【作者】 杨斌；

【导师】 曾维政；

【作者基本信息】 第三军医大学 ， 消化内科， 2010， 硕士

【摘要】 目的肝纤维化是各种慢性肝病发展到肝硬化的中间可逆阶段。在肝纤维化的病程中,肝星状细胞(hepatic stellate cells HSCs)的活化是其核心环节,因此对肝星状细胞的活化进行干预是临床治疗肝纤维化的发展方向。由于肝星状细胞的活化涉及众多的细胞信号传导通路,因此了解肝星状细胞的活化中各种信号通路的作用机制十分重要。引起肝星状细胞活化的病因很多,乙醛是酒精性肝病中作用于HSCs的致病因子,将乙醛作为HSCs的激活剂在各种实验中广泛运用。乙醛刺激大鼠HSCs活化的主要特点是增殖速度增快和包括α1胶原(α1 (I) collagen)在内的细胞基质分泌增多。在前期的实验中,红景天甙已经被证实可以影响肝星状细胞活化过程中的TGFβ-Smad和Rho-ROCK信号通路从而抑制HSCs的激活和过量细胞基质的产生,但是对其抑制HSCs增殖的具体分子机制仍然不是很明确。近年对Wnt信号通路的研究发现:它通过对目的基因MYC、CyclinD1等的转录调控影响细胞的增殖和转化,并与肝星状细胞的活化密切相关。因此了解红景天甙对活化的肝星状细胞中Wnt信号通路和细胞活性的影响对研究红景天甙的药理作用和治疗肝纤维化的分子机理具重要意义。本研究项目通过观察红景天甙处理乙醛刺激的大鼠肝星状细胞生长速度的变化以及细胞中Wnt信号通路中主要下游分子及目的基因mRNA表达和蛋白水平的改变,探讨Wnt信号通路在肝星状细胞活化中的作用。方法用红景天甙和乙醛处理体外培养的大鼠肝星状细胞,同时用正常培养细胞和单以乙醛刺激的细胞为对照[1],由MTT法绘制细胞生长曲线并对比,采用RT-PCR和Western-blot的方法分别从mRNA水平和蛋白水平观察Wnt信号通路相关指标β- catenin和细胞周期蛋白D1(cyclinD1)的表达变化,并用统计方法进行对比分析。结果从各组细胞生长曲线的对比可以清晰发现:单用乙醛刺激可使大鼠肝星状细胞增殖速度增快,红景天甙可以明显降低大鼠肝星状细胞在乙醛刺激后的生长和增殖速度。运用RT-PCR和Western-blot的方法,在单用乙醛组的大鼠肝星状细胞中,发现I型胶原[2]、Wnt信号通路中β- catenin(β-链蛋白)和细胞周期蛋白D1(cyclinD1)的mRNA表达和蛋白合成相对于正常细胞均增强(P<0.05),在红景天甙处理组的大鼠肝星状细胞中,细胞周期蛋白D1(cyclinD1)和β- catenin(β-链蛋白)的mRNA表达和蛋白合成相较于单用乙醛组均出现不同程度的下降(P<0.05)。结论Wnt信号通路的开放参与了乙醛刺激的肝星状细胞活化,红景天甙能有效降低大鼠肝星状细胞中Wnt信号通路的活性从而抑制乙醛刺激的大鼠肝星状细胞的活化和增殖。更多还原

【Abstract】 AIM: Hepatic fibrosis is the middle and reversible stage from chronic liver disease to cirrhosis . The activation of hepatic stellate cell is a core of hepatic fibrosis. It is a tendency for liver fibrosis treatment that interventing the hepatic stellate cell activation. A large number of cell signaling pathways participate in the activation of hepatic stellate cells , so understanding the mechanisms of these cell signaling pathways is very important. As the etiology of alcohol liver disease, acetaldehyde have been used widely as an activator of hepatic stellate cells in experiment . Rat hepatic stellate cells activated by acetaldehyde shows mainly that cell proliferation is accelerated andα1 (I) collagen secretion is increased. The salidroside have been shown to affect the hepatic stellate cell activation through the TGFβ-Smad and Rho-ROCK signaling pathway in the early experiments. In recent years, the Wnt signaling pathway was found to relate closely with activation of hepatic stellate cells. As the Wnt pathway target genes such as cyclin-D1 have close relationship with the cell proliferation, the influence of salidroside on Wnt pathway is very important for studing the molecular mechanism of salidroside treatment for liver fibrosis. In experiment, the role of Wnt signaling pathway and the effect of salidroside on Wnt pathway in the activation of rat hepatic stellate cells was studied by detecting mRNA and protein expression levels of downstream molecules’in Wnt signaling pathway.Methods The cryopreservating rat hepatic stellate cells was recoveryed and cultured in vitro. The acetaldehyde was used as a stimulant of hepatic stellate cell, meanwhile the rat hepatic stellate cells stimulated by acetaldehyde was treated with salidroside , using normal rat hepatic stellate cells and cells stimulated by acetaldehyde only as control. The cells growth curves were drawn using the method MTT.β-catenin、α1 (I) collagen and cyclinD1 were detected by reverse transcription PCR and West-blot.Results From the cell growth curves drawn, we found the proliferation of rat hepatic stellate cells stimulated by acetaldehyde only can be accelerated . By comparing the salidroside treatment can inhibit growth and proliferation rate of cells stimulated by acetaldehyde. We also found that mRNA and protein synthesis ofα1 (I) collagen, cyclinD1 andβ-catenin were enhanced in rat hepatic stellate cell stimulated by acetaldehyde[1] (P<0.05),however the mRNA expression and protein synthesis of cyclinD1、β-catenin were declined in the cells treated by salidroside than these in cells stimulated by acetaldehyde only (P<0.05) .Conclusion The opening Wnt signaling pathway may be involved in activation of hepatic stellate cells stimulated by acetaldehyde. The intervention of salidroside can reduce the proliferation of rat hepatic stellate cell through inhibiting the Wnt signaling pathway.更多还原