本文選題：BMSCs　切入點：MHCⅡ基因沉默　出處：《廣州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究表明,免疫原性改變可能是BMSCs(骨髓間充質(zhì)干細(xì)胞)同種異體移植后存活率低的原因之一,本課題設(shè)計低免疫原性BMSCs,觀察其是否能提高BMSCs移植后存活率,改善心臟心梗后狀態(tài),為干細(xì)胞的臨床應(yīng)用提供依據(jù)。方法:1,復(fù)蘇所購的從大鼠骨髓中提取的BMSCs,進(jìn)行細(xì)胞體外培養(yǎng),當(dāng)細(xì)胞融合達(dá)到70%-80%時,用0.25%胰酶消化,傳代,調(diào)整細(xì)胞狀態(tài),保持較好的細(xì)胞活性,為后期的慢病毒感染作準(zhǔn)備。2,取5代細(xì)胞,不同濃度嘌呤霉素共培養(yǎng)72h后,利用CCk-8測吸光值OD,選取細(xì)胞剛好全部死亡的最小嘌呤霉素濃度作為最佳的篩選濃度。3,將所設(shè)計的慢病毒以MOI=5、20、50、100感染BMSCs,在感染后72h,用熒光顯微鏡觀察細(xì)胞熒光表達(dá)情況,檢測感染效率,篩選獲得最佳感染效率的MOI值。4,選取生長狀態(tài)良好的BMSCs,以最佳感染效率的MOI感染BMSCs,72h后加入嘌呤霉素篩選一周,獲取穩(wěn)定的細(xì)胞株。在所得穩(wěn)定遺傳細(xì)胞株以及BMSCs中加入IFN-γ刺激,作用24h后提取細(xì)胞,RT-QPCR以及western blotting證實MHCⅡ基因的沉默。5,建立大鼠心肌梗死模型,結(jié)扎大鼠冠狀動脈前降支,1周后在心梗邊緣區(qū)進(jìn)行細(xì)胞移植。實驗分為4組,假手術(shù)組:冠脈僅穿線不結(jié)扎,AMI組,單純結(jié)扎冠脈,BMSCs組,移植BMSCs細(xì)胞,MHCⅡ組,移植MHCⅡ基因沉默的BMSCs。6,4周后行大鼠心臟彩超,測定大鼠心功能的改善情況;取心肌梗死組織,利用RT-QPCR技術(shù),測定SRY基因的表達(dá),間接推測BMSCs移植后的存活率;心臟組織病理切片,做HE,Msson染色,VEGF,F4/80的免疫組化,評價纖維化,血管的新生情況,巨噬細(xì)胞浸潤情況。7,最終數(shù)據(jù)處理均采用SPSS 16.0統(tǒng)計分析,均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,兩組間比較采用兩獨立樣本的t檢驗,多組比較采用單因素方差分析,P0.05提示差異具有統(tǒng)計學(xué)意義。結(jié)果:1,BMSCs形態(tài)多為長梭形,少數(shù)呈多角形,成功獲取了BMSCs2,慢病毒感染BMSCs,24h未見明顯的熒光蛋白表達(dá),48h方可見熒光蛋白表達(dá),72h熒光表達(dá)達(dá)到高峰。MOI=5,20時可見熒光蛋白表達(dá),但轉(zhuǎn)染陽性率較低,MOI=50,100時,可見明顯的熒光蛋白表達(dá)。慢病毒轉(zhuǎn)染時,細(xì)胞狀態(tài)稍差,綜合感染陽性率和病毒對細(xì)胞的影響,選擇MOI=50作為最佳的感染病毒滴度。3,用嘌呤霉素做穩(wěn)定細(xì)胞株的篩選,當(dāng)嘌呤霉素濃度為0,2,2.5μg/ml時,BMSCs未完全死亡,隨著嘌呤霉素濃度不斷升高,嘌呤霉素為3,3.5,4,4.5,5μg/ml時細(xì)胞完全死亡,且各組之間無明顯的統(tǒng)計學(xué)差異;3.0μg/ml為細(xì)胞恰好完全死亡的最低濃度,故選擇3.0μg/ml作為最佳的篩選濃度。4,篩選后的慢病毒感染BMSCs細(xì)胞,IFN-γ刺激下,MHCⅡ基因的mRNA表達(dá),基因沉默組低于未沉默組(p0.05),蛋白表達(dá)結(jié)果趨勢同RT-QPCR,MHCⅡ基因沉默組低于未沉默組(p0.05)。5,構(gòu)建大鼠心肌梗死模型,結(jié)扎冠狀動脈前降支后,結(jié)扎區(qū)域以下心肌變白,與非梗死區(qū)交界清晰,室壁運動減弱,心率增快。6,心梗模型4周后,心臟彩超結(jié)果顯示,干細(xì)胞移植組較單純心梗組大鼠心功能有一定的改善,且MHCⅡ基因沉默組心功能改善更為明顯。7,SRY基因的RT-QPCR結(jié)果:MHCⅡ基因沉默組SRY較單純BMSCs移植組SRY m RNA表達(dá)增高,MHCⅡ基因沉默可能提高BMSCs移植的存活率。8,心臟組織病理結(jié)果:HE及Masson染色結(jié)果顯示,心肌正常組織破壞。VEGF,F4/80結(jié)果:心梗區(qū)域,血管新生的情況及巨噬細(xì)胞浸潤程度,MHCⅡ基因沉默組較BMSCs移植組改善更為明顯。心肌梗死區(qū)域的心肌組織出現(xiàn)纖維化,結(jié)締組織增生,組織變薄,變軟,凹陷。結(jié)論:1,減低免疫原性能提高BMSCs的存活率。2,低免疫原性的BMSCs能更好的改善心臟心梗后狀況,其機制可能與巨噬細(xì)胞有關(guān)。
[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.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 趙小強;邵明;楊海平;孫玲;;骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)方法及其對CD8~+T淋巴細(xì)胞的免疫調(diào)控[J];中國組織工程研究;2017年05期

2 龔惠;陳志丹;鄒云增;;干細(xì)胞與心肌再生[J];上海大學(xué)學(xué)報(自然科學(xué)版);2016年03期

3 Ann De Becker;Ivan Van Riet;;Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?[J];World Journal of Stem Cells;2016年03期

4 何志裕;陸東風(fēng);;急性心肌梗死大鼠移植入人臍帶血間充質(zhì)干細(xì)胞后心肌組織檢測[J];血栓與止血學(xué);2015年05期

5 熊永泉;陸東風(fēng);何志裕;;基因修飾的骨髓間充質(zhì)干細(xì)胞移植對大鼠急性心肌梗死的療效觀察[J];中國醫(yī)學(xué)創(chuàng)新;2015年21期

6 鄒松平;王宇;李春雨;付雪丹;劉彥麗;;骨髓間充質(zhì)干細(xì)胞旁分泌對急性心肌梗死心肌的保護(hù)作用（英文）[J];中國組織工程研究;2014年23期

7 徐巧巖;夏琳;王吉昌;;低氧預(yù)處理骨髓間充質(zhì)干細(xì)胞耐受缺血缺氧的能力[J];中國組織工程研究;2013年49期

8 束波;劉志江;范芳;;基質(zhì)細(xì)胞衍生因子1α預(yù)處理大鼠骨髓間充質(zhì)干細(xì)胞的遷移[J];中國組織工程研究;2012年27期

9 吳祖常;吳國才;吳智明;熊丹;呂俊廷;楊志剛;;干細(xì)胞生長因子和粒細(xì)胞集落刺激因子與心肌梗死大鼠體內(nèi)骨髓間充質(zhì)干細(xì)胞的遷移[J];中國組織工程研究與臨床康復(fù);2011年36期

10 王承云;石磊;夏春;鄭欣鵬;張兵;林飛太;王少杰;;人骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)方法的改進(jìn)[J];中國組織工程研究與臨床康復(fù);2011年32期

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