同种异体移植的MHCⅡ基因沉默BMSCs对AMI大鼠心脏影响的研究

发布时间：2018-03-06 10:12

本文选题：BMSCs　切入点：MHCⅡ基因沉默　出处：《广州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究表明,免疫原性改变可能是BMSCs(骨髓间充质干细胞)同种异体移植后存活率低的原因之一,本课题设计低免疫原性BMSCs,观察其是否能提高BMSCs移植后存活率,改善心脏心梗后状态,为干细胞的临床应用提供依据。方法:1,复苏所购的从大鼠骨髓中提取的BMSCs,进行细胞体外培养,当细胞融合达到70%-80%时,用0.25%胰酶消化,传代,调整细胞状态,保持较好的细胞活性,为后期的慢病毒感染作准备。2,取5代细胞,不同浓度嘌呤霉素共培养72h后,利用CCk-8测吸光值OD,选取细胞刚好全部死亡的最小嘌呤霉素浓度作为最佳的筛选浓度。3,将所设计的慢病毒以MOI=5、20、50、100感染BMSCs,在感染后72h,用荧光显微镜观察细胞荧光表达情况,检测感染效率,筛选获得最佳感染效率的MOI值。4,选取生长状态良好的BMSCs,以最佳感染效率的MOI感染BMSCs,72h后加入嘌呤霉素筛选一周,获取稳定的细胞株。在所得稳定遗传细胞株以及BMSCs中加入IFN-γ刺激,作用24h后提取细胞,RT-QPCR以及western blotting证实MHCⅡ基因的沉默。5,建立大鼠心肌梗死模型,结扎大鼠冠状动脉前降支,1周后在心梗边缘区进行细胞移植。实验分为4组,假手术组:冠脉仅穿线不结扎,AMI组,单纯结扎冠脉,BMSCs组,移植BMSCs细胞,MHCⅡ组,移植MHCⅡ基因沉默的BMSCs。6,4周后行大鼠心脏彩超,测定大鼠心功能的改善情况;取心肌梗死组织,利用RT-QPCR技术,测定SRY基因的表达,间接推测BMSCs移植后的存活率;心脏组织病理切片,做HE,Msson染色,VEGF,F4/80的免疫组化,评价纤维化,血管的新生情况,巨噬细胞浸润情况。7,最终数据处理均采用SPSS 16.0统计分析,均数±标准差((?)±s)表示,两组间比较采用两独立样本的t检验,多组比较采用单因素方差分析,P0.05提示差异具有统计学意义。结果:1,BMSCs形态多为长梭形,少数呈多角形,成功获取了BMSCs2,慢病毒感染BMSCs,24h未见明显的荧光蛋白表达,48h方可见荧光蛋白表达,72h荧光表达达到高峰。MOI=5,20时可见荧光蛋白表达,但转染阳性率较低,MOI=50,100时,可见明显的荧光蛋白表达。慢病毒转染时,细胞状态稍差,综合感染阳性率和病毒对细胞的影响,选择MOI=50作为最佳的感染病毒滴度。3,用嘌呤霉素做稳定细胞株的筛选,当嘌呤霉素浓度为0,2,2.5μg/ml时,BMSCs未完全死亡,随着嘌呤霉素浓度不断升高,嘌呤霉素为3,3.5,4,4.5,5μg/ml时细胞完全死亡,且各组之间无明显的统计学差异;3.0μg/ml为细胞恰好完全死亡的最低浓度,故选择3.0μg/ml作为最佳的筛选浓度。4,筛选后的慢病毒感染BMSCs细胞,IFN-γ刺激下,MHCⅡ基因的mRNA表达,基因沉默组低于未沉默组(p0.05),蛋白表达结果趋势同RT-QPCR,MHCⅡ基因沉默组低于未沉默组(p0.05)。5,构建大鼠心肌梗死模型,结扎冠状动脉前降支后,结扎区域以下心肌变白,与非梗死区交界清晰,室壁运动减弱,心率增快。6,心梗模型4周后,心脏彩超结果显示,干细胞移植组较单纯心梗组大鼠心功能有一定的改善,且MHCⅡ基因沉默组心功能改善更为明显。7,SRY基因的RT-QPCR结果:MHCⅡ基因沉默组SRY较单纯BMSCs移植组SRY m RNA表达增高,MHCⅡ基因沉默可能提高BMSCs移植的存活率。8,心脏组织病理结果:HE及Masson染色结果显示,心肌正常组织破坏。VEGF,F4/80结果:心梗区域,血管新生的情况及巨噬细胞浸润程度,MHCⅡ基因沉默组较BMSCs移植组改善更为明显。心肌梗死区域的心肌组织出现纤维化,结缔组织增生,组织变薄,变软,凹陷。结论:1,减低免疫原性能提高BMSCs的存活率。2,低免疫原性的BMSCs能更好的改善心脏心梗后状况,其机制可能与巨噬细胞有关。
[Abstract]:Objective: the study shows that the change of immunogenicity may be BMSCs (mesenchymal stem cells) allograft survival after one of the reasons for the low rate, the low immunogenicity of BMSCs, observe whether the BMSCs can improve the survival rate of transplanted heart after myocardial infarction, improve the state, provide the basis for clinical application of stem cells. Methods: 1, the purchase of the extraction recovery from rat bone marrow BMSCs cells were cultured in vitro. When the cells were fused to 70%-80%, with 0.25% trypsin digestion, cell passage, adjust state, maintain good cell activity, for the period after the lentiviral infection for.2, take the 5 generation cells. Different concentrations of puromycin were co cultured with 72h, measuring a light absorption value of OD by CCk-8, the smallest puromycin selected cell death is just all the concentrations as the best screening concentration of.3, the slow virus BMSCs to MOI=5,20,50100, after infection with 72h. Fluorescence microscopy was used to observe the cell fluorescence expression, detecting the infection efficiency, screened the best infection efficiency of the MOI value of.4, select the good growth state of BMSCs infection, BMSCs infection with the best efficiency of MOI, 72h after addition of puromycin for a week, and obtain the stable cell lines. IFN- gamma in the stable cell lines and genetic BMSCs stimulation, 24h after extraction of RT-QPCR and Western cells, blotting confirmed that MHC II gene silencing.5, to establish the rat model of myocardial infarction, coronary artery ligation rat anterior descending cell transplantation in myocardial infarction border zone after 1 weeks. The rats were divided into 4 groups: sham operation group only wearline without coronary artery AMI group, ligation, ligation of coronary artery, BMSCs group, BMSCs transplantation, MHC group 2, transplantation of MHC II gene silencing BMSCs.6,4 weeks after echocardiography in rats, the improvement of measurement of cardiac function in rats; the myocardial infarction tissue by RT-QP CR technology, to detect the expression of SRY gene, indirect measurement of survival rate of BMSCs after transplantation; cardiac tissue biopsy, HE, VEGF, Msson staining, immunohistochemistry, F4/80 evaluation of fibrosis, new blood vessels,.7 macrophage infiltration, the final data were collected by statistical analysis of 16 SPSS, expressedasmean4 the standard deviation ((?) + s) said that the two groups were compared using two independent samples t test, were analyzed by single factor analysis of variance, P0.05 showed a statistically significant difference. Results: 1, BMSCs showed spindle shape, and a few more angular, successfully acquired BMSCs2, lentivirus BMSCs infection, expression of fluorescent protein expression of 48h 24h was not obvious, the fluorescent protein, the expression of visible fluorescent protein.MOI=5,20 reached peak expression of 72h fluorescence, but the positive rate was low, MOI=50100, visible fluorescent protein expression. The lentiviral transfection, cell status A little difference, the comprehensive influence of the infection rate and virus on the cell, MOI=50 is selected as the best virus titer.3 with puromycin screening stable cell line, when puromycin concentration is 0,2,2.5 g/ml, BMSCs is not completely dead, with puromycin concentration increased, puromycin for 3,3.5,4,4.5,5 g/ml total cell death, no statistically significant difference between the groups and 3 g/ml; the lowest concentration is exactly the cell death, so the choice of 3 g/ml as the best concentration of.4 screening, screening after lentivirus infected BMSCs cells, IFN- gamma stimulation, the expression of MHC gene mRNA, gene silencing group was lower than that of non silencing group (P0.05), the expression of the trend with RT-QPCR, MHC II gene silencing group was lower than that of non silent group (P0.05).5, construct the rat model of myocardial infarction, coronary artery ligation, ligation area following myocardial white, and Non infarcted border clear, ventricular wall motion decreased, heart rate increased by.6, 4 weeks after myocardial infarction model, echocardiography showed that stem cell transplantation group compared with myocardial infarction group rats heart function were improved, and the MHC II gene silencing group heart function improved more significantly in.7, SRY gene RT-QPCR results: MHC II gene silencing group SRY compared with BMSCs transplantation group SRY m increased the expression of RNA, MHC II gene silencing may improve the survival rate of transplanted BMSCs.8, heart pathology: HE and Masson staining showed that normal myocardial tissue damage.VEGF, F4/80 results: myocardial infarction area, degree of angiogenesis and macrophage infiltration. MHC II gene silencing group than in BMSCs group improved more obviously. Myocardial infarction area of myocardial fibrosis, connective tissue hyperplasia tissue, thin, soft, depression. Conclusion: 1. To reduce the immunogenicity of BMSCs could improve the survival rate of.2, low Immunogenic BMSCs can improve the status of myocardial infarction after cardiac infarction, and its mechanism may be related to macrophage.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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