【作者】 张海燕；

【导师】 臧宇辉；

【作者基本信息】 南京大学 ， 生物化学与分子生物学（专业学位）， 2013， 硕士

【摘要】 肝纤维化是多种肝病共同的病理过程,肝星状细胞(Hepatic Stellate Cells, HSCs)激活则是导致肝脏纤维化发生的关键步骤。HSCs激活和肝纤维化发生的过程受多种细胞因子和信号传导通路调控,其中转化生长因子TGF-β1是目前已知最重要和最强的促HSCs激活和纤维生成因子,TGF-β1主要通过TGF-β/Smad信号通路激活HSCs。微小核糖核酸(microRNA, miRNA)是一类长度约为21-25个核苷酸的非编码单链小核糖核酸分子,它们在进化上高度保守,广泛存在于动植物细胞中。近年研究发现,miRNA能够调控一系列肝纤维化相关基因的表达,在肝纤维化发生发展过程中扮演着极其重要的角色。虽然现有报道解释了HSC激活过程中KLF6在转录水平上调TGFBR1表达的机制,但是TGF-β受体的表达是否受转录后水平的调控迄今未有报道。本论文的研究发现在四氯化碳(CCl4)诱导的小鼠肝纤维化模型中,肝组织的miR-101表达水平显著降低,而KLF6和TGFBR1的表达水平明显升高。肝纤维化过程中分离原代HSCs,也发现HSCs中miR-101表达水平在CCl4注射开始后迅速下降,而TGFBR1和KLF6的表达水平在同期显著上升。荧光素酶报告基因检测和Weatern-blot均证实miR-101能够通过靶向KLF6和TGFBR1的3’UTR从而抑制其表达。我们构建了能够在动物体内表达miR-101的重组慢病毒,经尾静脉注射重组慢病毒,可以显著提高肝组织内miR-101的表达水平并下调小鼠肝组织内KLF6、TGFBR1的表达水平,而CCl4诱导的小鼠肝纤维化进程则受到明显的抑制。相反经尾静脉注射miR-101的反义核酸降低肝组织内miR-101的表达水平则会加重肝纤维化的程度。细胞实验的结果表明,miR-101能有效降低星状细胞中KLF6和TGFBR1的表达水平并抑制星状细胞的TGF-β/Smad信号传导。同时在活化的肝星状细胞中,miR-101可显著抑制其成纤维母细胞的生物学特性。综上所述,本论文通过动物体内、外的研究,初步发现了miR-101调控肝纤维化发生的作用机制,为从miRNA的角度了解肝纤维化发生的分子机制,提供了新的研究思路和实验依据。更多还原

【Abstract】 Liver fibrosis is a common outcome of chronic hepatic injuries or diseases. The activation of hepatic stellate cells (HSCs) is the key step of liver fibrosis. HSCs activation and liver fibrosis process are regulated by a variety of cytokines and signaling pathways. Of these, TGF-β1has been indentified as the key cytokine to promote the HSC activation through the Smad2/3pathway. MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules, which are21-25nucleotides in length. They are highly conserved in evolution and widely present in animal and plant cells. Recent studies show that miRNAs can regulate a series of hepatic fibrosis-related gene expression and play a very important role in the development of hepatic fibrosis.Although existing reports explain the mechanism of KLF6raising TGFBR1expression at transcription level in HSC activation process, there is no report about the regulation of TGFβ receptor expression at the transcription level so far. Here, we induced hepatic fibrosis in mouse by repetitive intraperitoneal injections of CCl4and detected marked downregulation of miR-101in the fibrotic liver, while the expression levels of KLF6and TGFBRl increased significantly. qRT-PCR results also revealed that miR-101level in the isolated HSCs dramatically decreased upon CCl4treatment. Meanwhile, TβRI and KLF6mRNA expression in the isolated HSCs significantly increased during the process of CCl4-induced liver fibrogenesis. Luciferase assay and western blot results showed that miR-101dramatically reduced TβRI and KLF6 expression by direct targeting the3’UTR of their mRNA. Lenti-miR-101, that can express miR-101in vivo, was intravenously injected into CCl4-treated mice via tail vein, and greatly reduced CCl4-induced liver fibrosis as demonstrated by HE, Sirius red and α-SMA staining. miR-101expression were greatly upregulated in Lenti-miR-101-infected mice. Meanwhile, hepatic TβRI and KLF6expression were significantly reduced, confirming negative regulation of TβRI and KLF6by miR-101in liver. Nevertheless, blocking hepatic miR-101expression by a chemically modified antisense oligonucleotide in a liver fibrosis model would aggravate fibrosis. Western-blot of HSCs revealed that miR-101markedly suppressed the upregulation of KLF6and TβRI induced by TGF-β1, and consequently reduced phosphorylation of Smad2. Moreover, HSCs transdifferentiation is greatly suppressed by miR-101In conclusion, we have identified the miR-101as a negative regulator of fibrotic TGF-β signaling in liver fibrosis by regulating TβRI production. These findings were further validated in vivo, where restored miR-101expression inhibited the liver fibrogenesis, indicating that miR-101may be a possible therapy for the treatment or reversion of liver fibrosis. Hence, the identification of miR-101and its target genes provide useful insights into mechanisms underlying liver fibrosis.更多还原