ASIC1a介导的自噬对高糖及PDGF诱导的HSC-T6活化的影响及相关机制研究

发布时间：2018-09-14 17:48

【摘要】：糖尿病(diabetes mellitus,DM)是一种以高血糖为主,伴随组织酸化的复杂的、多系统代谢性疾病,能够导致各种器官损坏和功能紊乱。肝纤维化(hepatic fibrosis,HF)是肝脏对各种慢性肝损伤的创伤修复反应,其实质是细胞外基质(extracellular matrix,ECM)的大量积累。而肝星状细胞(hepatic stellate cell,HSC)是肝脏中产生细胞外基质成分的最主要的细胞,静止的肝星状细胞只有被激活才能导致肝纤维化的发生。有研究表明,高血糖在慢性丙型肝炎患者中是纤维化进展的一项独立的协同因素,伴随着更高的促纤维化效应。高血糖是肝脏相关疾病发生发展的一个重要促进因子,近年来糖尿病合并肝纤维化发病率逐渐上升。深入研究高血糖对肝纤维化发生发展的影响、对肝纤维化病变过程中HSC细胞活化增殖的相关机制具有重要意义,研究备受国内外学者的关注。酸敏感离子通道1a(acid-sensing ion channels 1a,ASIC1a)是一类可由胞外H+激活的阳离子通道蛋白复合体,开放的通道对Na+、Ca2+具有通透性。ASIC1a通道的开放,会导致胞外的钙离子内流。有研究表明,细胞内钙离子内流会通过一系列信号通路导致自噬的发生。自噬在HSC活化过程中起着重要的作用。在肝脏损伤的情况下,静止的肝星状细胞会通过上调自噬,通过脂质代谢增加能量的生成而转变成活化的肝星状细胞从而导致肝纤维化的发生。课题组前期研究发现高血糖可诱发肝损伤,促进HSC活化增殖,加重肝纤维化,而此过程中ASIC1a在整体模型及细胞株水平上均存在高表达,进一步的研究发现ASIC1a参与了高血糖促进PDGF诱导的HSC活化增殖,加重肝纤维化的进展。然而,具体的机制仍不是很清楚。高血糖促进pdgf诱导的hsc活化增殖过程中是否通过促进asic1a的表达进而上调自噬,通过自噬促进hsc的活化增殖,加重肝纤维化的进展,相关研究目前国内外尚未见报道。为此,本研究在课题组前期研究基础上,以高糖及pdgf共同刺激hsc细胞,建立体外高糖合并肝纤维化双模型,探讨高糖环境下肝纤维化进程中asic1a介导的自噬对hsc细胞增殖活化的影响、相关机制。主要研究内容概括如下:1.糖尿病合并肝纤维化大鼠肝组织中自噬相关蛋白的表达在课题组前期通过链脲佐菌素和四氯化碳建立的大鼠模型上,he染色及masson染色观察实验组大鼠肝脏病理变化,结果发现糖尿病大鼠、肝纤维化大鼠肝组织均出现了较为显著的肝损伤,其中糖尿病合并肝纤维化组大鼠肝损伤最为严重。westernblot检测自噬相关蛋白lc3ii及beclin1、肝纤维化相关蛋白α-sma和collageni,结果发现糖尿病大鼠、肝纤维化大鼠及糖尿病合并肝纤维化大鼠肝组织较对照组大鼠肝组织lc3ii及beclin1、α-sma及collageni表达均增强,其中双模型组大鼠肝组织中lc3ii及beclin1、α-sma及collageni的表达最高,且较糖尿病和肝纤维化组差异具有统计学意义。结果显示自噬可能与糖尿病加剧肝纤维化病变过程有关。2.asic1a及自噬在高糖及pdgf刺激下hsc-t6中的变化为了检测asic1a及自噬在糖尿病合并肝纤维化细胞模型中的变化,参考课题组前期条件,以高糖(6000mg/l)刺激hsc-t6细胞24h后再给予pdgf(10ng/ml)刺激hsc-t6细胞24h,建立体外高糖合并肝纤维化模型,westernblot检测asic1a、自噬相关蛋白lc3ii及beclin1、肝纤维化相关蛋白α-sma和collageni的表达,ptflc-3质粒转染及mdc染色法观察各组细胞自噬的变化。结果发现,单纯高糖及pdgf刺激均能增强hsc-t6中asic1a、α-sma和collageni的表达,伴随着自噬的增强,其中以高糖合并pdgf双模型组最为明显,较高糖组和pdgf组差异均具有统计学意义。3.自噬阻断剂3-ma对高糖及pdgf刺激下hsc-t6增殖活化的影响为了观察自噬对hsc增殖活化的影响,给予3-ma阻断高糖及pdgf刺激下hsc的自噬,westernblot检测自噬相关蛋白lc3ii及beclin1、肝纤维化相关蛋白α-sma和collageni的表达。结果显示,3-ma能导致lc3ii、beclin1表达下调,同时α-sma和collageni明显降低,较高糖合并pdgf组差异具有统计学意义。4.自噬阻断剂3-ma对高糖及pdgf刺激下hsc-t6细胞周期的影响为了进一步观察自噬对hsc增殖活化的影响,在3-ma、高糖及pdgf共同刺激下,流式细胞术检测各组细胞周期的变化。结果表明,3-ma能使g0/g1期细胞比例增多,g2/m期细胞比例减少,抑制hsc的增殖,较高糖合并pdgf组差异具有统计学意义。5.amiloride对高糖及pdgf刺激下hsc-t6中自噬的影响为了观察高糖及pdgf刺激下hsc中asic1a是否影响自噬,以asic1a非特异性阻断剂阿米洛利(amiloride)阻断asic1a,然后以高糖及pdgf刺激hsc,westernblot检测asic1a、自噬相关蛋白lc3ii及beclin1、肝纤维化相关蛋白α-sma和collageni的表达,ptflc-3质粒转染及mdc染色法观察各组细胞自噬的变化。结果发现,amiloride能降低高糖及pdgf刺激下hsc中asic1a的表达,同时下调自噬相关蛋白lc3ii及beclin1、肝纤维化相关蛋白α-sma和collageni的表达,差异具有统计学意义。ptflc-3质粒转染及mdc染色法结果显示amiloride能导致高糖及pdgf刺激下hsc自噬减弱。6.asic1ashrna对高糖及pdgf刺激下hsc-t6自噬的影响为了进一步观察asic1a对自噬的影响,将特异性的asic1a-shrna转染到高糖及pdgf刺激的hsc-t6中。westernblot检测asic1a、自噬相关蛋白lc3ii及beclin1、肝纤维化相关蛋白α-sma和collageni的表达。结果发现,ASIC1a-ShRNA转染后高糖及PDGF刺激下HSC-T6中ASIC1a蛋白表达降低,同时LC3II及Beclin1、α-SMA和Collagen I的表达也降低,较高糖合并PDGF组差异具有统计学意义。7.高糖及PDGF刺激下HSC-T6中CaMKKβ/ERK通路蛋白表达的变化为观察高糖及PDGF刺激下HSC细胞中ASIC1a诱导自噬的可能通路,Western Blot检测CaMKKβ表达的变化及ERK的磷酸化水平。结果表明,单纯高糖及PDGF刺激均能增强HSC-T6中CaMKKβ的表达、ERK的磷酸化水平,以高糖合并PDGF双模型组最为明显,较高糖组和PDGF组差异均具有统计学意义。8.Amiloride对高糖及PDGF刺激下HSC-T6中CaMKKβ/ERK通路的影响为进一步观察高糖及PDGF刺激下,ASIC1a对HSC-T6中CaMKKβ/ERK通路蛋白表达的影响,以Amiloride、高糖及PDGF共同刺激HSC,Western Blot检测CaMKKβ表达的变化及ERK的磷酸化水平。结果显示,Amiloride能降低HSC-T6中CaMKKβ的表达,同时抑制ERK的磷酸化水平,较高糖合并PDGF组差异具有统计学意义。
[Abstract]:Diabetes mellitus (DM) is a complex, multi-system metabolic disease characterized by hyperglycemia and tissue acidification, which can lead to various organ damage and dysfunction. Hepatic fibrosis (HF) is the liver's wound-healing response to various chronic liver injuries. Its essence is extracellular matrix (E-matrix). The accumulation of CM. Hepatic stellate cell (HSC) is the most important cell that produces extracellular matrix components in the liver. Resting hepatic stellate cells can only be activated to induce liver fibrosis. Studies have shown that hyperglycemia is an independent co-factor in the progression of fibrosis in patients with chronic hepatitis C. Hyperglycemia is an important promoter of the development of liver-related diseases. In recent years, the incidence of diabetes mellitus complicated with hepatic fibrosis has gradually increased. In-depth study of the effect of hyperglycemia on the development of hepatic fibrosis has an important role in the mechanism of HSC cell activation and proliferation in the process of hepatic fibrosis. The acid-sensing channel 1a (ASIC1a) is a kind of cationic channel protein complex activated by extracellular H +. The open channel is permeable to Na +, Ca2 +. The opening of ASIC1a channel can lead to extracellular calcium influx. Autophagy plays an important role in the activation of HSC. In the case of liver injury, resting hepatic stellate cells can become activated hepatic stellate cells by up-regulating autophagy, increasing energy production through lipid metabolism and leading to liver fibrosis. Previous studies have shown that hyperglycemia can induce liver injury, promote HSC activation and proliferation, and aggravate liver fibrosis. In this process, ASIC1a is highly expressed in the whole model and cell line level. Further studies have found that ASIC1a is involved in hyperglycemia promoting PDGF-induced HSC activation and proliferation, and aggravating the progress of liver fibrosis. It is not clear whether hyperglycemia promotes the activation and proliferation of HSC induced by PDGF by promoting the expression of ASIC 1a and then up-regulation of autophagy, which promotes the activation and proliferation of HSC and aggravates the progress of hepatic fibrosis. So far, there are no reports on the related studies at home and abroad. Based on the previous studies of the research group, this study focused on high glucose and pdg. F co-stimulates HSC cells, establishes in vitro high glucose and hepatic fibrosis dual model, explores the effect of asic-1a-mediated autophagy on the proliferation and activation of HSC cells in the process of hepatic fibrosis under high glucose environment, and the related mechanisms. The main research contents are summarized as follows: 1. The expression of autophagy-related proteins in the liver tissues of diabetic rats with hepatic fibrosis is before the research group. On the rat model established by streptozotocin and carbon tetrachloride, HE staining and Masson staining were used to observe the pathological changes of the liver in the experimental group. The results showed that the liver tissues of diabetic rats and hepatic fibrosis rats were significantly damaged, and the liver injury in the diabetic rats with hepatic fibrosis group was the most serious. Autophagy-related proteins lc3ii and beclin1, liver fibrosis-related proteins alpha-SMA and CollagenI were measured. the results showed that the expression of lc3ii, beclin1, alpha-SMA and CollagenI in the liver tissue of diabetic rats, hepatic fibrosis rats and diabetic rats with hepatic fibrosis was higher than that of the control group. The results showed that autophagy may be related to the process of diabetes mellitus aggravating liver fibrosis. 2. The changes of autophagy and HSC-T6 stimulated by high glucose and PDGF in order to detect the expression of asic-1a and autophagy in diabetic liver fibrosis cell model. Referring to the pre-conditions of the research group, HSC-T6 cells were stimulated with high glucose (6000mg / l) for 24 hours and then PDGF (10ng / ml) was given to stimulate HSC-T6 cells for 24 hours to establish an in vitro model of high glucose with hepatic fibrosis. Western blot was used to detect the expression of asic1a, autophagy-related protein lc3ii and beclin1, liver fibrosis-related protein alpha-SMA and collageni, and ptflc-3 plasmid transduction. Autophagy was observed by staining and MDC staining. the results showed that high glucose and PDGF stimulation could enhance the expression of asic1a, a-SMA and CollagenI in hsc-t6, accompanied by the increase of autophagy. the high glucose combined with PDGF double model group was the most obvious, and the difference between the high glucose group and PDGF group was statistically significant. Effects of high glucose and PDGF stimulation on the proliferation and activation of HSC-T6 in order to observe the effect of autophagy on the proliferation and activation of hsc, 3-mA was given to block the autophagy of HSC stimulated by high glucose and pdgf, and the expressions of autophagy-related proteins lc3ii and beclin1, liver fibrosis-related proteins alpha-SMA and CollagenI were detected by Western blot. The effect of autophagy blocker 3-mA on the cell cycle of HSC-T6 stimulated by high glucose and PDGF in order to further observe the effect of autophagy on the proliferation and activation of hsc, flow cytometry was used to detect the cell cycle of each group under the co-stimulation of 3-ma, high glucose and pdgf. The results showed that 3-mA could increase the proportion of G0 / G1 phase cells, decrease the proportion of G2 / M phase cells and inhibit the proliferation of hsc. there was a significant difference between the high glucose group and PDGF group. 5. the effect of amiloride on autophagy in HSC-T6 stimulated by high glucose and pdgf. in order to observe whether asic-1a in HSC stimulated by high glucose and PDGF affected autophagy or not, the effect of asic-1a in HSC was non-specific. The expression of ASIC 1a, autophagy-related protein LC 3ii and Beclin 1, liver fibrosis-related protein alpha-SMA and collageni, ptflc-3 plasmid transfection and MDC staining were detected by Western blot. The results showed that amiloride could decrease the levels of high glucose and pdgf. The expression of ASIC1a in HSC was down-regulated by stimulation, and the expression of autophagy-related proteins lc3ii and beclin1, liver fibrosis-related proteins alpha-SMA and CollagenI were also down-regulated by stimulation. ptflc-3 plasmid transfection and MDC staining showed that amiloride could induce the decrease of HSC autophagy under high glucose and PDGF stimulation. 6. C-t6 autophagy in order to further observe the effect of ASIC1a on autophagy, specific asic1a-shrna was transfected into HSC-T6 stimulated by high glucose and pdgf. Western blot was used to detect the expression of asic1a, autophagy-related proteins lc3ii and beclin1, liver fibrosis-related proteins alpha-SMA and collageni. The expression of ASIC1a protein in HSC-T6 was decreased, while the expressions of LC3II and Beclin1, alpha-SMA and Collagen I were also decreased. There was a significant difference between the high glucose and PDGF groups. The results showed that both high glucose and PDGF stimulation could enhance the expression of CaMKK beta and the phosphorylation level of ERK in HSC-T6, especially in high glucose combined with PDGF double model group, and the difference between high glucose group and PDGF group was statistically significant. The effect of aMKK beta/ERK pathway on the expression of CaMKK beta/ERK pathway protein in HSC-T6 stimulated by high glucose and PDGF was further observed. Amiloride, high glucose and PDGF were used to stimulate HSC. Western Blot was used to detect the expression of CaMKK beta and the phosphorylation level of ERK. The phosphorylation level of ERK was significantly different from that of PDGF group.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2;R587.1

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