【摘要】：糖尿病(diabetes mellitus,DM)是一種以高血糖為主,伴隨組織酸化的復雜的、多系統(tǒng)代謝性疾病,能夠?qū)е赂鞣N器官損壞和功能紊亂。肝纖維化(hepatic fibrosis,HF)是肝臟對各種慢性肝損傷的創(chuàng)傷修復反應,其實質(zhì)是細胞外基質(zhì)(extracellular matrix,ECM)的大量積累。而肝星狀細胞(hepatic stellate cell,HSC)是肝臟中產(chǎn)生細胞外基質(zhì)成分的最主要的細胞,靜止的肝星狀細胞只有被激活才能導致肝纖維化的發(fā)生。有研究表明,高血糖在慢性丙型肝炎患者中是纖維化進展的一項獨立的協(xié)同因素,伴隨著更高的促纖維化效應。高血糖是肝臟相關(guān)疾病發(fā)生發(fā)展的一個重要促進因子,近年來糖尿病合并肝纖維化發(fā)病率逐漸上升。深入研究高血糖對肝纖維化發(fā)生發(fā)展的影響、對肝纖維化病變過程中HSC細胞活化增殖的相關(guān)機制具有重要意義,研究備受國內(nèi)外學者的關(guān)注。酸敏感離子通道1a(acid-sensing ion channels 1a,ASIC1a)是一類可由胞外H+激活的陽離子通道蛋白復合體,開放的通道對Na+、Ca2+具有通透性。ASIC1a通道的開放,會導致胞外的鈣離子內(nèi)流。有研究表明,細胞內(nèi)鈣離子內(nèi)流會通過一系列信號通路導致自噬的發(fā)生。自噬在HSC活化過程中起著重要的作用。在肝臟損傷的情況下,靜止的肝星狀細胞會通過上調(diào)自噬,通過脂質(zhì)代謝增加能量的生成而轉(zhuǎn)變成活化的肝星狀細胞從而導致肝纖維化的發(fā)生。課題組前期研究發(fā)現(xiàn)高血糖可誘發(fā)肝損傷,促進HSC活化增殖,加重肝纖維化,而此過程中ASIC1a在整體模型及細胞株水平上均存在高表達,進一步的研究發(fā)現(xiàn)ASIC1a參與了高血糖促進PDGF誘導的HSC活化增殖,加重肝纖維化的進展。然而,具體的機制仍不是很清楚。高血糖促進pdgf誘導的hsc活化增殖過程中是否通過促進asic1a的表達進而上調(diào)自噬,通過自噬促進hsc的活化增殖,加重肝纖維化的進展,相關(guān)研究目前國內(nèi)外尚未見報道。為此,本研究在課題組前期研究基礎(chǔ)上,以高糖及pdgf共同刺激hsc細胞,建立體外高糖合并肝纖維化雙模型,探討高糖環(huán)境下肝纖維化進程中asic1a介導的自噬對hsc細胞增殖活化的影響、相關(guān)機制。主要研究內(nèi)容概括如下:1.糖尿病合并肝纖維化大鼠肝組織中自噬相關(guān)蛋白的表達在課題組前期通過鏈脲佐菌素和四氯化碳建立的大鼠模型上,he染色及masson染色觀察實驗組大鼠肝臟病理變化,結(jié)果發(fā)現(xiàn)糖尿病大鼠、肝纖維化大鼠肝組織均出現(xiàn)了較為顯著的肝損傷,其中糖尿病合并肝纖維化組大鼠肝損傷最為嚴重。westernblot檢測自噬相關(guān)蛋白lc3ii及beclin1、肝纖維化相關(guān)蛋白α-sma和collageni,結(jié)果發(fā)現(xiàn)糖尿病大鼠、肝纖維化大鼠及糖尿病合并肝纖維化大鼠肝組織較對照組大鼠肝組織lc3ii及beclin1、α-sma及collageni表達均增強,其中雙模型組大鼠肝組織中l(wèi)c3ii及beclin1、α-sma及collageni的表達最高,且較糖尿病和肝纖維化組差異具有統(tǒng)計學意義。結(jié)果顯示自噬可能與糖尿病加劇肝纖維化病變過程有關(guān)。2.asic1a及自噬在高糖及pdgf刺激下hsc-t6中的變化為了檢測asic1a及自噬在糖尿病合并肝纖維化細胞模型中的變化,參考課題組前期條件,以高糖(6000mg/l)刺激hsc-t6細胞24h后再給予pdgf(10ng/ml)刺激hsc-t6細胞24h,建立體外高糖合并肝纖維化模型,westernblot檢測asic1a、自噬相關(guān)蛋白lc3ii及beclin1、肝纖維化相關(guān)蛋白α-sma和collageni的表達,ptflc-3質(zhì)粒轉(zhuǎn)染及mdc染色法觀察各組細胞自噬的變化。結(jié)果發(fā)現(xiàn),單純高糖及pdgf刺激均能增強hsc-t6中asic1a、α-sma和collageni的表達,伴隨著自噬的增強,其中以高糖合并pdgf雙模型組最為明顯,較高糖組和pdgf組差異均具有統(tǒng)計學意義。3.自噬阻斷劑3-ma對高糖及pdgf刺激下hsc-t6增殖活化的影響為了觀察自噬對hsc增殖活化的影響,給予3-ma阻斷高糖及pdgf刺激下hsc的自噬,westernblot檢測自噬相關(guān)蛋白lc3ii及beclin1、肝纖維化相關(guān)蛋白α-sma和collageni的表達。結(jié)果顯示,3-ma能導致lc3ii、beclin1表達下調(diào),同時α-sma和collageni明顯降低,較高糖合并pdgf組差異具有統(tǒng)計學意義。4.自噬阻斷劑3-ma對高糖及pdgf刺激下hsc-t6細胞周期的影響為了進一步觀察自噬對hsc增殖活化的影響,在3-ma、高糖及pdgf共同刺激下,流式細胞術(shù)檢測各組細胞周期的變化。結(jié)果表明,3-ma能使g0/g1期細胞比例增多,g2/m期細胞比例減少,抑制hsc的增殖,較高糖合并pdgf組差異具有統(tǒng)計學意義。5.amiloride對高糖及pdgf刺激下hsc-t6中自噬的影響為了觀察高糖及pdgf刺激下hsc中asic1a是否影響自噬,以asic1a非特異性阻斷劑阿米洛利(amiloride)阻斷asic1a,然后以高糖及pdgf刺激hsc,westernblot檢測asic1a、自噬相關(guān)蛋白lc3ii及beclin1、肝纖維化相關(guān)蛋白α-sma和collageni的表達,ptflc-3質(zhì)粒轉(zhuǎn)染及mdc染色法觀察各組細胞自噬的變化。結(jié)果發(fā)現(xiàn),amiloride能降低高糖及pdgf刺激下hsc中asic1a的表達,同時下調(diào)自噬相關(guān)蛋白lc3ii及beclin1、肝纖維化相關(guān)蛋白α-sma和collageni的表達,差異具有統(tǒng)計學意義。ptflc-3質(zhì)粒轉(zhuǎn)染及mdc染色法結(jié)果顯示amiloride能導致高糖及pdgf刺激下hsc自噬減弱。6.asic1ashrna對高糖及pdgf刺激下hsc-t6自噬的影響為了進一步觀察asic1a對自噬的影響,將特異性的asic1a-shrna轉(zhuǎn)染到高糖及pdgf刺激的hsc-t6中。westernblot檢測asic1a、自噬相關(guān)蛋白lc3ii及beclin1、肝纖維化相關(guān)蛋白α-sma和collageni的表達。結(jié)果發(fā)現(xiàn),ASIC1a-ShRNA轉(zhuǎn)染后高糖及PDGF刺激下HSC-T6中ASIC1a蛋白表達降低,同時LC3II及Beclin1、α-SMA和Collagen I的表達也降低,較高糖合并PDGF組差異具有統(tǒng)計學意義。7.高糖及PDGF刺激下HSC-T6中CaMKKβ/ERK通路蛋白表達的變化為觀察高糖及PDGF刺激下HSC細胞中ASIC1a誘導自噬的可能通路,Western Blot檢測CaMKKβ表達的變化及ERK的磷酸化水平。結(jié)果表明,單純高糖及PDGF刺激均能增強HSC-T6中CaMKKβ的表達、ERK的磷酸化水平,以高糖合并PDGF雙模型組最為明顯,較高糖組和PDGF組差異均具有統(tǒng)計學意義。8.Amiloride對高糖及PDGF刺激下HSC-T6中CaMKKβ/ERK通路的影響為進一步觀察高糖及PDGF刺激下,ASIC1a對HSC-T6中CaMKKβ/ERK通路蛋白表達的影響,以Amiloride、高糖及PDGF共同刺激HSC,Western Blot檢測CaMKKβ表達的變化及ERK的磷酸化水平。結(jié)果顯示,Amiloride能降低HSC-T6中CaMKKβ的表達,同時抑制ERK的磷酸化水平,較高糖合并PDGF組差異具有統(tǒng)計學意義。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575.2;R587.1

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