间充质干细胞条件培养基治疗肝细胞氧化应激损伤中miRNA的表达与调控机制

发布时间：2018-08-01 12:45

【摘要】：目的:1.人脐带来源的间充质干细胞条件培养基(MSC-CM)对肝细胞氧化应激损伤的研究。2.MSC-CM治疗肝细胞氧化应激损伤过程中微小RNA(microRNA,miRNA)的差异性表达,探讨相关miRNA的调控机制。方法:1.人脐带来源的间充质干细胞(hUC-MSCs)的分离、培养、鉴定及MSC-CM的制备1.1从脐带中分离培养细胞;显微镜观察细胞形态;通过流式细胞学表型鉴定和诱导分化能力检测确定上述细胞具备间充质干细胞(MSCs)的特性。1.2MSCs融合至80%更换培养基,继续培养24 h后收集培养基,离心、过滤获取MSC-CM。2.MSC-CM修复肝细胞氧化应激损伤的研究人正常肝细胞株(L02)经1 mMH_2O_2作用3h,构建肝细胞氧化应激损伤模型;损伤模型经MSC-CM修复24 h制备肝细胞氧化应激损伤修复模型。使用凋亡、线粒体膜电位(MMP)、周期、细胞活性与凋亡相关蛋白等实验研究MSC-CM对肝细胞氧化应激损伤模型的影响。将L02细胞分为3组,即损伤组(H_2O_2)、损伤修复(H_2O_2+MSC-CM)与正常对照组(Control)。2.1凋亡与MMP检测:采用AnnexinV/PI双染、JC-1单染和流式细胞术检测MSC-CM对L02细胞凋亡和MMP的影响。2.2细胞周期:使用PI染色和流式细胞术检测MSC-CM对L02细胞周期的影响。2.3细胞活性检测:采用CCK8法检测实验组L02细胞的吸光度值并绘制增值曲线,研究MSC-CM对L02细胞活性的影响。2.4Western blot方法检测L02细胞内凋亡相关蛋白Bcl-2,Bax和BMF的表达。2.5凋亡形态学检测:Hoechst染色方法检测L02细胞在损伤与修复过程中细胞核的变化。3.筛选差异性表达miRNA及miRNA功能学验证3.1根据MSCs对肝硬化损伤模型拯救机制所行的miRNA微阵列基因芯片杂交分析结果筛选了在肝硬化损伤与修复过程中显著差异性表达的21种miRNA,RT-qPCR验证在L02细胞氧化应激损伤与修复过程中miRNA的差异性表达并筛选出差异性表达显著的4种miRNA(miR143,miR145,miR301a和let7a)。3.2应用生物信息学在线分析软件预测受miRNA调控的靶基因,根据文献确定和凋亡、周期、增殖及细胞代谢相关的靶基因,使用western blot方法验证在肝细胞氧化应激损伤与修复过程中靶基因的表达。3.3miR143功能学验证的研究:微阵列与RT-qPCR显示miR143的差异性表达最为显著。转染miR143的模拟物与抑制物,上调或下调L02细胞内miR143的表达,同时设置转染miRNA的阴性对照组(NC与inhibitor NC)与未经转染的L02细胞(Control)。转染miR143模拟物即miR143在L02细胞内过表达(mimics);转染miR143抑制物即下调miR143的表达(inhibitors)。3.3.1RT-qPCR根据miR143差异性表达验证miR143是否成功转染入L02细胞。转染60 h后行凋亡、细胞活性、MMP、周期及其靶蛋白等检测,研究miR143的功能。采用流式细胞术研究miR143对L02细胞凋亡、MMP与周期的影响;CCK8方法检测miR143对肝细胞活性的影响;western blot方法检测miR143对靶蛋白HK2和ADRB1的影响。3.3.2肝细胞氧化应激损伤与修复过程中miR143功能学验证的研究:转染miR143的L02细胞在miR143转染60 h,经0.8 mM H_2O_2损伤2 h制备肝细胞损伤模型,结束损伤后L02细胞自我修复4-6h行凋亡,周期,细胞活性,预测靶蛋白与凋亡相关蛋白等检测。结果:1.hUC-MSCs的分离、培养与鉴定及MSC-CM的制备:成功分离培养出大量贴壁、集落样生长的长梭形或纺锤样的纤维细胞;细胞表面标志物CD44,CD73,CD90与CD105阳性表达,而CD31,CD34与CD45阴性表达;诱导分化鉴定示上述成纤维细胞可向成骨,成软骨和成脂肪细胞方向定向分化。上述细胞具有MSCs的生物学特性,收集MSC-CM。2.MSC-CM修复肝细胞氧化应激损伤的研究2.1凋亡与MMP检测:H_2O_2增加细胞凋亡率与MMP去极化比率,MSC-CM会降低上述变化。2.2细胞周期:H_2O_2会降低肝细胞G0/G1期比例,升高S+G2/M期比例;MSC-CM可逆转上述变化。2.3细胞活性检测:氧化应急损伤降低细胞活性,加入MSC-CM其明显上升。2.4Westernblot检测:H_2O_2组Bcl-2/Bax比值降低,加入MSC-CM其明显上升。2.5凋亡形态学检测:H_2O_2组细胞核为致密浓染或多相碎块状致密浓染,有时可见新月体;Control组与MSC-CM修复组无明显差别。3.差异性表达miRNA的筛选与功能学验证的研究3.1RT-qPCR 筛选出 miR143,miR145,miR301a 与 let7a。H_2O_2 损伤后上述 miRNA表达升高,MSC-CM救治后表达降低。3.2生物信息学软件预测靶基因,miR143调控的靶基因为HK2和ADRB1;miRNA145、miRNA301a与let7a调控的靶基因分别为DAB2,NR2C2和ESR2。Western blot分析示靶蛋白的表达均在H_2O_2损伤后降低,MSC-CM作用后升高。3.3 miR143功能学验证的研究3.3.1 RT-qPCR示mimics组miR143的表达显著上调,inhibitor组其表达明显下调。miR143成功转染入L02细胞且差异性表达。与inhibitor组相比,miR143的过表达(mimics)明显促进细胞凋亡、MMP的去极化,降低细胞活性;miR143的过表达可引起细胞周期阻滞。除此,mimics组中靶蛋白HK2和ADRB1的表达低于 inhibitor 组。3.3.2肝细胞氧化应激损伤过程中miR143功能学验证的研究:miR143的过表达显著增加损伤L02细胞的凋亡比例,降低细胞活性,细胞周期阻滞更加显著,靶蛋白HK2、ADRB1与Bcl-2/Bax的表达下降;miR143的低表达可降低损伤L02细胞的凋亡比率,增加细胞活性,解除周期阻滞,靶蛋白与Bcl-2/Bax表达上调;损伤条件下,miR143对凋亡,周期及靶蛋白的影响更加显著。结论:1.成功分离培养出hUC-MSCs,获取MSC-CM。2.MSC-CM通过调节凋亡、影响周期与细胞活性逆转并修复肝细胞氧化应激损伤。3.在肝细胞氧化应激损伤与修复中,miRNA发挥重要作用。MiR143通过调节凋亡、周期、细胞活性及靶蛋白HK2、ADRB1的表达等影响肝细胞氧化应激损伤的修复。MiR145、miRNA301a与let7a分别通过调控的靶蛋白DAB2、NR2C2和ESR2的表达参与肝细胞氧化应激损伤与修复进程。
[Abstract]:Objective: Study on the oxidative stress damage in hepatocytes by 1. human umbilical cord derived mesenchymal stem cell conditioned medium (MSC-CM);.2.MSC-CM differential expression of RNA (microRNA, miRNA) in the process of oxidative stress injury of hepatocytes, and to explore the regulation mechanism of related miRNA. Methods: the separation of mesenchymal stem cells (hUC-MSCs) from 1. human umbilical cord derived mesenchymal stem cells (hUC-MSCs) Culture, identification and preparation of MSC-CM 1.1 from the umbilical cord, the cells were isolated from the umbilical cord, and the morphology of the cells was observed under the microscope. The characteristics of the cells with mesenchymal stem cells (MSCs) were fused to 80% replacement medium by flow cytological phenotype identification and induction of differentiation, and the culture medium was collected after 24 h, and the culture medium was collected, centrifugation and filtration. The human normal liver cell line (L02) was repaired by MSC-CM.2.MSC-CM to repair the oxidative stress of liver cells (L02), the oxidative stress damage model of liver cells was constructed by 1 mMH_2O_2 action, and the damage model was repaired by MSC-CM for 24 h to prepare the repair model of oxidative stress injury of hepatocytes. Apoptosis, linear granular membrane potential (MMP), cycle, cell activity and apoptosis related eggs were used. The effect of MSC-CM on oxidative stress damage model of hepatocytes was studied in white test. L02 cells were divided into 3 groups, namely, damage group (H_2O_2), damage repair (H_2O_2+MSC-CM) and normal control group (Control).2.1 apoptosis and MMP detection: AnnexinV/PI double staining, JC-1 single staining and flow cytometry were used to detect MSC-CM to L02 cell apoptosis and MMP Cycle: PI staining and flow cytometry were used to detect the effect of MSC-CM on the cell cycle of L02 cells. The activity of.2.3 cells was detected by CCK8. The effect of MSC-CM on the activity of L02 cells was studied by MSC-CM. The.2.4Western blot method was used to detect the apoptosis related protein Bcl-2. 5 apoptotic morphological detection: Hoechst staining method detection of cell nuclei changes in L02 cells during injury and repair process.3. screening differential expression miRNA and miRNA functional verification 3.1 according to the miRNA microarray gene chip hybridization analysis of MSCs for liver cirrhosis damage model screening results in the process of liver cirrhosis injury and repair process 21 kinds of significant differential expression of miRNA, RT-qPCR verify the differential expression of miRNA during oxidative stress injury and repair of L02 cells and select 4 kinds of miRNA (miR143, miR145, miR301a and let7a).3.2 application bioinformatics online analysis software to pretest the target genes regulated by miRNA, and determine and wither according to literature. Target genes related to death, cycle, proliferation and cell metabolism, and using the Western blot method to verify the.3.3miR143 functional verification of target gene expression in the process of oxidative stress injury and repair of hepatocytes: microarrays and RT-qPCR showed that the difference expression of miR143 was the most significant. MiR143 mimics and inhibitors, up or down L02 The expression of intracellular miR143, and the negative control group transfected with miRNA (NC and inhibitor NC) and untransfected L02 cells (Control). The transfection of miR143 mimics is the overexpression of miR143 in L02 cells (mimics). L02 cells were transfected successfully. After transfection of 60 h, apoptosis, cell activity, MMP, cycle and target protein were detected to study the function of miR143. Flow cytometry was used to study the effect of miR143 on L02 cell apoptosis, MMP and cycle; CCK8 method was used to detect the effect of miR143 on the activity of liver cells; Western blot method was used to detect miR143 against target protein The study of miR143 functional verification during the oxidative stress injury and repair of.3.3.2 liver cells: transfection of miR143 L02 cells to 60 h in miR143 and 2 h by 0.8 mM H_2O_2, and the apoptosis, cycle, cell activity, and prediction of target protein and apoptosis related protein after the injury of L02 cells after the injury. Results: the separation, culture and identification of 1.hUC-MSCs, and the preparation of MSC-CM: a large number of spindle shaped or spindle like fibroblasts were successfully isolated and cultured; the surface markers of CD44, CD73, CD90 and CD105 were positive, and CD31, CD34 and CD45 negative expression, and the induction of differentiation showed that the above fibroblasts were osteogenic. The chondrogenic and adipocyte oriented differentiation. These cells have the biological characteristics of MSCs, collecting MSC-CM.2.MSC-CM to repair oxidative stress damage of liver cells 2.1 apoptosis and MMP detection: H_2O_2 increases the rate of apoptosis and MMP depolarization ratio, MSC-CM will reduce the above-mentioned.2.2 cell cycle: H_2O_2 will reduce the G0/G1 phase of liver cells Proportion, increase the proportion of S+G2/M phase, MSC-CM can reverse the above changes of.2.3 cell activity detection: oxidation emergency injury to reduce cell activity, adding MSC-CM to significantly increase.2.4Westernblot detection: H_2O_2 group Bcl-2/Bax ratio decreased, adding MSC-CM to increase the.2.5 apoptosis morphological detection: H_2O_2 group nuclear dense concentrated or polyphase fragments Dense dense staining and sometimes visible crescent, Control group and MSC-CM repair group had no significant difference in.3. differential expression miRNA screening and functional verification research 3.1RT-qPCR screened miR143, miR145, miR301a and let7a.H_2O_2 injury after the above miRNA expression increased, MSC-CM after treatment to reduce.3.2 bioinformatics software prediction target base The target genes regulated by miR143, HK2 and ADRB1, miRNA145, miRNA301a and let7a are the target genes for DAB2, NR2C2 and ESR2.Western blot respectively. Compared with group inhibitor, the overexpression of miR143 (mimics) significantly promoted apoptosis, MMP depolarization, and reduced cell activity, and the overexpression of miR143 could cause cell cycle arrest compared with the inhibitor group. In addition, the expression of HK2 and ADRB1 of the target protein in mimics group was lower than that of inhibitor group.3.. 3.2 the study of miR143 functional verification during oxidative stress injury of hepatocytes: the overexpression of miR143 significantly increased the apoptosis ratio of L02 cells, reduced cell activity, cell cycle arrest more significantly, the expression of target protein HK2, ADRB1 and Bcl-2/Bax decreased, and the low surface reach of miR143 could reduce the apoptosis ratio of damaged L02 cells and increase the cells. Activity, release cycle block, target protein and Bcl-2/Bax expression up-regulated; under damage conditions, miR143 has more significant effect on apoptosis, cycle and target protein. Conclusion: 1. successfully isolated and cultured hUC-MSCs, obtained MSC-CM.2.MSC-CM by regulating apoptosis, reversing cycle and cell activity and repairing oxidative stress injury of liver cells.3. in liver cell oxygen During the damage and repair of chemical stress, miRNA plays an important role in the repair of.MiR143 by regulating apoptosis, cycle, cell activity and the expression of target protein HK2, ADRB1, and other effects of.MiR145, miRNA301a and let7a through regulated target protein DAB2, NR2C2 and ESR2 expressions involved in oxidative stress injury and repair of liver cells. Cheng.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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