【摘要】：肝纖維化是各種致病因素作用于肝臟，導致細胞外基質(zhì)(ECM)在肝臟內(nèi)過量沉積的損傷修復過程，活化的肝星狀細胞(HSC)是產(chǎn)生ECM的主要來源細胞。文獻報道甲基化CpG結(jié)合蛋白2(MeCP2)在HSC活化增殖過程中起促進作用。進一步研究MeCP2在肝纖維化形成中的分子機制，使用小干擾RNA技術(shù)和5-雜氮-2-脫氧胞苷(5-AzadC)，探討對HSC細胞活化增殖的影響，有利于發(fā)現(xiàn)潛在治療HSC細胞活化增殖的新靶點。本實驗擬以TGF-β1刺激誘導HSC活化，重點觀察Hedgehog(Hh)信號轉(zhuǎn)導通路在HSC活化增殖中的作用，及MeCP2對Hedgehog(Hh)信號轉(zhuǎn)導通路的影響。研究內(nèi)容主要包括以下三個部分： 1.大鼠肝纖維化組織中Shh、PTCH1、Gli1的表達變化 大鼠皮下注射CCl4制備SD大鼠肝纖維化模型，CCl4處理組(12W)和正常組各20只。自造模開始，給予CCl4花生油(體積比1:1)1ml/kg劑量皮下注射模型組SD大鼠，，2次/周，總計12W；給予正常組皮下注射1ml/kg劑量花生油。制備模型完畢，取出肝臟組織用于Masson膠原染色和HE染色檢測，觀察大鼠肝組織中Shh、PTCH1、Gli1的表達情況，同時，檢測在TGF-β1體外刺激誘導活化HSC過程中Shh、PTCH1、Gli1的表達情況。研究發(fā)現(xiàn)，CCl4成功誘導肝纖維化疾病模型，PTCH1的表達在肝纖維化模型中顯著減少，Shh、Gli1的表達逐漸增高。 2. DNA甲基化抑制劑和MeCP2對肝星狀細胞活化增殖的影響 利用DNA甲基化抑制劑5-AzadC和RNA干擾沉默MeCP2以觀察對HSC-T6細胞增殖的影響。HSC-T6體外培養(yǎng)，應用5ng/ml TGF-β1刺激細胞24h后，然后加入5-AzadC孵育48h，MTT實驗觀察5-AzadC對肝星狀細胞增殖活性的影響，流式細胞術(shù)檢測HSC-T6細胞周期的變化，QRT-PCR實驗檢測Col1A1、α-SMA的mRNA表達變化；另外，借助免疫組化和QRT-PCR實驗觀察MeCP2、α-SMA在肝纖維化模型中的表達情況。依照小RNA設(shè)計原則合成MeCP2的小干擾RNA(RNAi)，借助LipofectamineTM2000脂質(zhì)體瞬時轉(zhuǎn)染進入HSC-T6細胞里，流式細胞術(shù)檢測HSC-T6細胞周期的變化。檢測結(jié)果發(fā)現(xiàn)：應用5-AzadC處理HSC-T6后α-SMA、CollagenⅠ的表達水平明顯降低；提示靶向沉默MeCP2表達和5-AzadC處理后，能夠有效抑制HSC-T6的活化增殖。以上結(jié)果表明，MeCP2和DNA甲基化在肝纖維化形成和HSC活化過程中發(fā)揮了重要作用。 3. MeCP2對PTCH1表達的調(diào)控機制研究 前期研究發(fā)現(xiàn)，應用DNA甲基化特異性抑制劑5-AzadC處理HSC可明顯抑制其活化增殖，并可促進PTCH1在活化的HSC中的表達。提示在HSC活化過程中DNA甲基化參與調(diào)控PTCH1的表達。據(jù)此推測，PTCH1表達下降可能與PTCH1啟動子區(qū)域發(fā)生甲基化有關(guān)。文獻報道，MeCP2作為DNA甲基化重要的結(jié)合蛋白參與調(diào)控HSC活化增殖。利用小干擾RNA沉默MeCP2表達后，可明顯提高PTCH1的表達水平，提示MeCP2與PTCH1表達沉默密切相關(guān)。這些實驗結(jié)果進一步提示，PTCH1表達下降與PTCH1啟動子區(qū)域甲基化有關(guān)，為臨床防治肝纖維化的潛在治療作用靶點。
[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 偽-SMA; In addition, the expression of MeCP2, 偽-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 偽-SMA,Collagen 鈪

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