MeCP2调控PTCH1表达对大鼠HSC活化增殖的影响
发布时间：2018-11-19 08:29
【摘要】：肝纤维化是各种致病因素作用于肝脏，导致细胞外基质(ECM)在肝脏内过量沉积的损伤修复过程，活化的肝星状细胞(HSC)是产生ECM的主要来源细胞。文献报道甲基化CpG结合蛋白2(MeCP2)在HSC活化增殖过程中起促进作用。进一步研究MeCP2在肝纤维化形成中的分子机制，使用小干扰RNA技术和5-杂氮-2-脱氧胞苷(5-AzadC)，探讨对HSC细胞活化增殖的影响，有利于发现潜在治疗HSC细胞活化增殖的新靶点。本实验拟以TGF-β1刺激诱导HSC活化，重点观察Hedgehog(Hh)信号转导通路在HSC活化增殖中的作用，及MeCP2对Hedgehog(Hh)信号转导通路的影响。研究内容主要包括以下三个部分： 1.大鼠肝纤维化组织中Shh、PTCH1、Gli1的表达变化 大鼠皮下注射CCl4制备SD大鼠肝纤维化模型，CCl4处理组(12W)和正常组各20只。自造模开始，给予CCl4花生油(体积比1:1)1ml/kg剂量皮下注射模型组SD大鼠，，2次/周，总计12W；给予正常组皮下注射1ml/kg剂量花生油。制备模型完毕，取出肝脏组织用于Masson胶原染色和HE染色检测，观察大鼠肝组织中Shh、PTCH1、Gli1的表达情况，同时，检测在TGF-β1体外刺激诱导活化HSC过程中Shh、PTCH1、Gli1的表达情况。研究发现，CCl4成功诱导肝纤维化疾病模型，PTCH1的表达在肝纤维化模型中显著减少，Shh、Gli1的表达逐渐增高。 2. DNA甲基化抑制剂和MeCP2对肝星状细胞活化增殖的影响 利用DNA甲基化抑制剂5-AzadC和RNA干扰沉默MeCP2以观察对HSC-T6细胞增殖的影响。HSC-T6体外培养，应用5ng/ml TGF-β1刺激细胞24h后，然后加入5-AzadC孵育48h，MTT实验观察5-AzadC对肝星状细胞增殖活性的影响，流式细胞术检测HSC-T6细胞周期的变化，QRT-PCR实验检测Col1A1、α-SMA的mRNA表达变化；另外，借助免疫组化和QRT-PCR实验观察MeCP2、α-SMA在肝纤维化模型中的表达情况。依照小RNA设计原则合成MeCP2的小干扰RNA(RNAi)，借助LipofectamineTM2000脂质体瞬时转染进入HSC-T6细胞里，流式细胞术检测HSC-T6细胞周期的变化。检测结果发现：应用5-AzadC处理HSC-T6后α-SMA、CollagenⅠ的表达水平明显降低；提示靶向沉默MeCP2表达和5-AzadC处理后，能够有效抑制HSC-T6的活化增殖。以上结果表明，MeCP2和DNA甲基化在肝纤维化形成和HSC活化过程中发挥了重要作用。 3. MeCP2对PTCH1表达的调控机制研究 前期研究发现，应用DNA甲基化特异性抑制剂5-AzadC处理HSC可明显抑制其活化增殖，并可促进PTCH1在活化的HSC中的表达。提示在HSC活化过程中DNA甲基化参与调控PTCH1的表达。据此推测，PTCH1表达下降可能与PTCH1启动子区域发生甲基化有关。文献报道，MeCP2作为DNA甲基化重要的结合蛋白参与调控HSC活化增殖。利用小干扰RNA沉默MeCP2表达后，可明显提高PTCH1的表达水平，提示MeCP2与PTCH1表达沉默密切相关。这些实验结果进一步提示，PTCH1表达下降与PTCH1启动子区域甲基化有关，为临床防治肝纤维化的潜在治疗作用靶点。
[Abstract]:Hepatic fibrosis is a process of damage and repair caused by various pathogenic factors acting on the liver, resulting in excessive deposition of extracellular matrix (ECM) in the liver. Activated hepatic stellate cell (HSC) is the main source of ECM. It is reported that methylated CpG binding protein 2 (MeCP2) promotes the activation and proliferation of HSC. To further study the molecular mechanism of MeCP2 in the formation of hepatic fibrosis, the effects of small interfering RNA technique and 5-aza-2-deoxycytidine (5-AzadC) on the activation and proliferation of HSC cells were investigated. It is helpful to find new targets for the potential therapy of HSC cell activation and proliferation. In this study, HSC activation was induced by TGF- 尾 1 stimulation, and the role of Hedgehog (Hh) signal transduction pathway in HSC activation and proliferation, and the effect of MeCP2 on Hedgehog (Hh) signal transduction pathway were observed. The research includes the following three parts: 1. Changes of Shh,PTCH1,Gli1 expression in Rat liver Fibrosis Model of SD Rats was established by subcutaneous injection of CCl4, 20 rats in CCl4 treated group (12 W) and 20 in normal group. Since the establishment of the model, CCl4 peanut oil (1:1 by volume) was given subcutaneously to the SD rats in the model group, twice a week, for a total of 12 ws.The normal group was subcutaneously injected with 1ml/kg peanut oil. After the model was made, the liver tissue was taken out for Masson collagen staining and HE staining to observe the expression of Shh,PTCH1,Gli1 in rat liver tissue. Meanwhile, Shh,PTCH1, was detected during the process of HSC activation induced by TGF- 尾 1 in vitro. Expression of Gli1. It was found that the expression of PTCH1 decreased significantly and the expression of Shh,Gli1 increased gradually in the model of hepatic fibrosis induced by CCl4. 2. Effects of DNA methylation inhibitor and MeCP2 on the activation and proliferation of hepatic stellate cells the effects of DNA methylation inhibitor 5-AzadC and RNA interference silencing MeCP2 on the proliferation of HSC-T6 cells were observed in vitro HSC-T6 culture. The effects of 5ng/ml TGF- 尾 1 on the proliferation of hepatic stellate cells were observed by 5ng/ml TGF- 尾 1 stimulation for 24 h and then incubated with 5-AzadC for 48 h. The changes of HSC-T6 cell cycle were detected by flow cytometry, and Col1A1, was detected by QRT-PCR assay. The change of mRNA expression of 伪-SMA; In addition, the expression of MeCP2, 伪-SMA in liver fibrosis model was observed by immunohistochemistry and QRT-PCR experiment. The small interfering RNA (RNAi), that synthesized MeCP2 according to the principle of small RNA design was transiently transfected into HSC-T6 cells by LipofectamineTM2000 liposome. The changes of HSC-T6 cell cycle were detected by flow cytometry. The results showed that the expression level of 伪-SMA,Collagen 鈪
本文编号：2341701
[Abstract]:Hepatic fibrosis is a process of damage and repair caused by various pathogenic factors acting on the liver, resulting in excessive deposition of extracellular matrix (ECM) in the liver. Activated hepatic stellate cell (HSC) is the main source of ECM. It is reported that methylated CpG binding protein 2 (MeCP2) promotes the activation and proliferation of HSC. To further study the molecular mechanism of MeCP2 in the formation of hepatic fibrosis, the effects of small interfering RNA technique and 5-aza-2-deoxycytidine (5-AzadC) on the activation and proliferation of HSC cells were investigated. It is helpful to find new targets for the potential therapy of HSC cell activation and proliferation. In this study, HSC activation was induced by TGF- 尾 1 stimulation, and the role of Hedgehog (Hh) signal transduction pathway in HSC activation and proliferation, and the effect of MeCP2 on Hedgehog (Hh) signal transduction pathway were observed. The research includes the following three parts: 1. Changes of Shh,PTCH1,Gli1 expression in Rat liver Fibrosis Model of SD Rats was established by subcutaneous injection of CCl4, 20 rats in CCl4 treated group (12 W) and 20 in normal group. Since the establishment of the model, CCl4 peanut oil (1:1 by volume) was given subcutaneously to the SD rats in the model group, twice a week, for a total of 12 ws.The normal group was subcutaneously injected with 1ml/kg peanut oil. After the model was made, the liver tissue was taken out for Masson collagen staining and HE staining to observe the expression of Shh,PTCH1,Gli1 in rat liver tissue. Meanwhile, Shh,PTCH1, was detected during the process of HSC activation induced by TGF- 尾 1 in vitro. Expression of Gli1. It was found that the expression of PTCH1 decreased significantly and the expression of Shh,Gli1 increased gradually in the model of hepatic fibrosis induced by CCl4. 2. Effects of DNA methylation inhibitor and MeCP2 on the activation and proliferation of hepatic stellate cells the effects of DNA methylation inhibitor 5-AzadC and RNA interference silencing MeCP2 on the proliferation of HSC-T6 cells were observed in vitro HSC-T6 culture. The effects of 5ng/ml TGF- 尾 1 on the proliferation of hepatic stellate cells were observed by 5ng/ml TGF- 尾 1 stimulation for 24 h and then incubated with 5-AzadC for 48 h. The changes of HSC-T6 cell cycle were detected by flow cytometry, and Col1A1, was detected by QRT-PCR assay. The change of mRNA expression of 伪-SMA; In addition, the expression of MeCP2, 伪-SMA in liver fibrosis model was observed by immunohistochemistry and QRT-PCR experiment. The small interfering RNA (RNAi), that synthesized MeCP2 according to the principle of small RNA design was transiently transfected into HSC-T6 cells by LipofectamineTM2000 liposome. The changes of HSC-T6 cell cycle were detected by flow cytometry. The results showed that the expression level of 伪-SMA,Collagen 鈪
本文编号：2341701
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