IL-22与miR-200a交互作用抑制肝纤维化的研究

发布时间：2018-03-05 16:01

本文选题：肝纤维化　切入点：白介素-22　出处：《广西医科大学》2017年博士论文　论文类型：学位论文

【摘要】：肝纤维化是各种慢性肝病向肝硬化发展所共有的病理改变和必经途径,以细胞外基质异常增生和在肝内过量沉积为病理特征。肝星状细胞(Hepatic stellate cells,HSCs)的异常激活和分泌致纤维化因子是肝纤维化发生的重要原因。研究表明,多种细胞因子和mi RNAs在肝纤维化的发生和发展过程中扮演着重要角色?IL-22可由多种免疫细胞分泌,通过与细胞表面的IL-22受体结合后,激活细胞内信号传导与转录激活因子(signal transducer and activator of transcription,STAT)发挥调控作用。mi RNAs通过与靶m RNA完全或不完全配对形成沉默复合体,在转录后水平对基因转录进行负调控。mi R-200a在肝纤维化患者和大小鼠肝纤维模型中均表达下调,并通过抑制靶基因发挥抑制HSCs活性的作用。我们前期研究已发现IL-22可以抑制HSCs活性和发挥抗小鼠肝纤维化作用,但IL-22是否与mi R-200a有交互作用关系?如果有,相关细胞因子变化及其相互关系如何?目前无相关研究?为进一步了解IL-22和mi R-200a在抗肝纤维化作用的机制,并阐明IL-22和mi R-200a在肝纤维化中的交互作用(cross-talk)关系,进而探索出肝纤维化新的治疗靶点,本研究应用大鼠HSCs和肝纤维化模型,分别给予IL-22和mi R-200a干预,检测相关下游因子的改变情况,观察两者在HSCs和肝纤维化模型中的交互作用。本研究将从以下三方面进行研究:第一部分IL-22经STAT3通路抑制HSCs活性和抗肝纤维作用研究目的:观察IL-22干预HSCs后,细胞活性的改变情况,并观察IL-22对大鼠肝纤维化模型的作用,探讨IL-22抑制HSCs活性和抗肝纤维作用的机制。方法:培养大鼠HSCs细胞系(HSCs-T6)至对数生长期,分别使用250pg/mL、500pg/m L、750pg/m L和1000pg/m L浓度的IL-22加入培养基中干预48小时;分别采用0、2、5ng/m L浓度的TGF-β1加入培养基24小时激活HSCs;CCK8法检测细胞增殖率,流式细胞术检测细胞凋亡率,ELISA检测细胞培养上清液中的α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和胶原蛋白I(Col I)表达水平,western-blot法检测细胞中的STAT3和p-STAT3蛋白表达水平;采用CCL4腹腔注射大鼠8周构建大鼠肝纤维化模型,在给予IL-22腹腔注射1周,处死大鼠,收集肝脏组织,HE和Masson染色观察小鼠肝脏病理改变,根据Ishak评分系统评估肝纤维化的程度;免疫组织化学染色检测肝脏组织中α-SMA,western-blot法检测肝组中的STAT3和p-STAT3蛋白表达水平。结果:随着IL-22浓度的升高,HSCs增殖率下降,到750 pg/m L时,与无IL-22干预相比差异有统计学意义(P0.05)。但随着IL-22浓度的升高,HSCs凋亡率无显著改变。ELIAS法检测培养上清液中的α-SMA和Col I随着IL-22浓度的升高而下降,到750pg/m L时,α-SMA水平与无IL-22干预相比差异有统计学意义(P0.05);随着TGF-β1浓度的升高,HSCs增殖率上升,到5 ng/m L时,HSCs的增殖率与无TGF-β1干预相比差异有统计学意义(P0.05)。STAT3蛋白表达水平随着TGF-β1浓度的升高无显著改变,而p-STAT3蛋白表达水平随着TGF-β1浓度的升高而下降,到5ng/m L时,p-STAT3蛋白表达水平比无TGF-β1干预时显著下降,差异有统计学意义(P0.05);采用TGF-β1预处理HSCs,再使用IL-22分别干预后,STAT3蛋白表达水平无显著改变,而p-STAT3蛋白表达水平显著上升(P0.05);大鼠模型实验中,与未进行IL-22干预相比,IL-22干预后,大鼠肝纤维化明显改善,肝脏Ishak组织学评分显示差异有统计学意义(P0.05),肝组织中的STAT3蛋白表达水平无显著改变,而p-STAT3蛋白表达水平显著上升(P0.05)。结论:IL-22以浓度依赖性抑制HSCs增殖和活性,但对HSCs凋亡无显著影响;IL-22可以有效的抑制HSCs活性和抗肝纤维化作用,其机制是通过激活STAT3磷酸化信号通路表达实现的。第二部分miR-200a靶向β-catenin抑制HSCs增殖与活性目的:检测miR-200a在HSCs和肝纤维化组织的表达情况,并观察过表达miR-200a对HSCs的影响;验证miR-200a对β-catenin直接调控作用,并在HSCs验证,阐明miR-200a靶向β-catenin抑制HSCs增殖与活性的机制。方法:培养HSCs至对数培养期,分别采用0、5ng/m L浓度的TGF-β1加入培养基中干预24小时,采用荧光定量PCR法检测HSCs中的miR-200a表达情况;采用慢病毒包装的miR-200a mimics转染HSCs,观察过表达HSCs中的miR-200a对HSCs的影响;CCK8法检测细胞增殖率,流式细胞术检测细胞凋亡率,ELISA检测细胞培养上清液中的α-SMA表达水平,western-blot法检测细胞中的β-catenin蛋白表达水平。使用在线数据库Targetscan预测miR-200a的靶基因,在使用双荧光素酶报告系统验证miR-200a对β-catenin直接调控作用,结果:TGF-β1激活后,HSCs中的miR-200a表达水平显著下降,与无TGF-β1激活相比差异有统计学意义(P0.05)。使用慢病毒包装的大鼠miR-200a mimics培养基转染HSCs,与未进行miR-200a转染的HSCs相比,转染了miR-200a的HSCs中,miR-200a表达升高打8倍以上;CCK8法发现HSCs增殖率下降,与未进行miR-200a mimics相比差异有统计学意义(P0.05)。ELIAS法发现miR-200a mimics转染后,HSCs培养上清液中的α-SMA水平下降,与未进行miR-200amimics相比差异有统计学意义(P0.05)。双荧光素酶系统显示转染了miR-200a mimics和β-catenin质粒后,细胞中的荧光素酶活性显著下降(P0.05);western-blot法显示HSCs转染miR-200a mimics后,细胞中的β-catenin蛋白表达水平下降(P0.05)。结论:miR-200a在HSCs激活过程中表达下降,过表达miR-200a可以抑制HSCs增殖和活性;双荧光素酶系统验证了β-catenin是miR-200a的直接靶基因;miR-200a通过靶向调控β-catenin抑制HSCs增殖和活性。第三部分IL-22与miR-200a在HSCs和肝纤维组织中交互作用研究目的:观察IL-22干预HSCs和大鼠肝纤维化模型以及抑制miR-200a后,HSCs和肝纤维化组织中的IL-22和miR-200a下游因子的表达情况,阐明IL-22与miR-200a在HSCs和肝纤维组织中交互作用机制。方法:IL-22干预HSCs后细胞,荧光定量PCR和western-blot分别检测细胞中miR-200a和β-catenin表达的情况;慢病毒转染miR-200a inhibitors至HSCs,抑制HSCs中的miR-200a表达,再给予IL-22干预,观察抑制miR-200a后,IL-22对HSCs的影响,ELISA检测细胞培养上清液中的α-SMA表达水平;western-blot法检测细胞中的STAT3和p-STAT3蛋白表达水平。采用CCL4腹腔注射大鼠4周构建大鼠肝纤维化模型,在给予IL-22腹腔注射,同时给予尾静脉注射慢病毒包装的miR-200a inhibitors,1周后处死大鼠,收集肝脏组织,抑制大鼠肝纤维化模型中的miR-200a,HE和Masson染色观察小鼠肝脏病理改变并根据Ishak评分系统评估肝纤维化的程度;免疫组织化学染色检测肝脏组织中α-SMA,western-blot法检测肝组中的β-catenin、STAT3和p-STAT3蛋白表达水平,观察抑制miR-200a后,IL-22对大鼠肝纤维化的影响;结果:TGF-β1加入培养基中激活HSCs活性,在给予IL-22干预,发现与未进行IL-22干预相比,IL-22干预后HSCs中的miR-200a水平上升,而β-catenin蛋白水平下降;转染了miR-200a inhibitors的HSCs中β-catenin蛋白表达水平显著升高(P0.05),STAT3蛋白表达水平无显著改变,但p-STAT3的蛋白表达水平明显下降(P0.05);大鼠肝纤维化模型构建成功后,分为IL-22和miR-200a inhibitors单独注射组和IL-22+miR-200a inhibitors注射组,转染了miR-200a inhibitors的肝组织中的miR-200a表达比未转染miR-200a inhibitors的低,IL-22的抗肝纤维化作用被miR-200a inhibitors抑制,而β-catenin蛋白表达水平也较升高(P0.05),STAT3蛋白表达水平无显著改变,但p-STAT3的蛋白表达水平明显下降(P0.05)。结论:IL-22可以上调HSCs中的miR-200a表达量,进而下调β-catenin表达水平;抑制miR-200a表达量可以抑制IL-22对HSCs的作用,并抑制STAT3磷酸化水平;IL-22改善大鼠肝纤维化作用可被miR-200a inhibitors抑制,STAT3磷酸化水平也被抑制。
[Abstract]:Hepatic fibrosis is a chronic liver disease to liver cirrhosis is the common pathological change and necessary way, with abnormal proliferation and excessive extracellular matrix deposition in the liver. The pathological feature of hepatic stellate cells (Hepatic stellate cells, HSCs) of the abnormal activation and secretion of fibrosis factor is an important cause of liver fibrosis. Research shows that a variety of cytokines and MI RNAs plays an important role in the occurrence and development of liver fibrosis? IL-22 can be secreted by a variety of immune cells with IL-22 receptors on cell surface, intracellular signaling and transcription factor activation (signal transducer and activator of transcription, STAT.Mi) play a role in regulating RNAs through the target m RNA complete or incomplete pairing of silencing complex, at the post transcriptional level of gene transcription of the negative regulation of.Mi R-200a in liver fibrosis patients and The fiber model in mouse liver were down regulated, and exert inhibitory effect on HSCs activity by inhibition of target gene. Our previous studies have found that IL-22 can inhibit HSCs activity and play a preventive effect against liver fibrosis in mice, but whether IL-22 and MI R-200a have interaction relationship? If there is, how the changes of related cytokines and their relationship are not? Study? In order to further understand the mechanism of IL-22 and MI R-200a in anti hepatic fibrosis, and to elucidate the interaction of IL-22 and MI R-200a in hepatic fibrosis (cross-talk), and to explore a new therapeutic target for liver fibrosis, this research applied HSCs and liver fibrosis rat model, treated with IL-22 and MI R-200a the intervention respectively, the change detection of downstream factors, interaction was observed between HSCs and hepatic fibrosis in the model. This research will conduct the research from the following three aspects: the first part IL- 22 via the STAT3 pathway to inhibit the activity of HSCs and to study the effect of anti hepatic fibrosis Objective: To observe the effect of IL-22 HSCs after the intervention, the change of cell activity, and to observe the effect of IL-22 on rat liver fibrosis model, to explore the mechanism of IL-22 inhibition and anti liver fiber HSCs activity. Methods: cultured rat HSCs cell line (HSCs-T6) to the logarithmic growth phase, respectively, using 250pg/mL, 500pg/m L, 750pg/m L and 1000pg/m L concentration of IL-22 into the culture medium 48 hour intervention; using 0,2,5ng/m L concentration of TGF- beta 1 is added to the medium 24 hours to activate HSCs; CCK8 method to detect cell proliferation rate, cell apoptosis was detected by flow cytometry, ELISA assay the culture supernatant of alpha smooth muscle actin (-smooth muscle alpha actin, alpha -SMA) and collagen I (Col I) expression level of STAT3 and p-STAT3 protein expression in Western-blot cell was detected by using intraperitoneal injection of CCL4 large; In 8 weeks to build rat liver fibrosis model in IL-22 was given by intraperitoneal injection for 1 weeks, the rats were killed to collect liver tissue, liver pathological changes were observed in mice HE and Masson staining, according to the Ishak scoring system to assess the degree of liver fibrosis; immunohistochemical staining of liver tissue in alpha -SMA, Western-blot detection of liver group the expression of STAT3 and p-STAT3 protein level. Results: with the increase of IL-22 concentration, decreased the proliferation of HSCs to 750 pg/m L, and there was significant difference compared with IL-22 without intervention (P0.05). But with the increasing of IL-22 concentration, apoptosis rate of HSCs had no significant change in supernatant were determined by.ELIAS method in a -SMA and Col I with the increase of IL-22 concentration decreased to 750pg/m L, there was statistical significance level a -SMA with and without IL-22 intervention compared to the difference (P0.05); with the increase of TGF- beta 1 concentration, the rate of rise of the proliferation of HSCs ng/m to 5 L, the proliferation of HSCs The rate of TGF- beta 1 compared with no intervention was statistically significant (P0.05) the expression level of.STAT3 protein with TGF- beta 1 concentration did not change significantly, while the expression level of p-STAT3 protein decreased with increasing concentration of TGF- beta 1, 5ng/m to L, the expression level of p-STAT3 protein than non TGF- beta 1 intervention decreased significantly. The difference was statistically significant (P0.05); the TGF- beta 1 pretreatment HSCs, then use IL-22 respectively after the intervention, the expression level of STAT3 protein did not change significantly, while p-STAT3 protein expression level increased significantly (P0.05); experimental rat model, compared with no IL-22 intervention, the intervention of IL-22, hepatic fibrosis rats significantly improvement of liver Ishak histological scores revealed a statistically significant difference (P0.05) in liver tissue, the expression level of STAT3 protein did not change significantly, while p-STAT3 protein expression level increased significantly (P0.05). Conclusion: IL-22 in a concentration dependent inhibition of HSCs The proliferation and activity, but had no significant effect on HSCs apoptosis; IL-22 can inhibit HSCs activity and anti hepatic fibrosis, its mechanism is activated through phosphorylation of STAT3 signaling pathway expression. The second part of the miR-200a targeting beta -catenin inhibit the proliferation and activity of HSCs Objective: to detect the expression of HSCs and miR-200a in hepatic fibrosis the expression of miR-200a, and observe the effect on HSCs; miR-200a verification of direct regulation of beta -catenin, and verified in HSCs, to clarify the mechanism of targeting miR-200a beta -catenin inhibit the proliferation and activity of HSCs. Methods: HSCs cultured to the logarithmic incubation period, respectively using 0,5ng/m L concentration of TGF- beta 1 into the culture medium 24 intervention hours, the fluorescent quantitative PCR method was used to detect HSCs miR-200a expression in mimics transfected by miR-200a; HSCs lentivirus packaging, observed the expression of HSCs in miR-200a on the impact of HSCs; CCK8 assay Measuring the rate of cell proliferation, cell apoptosis was detected by flow cytometry, alpha -SMA in the supernatant of ELISA cell culture to detect expression levels, the expression level of Western-blot cells detected in beta -catenin protein. The use of online database Targetscan prediction of miR-200a target genes, using dual luciferase reporter system to verify the miR-200a of beta -catenin direct regulation results TGF- beta 1 after activation, the expression level of HSCs in miR-200a was significantly decreased, and TGF- beta 1 activation had significant difference (P0.05). The use of lentiviral packaging miR-200a mimics culture medium was transfected into HSCs rats, compared with no miR-200a transfected HSCs, HSCs transfected miR-200a, the expression of miR-200a increase hit more than 8 times; found that the decline in the rate of HSCs proliferation by CCK8, compared with no miR-200a mimics had significant difference (P0.05) of.ELIAS showed that the miR-200a mimics after transfection, HSCs The culture supernatant of alpha -SMA decreased, compared with no miR-200amimics difference was statistically significant (P0.05). Dual luciferase system display miR-200a transfected with -catenin plasmid mimics and beta cells, luciferase activity was significantly decreased (P0.05); HSCs miR-200a mimics Western-blot showed that after transfection, the expression level of -catenin protein beta decreased (P0.05). Conclusion: miR-200a in activation of HSCs expression decreased in the process, overexpression of miR-200a can inhibit the proliferation and activity of HSCs; dual luciferase reporter system to verify the beta -catenin is a direct target gene miR-200a; miR-200a inhibits the proliferation and activity of HSCs to -catenin by regulation of beta target. The aim of the third part IL-22 interacts with miR-200a in HSCs and liver fibrous tissue in the study: observe the effect of IL-22 and HSCs in hepatic fibrosis of rats and the inhibition of miR-200a, HSCs and liver fibrosis tissues IL The expression of -22 and miR-200a downstream factor, IL-22 and miR-200a in HSCs and elucidate the interaction mechanism of hepatic fibrosis. Methods: IL-22 HSCs treated cells, fluorescence quantitative PCR and Western-blot were used to detect the expression of miR-200a and -catenin in the beta cell; lentiviral transfection of miR-200a inhibitors to HSCs, the inhibition of HSCs miR-200a expression. Then given IL-22 intervention, observe the inhibition effect of IL-22 on miR-200a, HSCs, -SMA alpha ELISA in the supernatant of cell culture to detect the expression level of cells was detected by Western-blot; the expression of STAT3 and p-STAT3 protein levels. By intraperitoneal injection of CCL4 in rats for 4 weeks to construct rat liver fibrosis model in IL-22 was given by intraperitoneal injection at the same time. Given intravenous injection of lentivirus packaging miR-200a inhibitors, rats were killed after 1 weeks, collect the liver tissue, inhibit liver fibrosis in a rat model of miR-200a, HE and Masson staining To observe the pathological changes of liver in mice and color grading system to assess the degree of liver fibrosis by Ishak; immunohistochemical staining of liver tissue in alpha -SMA, Western-blot detection of liver in group -catenin beta, STAT3 and p-STAT3 protein expression observed after inhibition of miR-200a, effects of IL-22 on hepatic fibrosis in rats; results: TGF- beta 1 to activate the activity of HSCs in medium, in the given IL-22 intervention, and found no IL-22 intervention compared to HSCs increased the level of miR-200a in IL-22 after the intervention, and the beta -catenin protein level; the expression of -catenin protein in miR-200a transfected with inhibitors was significantly increased in HSCs (P0.05), the expression level of STAT3 protein had no significant change. But the expression of p-STAT3 protein was significantly decreased (P0.05); the successful construction of rat liver fibrosis model, divided into IL-22 and miR-200a inhibitors and IL-22+miR-200a inhibit single injection group Ors injection group, liver tissue miR-200a transfected with inhibitors in miR-200a expression than non transfected miR-200a inhibitors low, IL-22 anti fibrosis effect by miR-200a inhibition of inhibitors beta -catenin protein expression level was also increased (P0.05), the expression level of STAT3 protein did not change significantly, but the expression of p-STAT3 protein was significantly decreased (P0.05). Conclusion: IL-22 can be up-regulated in HSCs miR-200a, and downregulation of -catenin expression; inhibition of miR-200a expression can inhibit effect of IL-22 on HSCs, and the inhibition of the phosphorylation of STAT3; IL-22 improved hepatic fibrosis in rats by miR-200a inhibitors inhibited the phosphorylation of STAT3 was inhibited.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.2

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