabolism function in the body.Hepatocytes express a large number of hepatocyte-specific genes encoding plasma proteins, clotting factors and enzymes involved in detoxification,gluconeogenesis,glycogen synthesis,and glucose,fat,and cholesterol metabolism.Functional analysis of numerous hepatocyte-specific DNA regulatory regions reveals that they are composed of multiple cis-acting DNA sequences that bind to different families of liver-enriched transcription factors.The protein interactions between multiple liver-enriched transcriptional factors provide synergistic transcriptional stimulation and maintenance to hepatocyte-specific genes expression.Hepatocyte nuclear factor 4 alpha(HNF4α) binding to promoter and enhancer regions of numerous hepatocyte-specific genes directly regulates the expression of nearly forty percent of hepatocyte-specific genes.Furthermore,HNF4αregulates the transcriptional activity of other families of liver-enriched transcriptional factors,thus regulating hepatocyte function indirectly.Therefore,HNF4α,which locates at the upstream of the liver-enriched transcriptional regulation network,is a critical transcriptional factor to the expression of hepatocyte-spacific genes.Although a large number of studies refer to the function of HNF4αin liver development and maintenance of mature hepatocyte functions and phenotypes,its expression and role in the differentiation of hepatocyte in vitro remains unknown so far.Bone marrow stem cells(BMSCs) can differentiate into liver precursor cells and hepatocytes.In addition,they can easily be acquired,cultured,suncultured and proliferated in vitro.Therefore,BMSCs are stable resource for the research of hepatocytes differentiation in vitro.The mechanism of differentiation of bone marrow-derived hepatocytes remains unknown so far.The main purpose of this research is to elucidate the effect and mechanism of HNF4αduring the differentiation process of bone marrow-derived hepatocytes.This study may provide a rationale for the clinical application of promotion of hepatocyte differentiation and regeneration.Methods1.Bone marrow stem cells were isolated from rat femurs and induced to mature hepatocytes in vitro.Whether the induced cells were differentiatied into mature hapatocyte in vitro was identified through the morphological phenotype,ALB immunohistochemical staining,mRNA expression of hepatocyte- specific gene ALB and AFP tested by semi-quantitative RT-PCR,the production of ALB,urea and ALT detected by biochemical test and the function of glycogen synthesis and storage by the PAS staining.2.Bone marrow stem cells were isolated from rat femurs and induced to mature hepatocytes in vitro.The mRNA expression levels of hepatocyte nuclear factor 4α(HNF4α),CCAAT enhancer binding proteinα(C/EBPα) andβ(C/EBPβ) were compared between induced and non-induced cultures using semi-quantitative RT-PCR. The distribution pattern of HNF4αwas detected by immunofluorescence staining and observed by fluorescence microscope.3.We transfected HNF4αsmall interfering RNA into differentiated cells to depress the expression of HNF4αmRNA in three different time points in induction culture of rat bone marrow mesenchymal stem cells.The effect of HNF4αdepression was detected by Realtime RT-PCR and immunofluorescence staining.At the same time points,the effect of differentiated cells function after HNF4αsiRNA transfection was evaluated by ALB immunohistochemical staining performance,mRNA expression of hepatocyte-specific gene ALB,AFP and transcriptional factor HNF1αtested by Realtime RT-PCR,the production of ALB and urea detected by biochemical test,and the ability of glycogen synthesis and storage by the PAS staining.Results1.Rat bone marrow cells perform mature hapatocyte morphorlogy after 21 day induction culture in vitro.We found that the expressions of several hepatocyte specific markers in induction cultures,for example,ALB and AFP mRNA were much higher than those in the non-induced cells.The ALB mRNA expression was increased throughout the whole culture.At the protein level,we found clear positive staining of ALB in the cytoplasm of inducted cells.Furthermore,the production of ALB,urea and ALT and glocogen synthesis and storage of induced cells were much higher in the induced cells.2.Transcriptional factors,HNF4α,C/EBPαand C/EBPβ,were expressed in the induced cells during the culture process.The mRNA expression levels of HNF4αand C/EBPαwere significantly higher in induced cells than those in non-induced cells in the early stage,whereas C/EBPβexpression was significantly up-regulated in induced cultures at the late stage(p＜0.05).Immunofluorescence staining showed that HNF4αwas located in the cell nucleus of differentiated cells.3.In three different time points of induced differentiation of hepatocyte in vitro, the transfection of HNF4αsiRNA resulted in nearly forty percent depression in the mRNA expression of HNF4α.Due to the downregulation of HNF4α,the hepatocyte-specific genes,AFP and HNF1α,the production of urea and ability of glocogen synthesis and storage in the siRNA transfected cells were much lower than those in the normal differnentiated cells at the same time points.Besides,the expression of another hepatocyte marker ALB mRNA and protein were down-regulated by HNF4αdepression at the early stage of the induced differentiation.But at the late stage of the differentiation,the expression of ALB was not influenced by the reduction of HNF4α.Conclusions1.The mesenchymal stem cells from the rat bone marrow can differentiate into hepatocyte-like cells which have function and morphorlogy of mature hepatocyte in vitro when cultured in the presence of epithelial cell growthfactor(EGF), basic-fibroblast-growth factor(bFGF),hepatocyte growth factor(HGF),Insulin and Dexamethasone.2.The characteristical time-dependent expression of transcriptional factors HNF4α,C/EBPαand C/EBPβduring the hepatocyte differentiation by bone marrow stem cells demonstrates that the expressions of these transcriptional factors are closely related to the initiation and maintenance of hepatocyte differentiation.3.HNF4αregulates the expression of hepatocyte-spacific genes and physiological functions of bone marrow-derived hepatocytes directly and indirectly.HNF4αplays a critical role in the differentiation of functional hepatocyte-like cells from rat bone marrow mesenchymal stem cells in vitro.更多还原