【作者】 李莉；

【导师】 田杰；

【作者基本信息】 重庆医科大学 ， 儿科学， 2008， 博士

【摘要】 目的:筛选本课题组前期对Gcn5(组蛋白乙酰基转移酶辅激酶)基因设计的7条质粒片段中能高效抑制Gcn5表达的shRNA序列;探讨干扰Gcn5表达后是否能够引起体内组蛋白乙酰化修饰平衡的改变;观测下调组蛋白乙酰化修饰状态对体内、外MSCs(骨髓间充质干细胞)定向分化为心肌细胞的影响,从而探讨组蛋白低乙酰化状态对MSCs定向分化的调控机理;建立通过干预组蛋白乙酰化平衡状态来调节MSCs定向分化的“基因调节开关”的理论与实验技术平台。方法:采用不同组合质粒片段干扰体外培养的MSCs,通过观察细胞形态变化,ELISA技术检测MSCs细胞核蛋白HAT(组蛋白乙酰化酶)活性状态,Western-blotting分析MSCs细胞核Gcn5蛋白表达,荧光显微镜观察质粒转染细胞荧光表达量,流式细胞技术检测质粒转染效率,筛选有效质粒片段;利用筛选出的有效shRNA转染MSCs,通过ELISA技术检测MSCs细胞核HAT活性状态,验证通过沉默Gcn5基因是否能够下调组蛋白乙酰化修饰水平。利用shRNA-ZJ3转染单纯MSCs或经5-aza(5-氮杂胞苷)处理前后的MSCs,通过观察细胞形态变化, CHIP技术分析乙酰化的组蛋白H3肽链上的相关基因(Gcn5;GATA-4)表达,透射电镜观测细胞超微结构的变化,分析下调组蛋白乙酰化修饰状态是否能够干预MSCs体外特化心肌细胞的过程;移植经有效shRNA转染的MSCs至大鼠心肌组织,2W后检测移植区心肌组织中HAT活性状态,Gcn5、GATA-4基因及Gcn5、MHC、Cx43蛋白的表达状况,探讨心肌微环境中组蛋白乙酰化修饰对MSCs特化心肌细胞的调控机理。结果:1、观察各种质粒转染的MSCs形态变化显示经shRNA-ZJ3经各种预处理的MSCs较正常MSCs形态变化最大,生长比较缓慢;shRNA-ZJ3转染的MSCs细胞核HAT活性及Gcn5蛋白表达量均明显降低(P<0.05);荧光显微镜观察GFP表达量及流式细胞技术检测显示转染效率大于20%。2、经shRNA-ZJ3转染的MSCs细胞核内HAT活性较正常MSCs及shRNA-HK(通用阴性质粒对照)转染的MSCs明显降低(P<0.05)。3、5-aza诱导的MSCs细胞生长旺盛,单纯shRNA-ZJ3转染或5-aza诱导前后经shRNA-ZJ3转染的MSCs生长迟滞,形态瘦小;细胞核蛋白HAT测定显示:5-aza诱导组乙酰化水平最高,单纯转染组(TR组)、诱导后转染组(IN-TR组)以及转染后诱导组(TR-IN组)与正常组比较HAT活性均有显著性降低(P<0.05),TR组分别与Normal组、HK组及IN组比较HAT活性降低亦有显著性(P<0.05);经5-aza诱导后的MSCs(阳性对照组)与高乙酰化水平组蛋白H3相交联的Gcn5及GATA-4表达较正常组(正常培养MSCs组)、阴性对照组(HK转染组)及实验组(5-aza诱导后shRNA-ZJ3转染组)均有显著性增高(P<0.05);实验组中与高乙酰化水平组蛋白H3相交联的Gcn5表达较正常对照组、阴性对照组及阳性对照组均有显著性的降低(P<0.05);电镜检测细胞超微结构发现经5-aza诱导的MSCs增生活跃,外形长条化,胞浆出现肌丝结构,shRNA-ZJ3转染的MSCs细胞核中异染色质比例增大,部分细胞出现凋亡。4、体内移植区心肌组织内HAT活性检测发现实验组(shRNA-ZJ3转染MSCs移植组)乙酰化酶活性较正常组(正常心肌组织)、阴性对照组1(shRNA-HK转染组)及阴性对照组2(DAPI标记MSCs移植组)均有显著性降低(P<0.05);实验组中可检测到Gcn5及GATA-4基因的低表达,较其它三个对照组表达量均有显著性降低(P<0.05)。结论:1、前期课题组构建的7条质粒片段中shRNA-ZJ3能够有效干预Gcn5基因的表达。2、shRNA-ZJ3阻断Gcn5基因表达后能够下调细胞核组蛋白乙酰化修饰水平,导致组蛋白乙酰化失衡状态的出现。3、组蛋白乙酰化修饰平衡参与调控MSCs体外特化心肌细胞的过程。4、心肌微环境中MSCs的组蛋白乙酰化活性状态与分化活性及分化启动密切相关。5、组蛋白乙酰化修饰参与调节MSCs在体内心肌微环境中特化为心肌细胞的过程。低组蛋白乙酰化修饰导致的静默状态的MSCs无法进入定向分化进程。6、成功建立通过干预组蛋白乙酰化平衡调节MSCs定向分化的“基因调控开关”的理论与实验技术平台。更多还原

【Abstract】 Objective:The study is to screen valid plasmid targeted to Gcn5 among the constructed recombinant plasmids; and to detect weather it could silence Gcn5 gene expression. To confirm the effective plasmid which could cause low-grade acetylation. Then to explore the disbalance of histone acetylizad modification result in the effective plasmid could regulate MSCs differentiation in vivo and in vitro. Finally establish a technique platform for the switch which regulates the gene expression in the process of MSCs differentiation.Methods and materials:1. Screen an effective plasmid, transfect different plasmids into MSCs, and then investigate the cell morphologic change, HAT activity and the expression of Gcn5.2. Confirm weather the plasmid ZJ3 could cut down the level of histone acetylation, transfect the MSCs with shRNA-ZJ3 and then detect the HAT activity of MSCs.3. Analysis weather the low-grade acetylation of histone could regulate the MSCs differentiation, transfect the MSCs induced by 5-aza or not with shRNA-ZJ3 and then detect the cell morphologic change, HAT activity, the expression of Gcn5 and GATA-4 cross-linked to H3 peptide chain, and the ultra microstructure of MSCs.4. Transfect the MSCs induced by 5-aza with shRNA-ZJ3, and transplant MSCs into myocardium which marked with GFP or DAPI. Two weeks later, collect the myocardium transplanted by MSCs, and detect the HAT activity, expression of gene Gcn5 and GATA-4 and the expression of protein Cx43, MHC, and Gcn5.Results:1. In MSCs transfected by different plasmids, some of MSCs which was transfected by shRNA-ZJ3 has a significant change in cell morphologic, low-grade activity of HAT and low-expression of protein Gcn5.2. HAT activity of MSCs transfected by shRNA-ZJ3 is lower than normal MSCs.3. MSCs induced by 5-aza grow vigorously, but the MSCs transfected by shRNA-ZJ3 grow flaggingly. HAT activity of MSCs induced by 5-aza shows at high-grade; however in the MSCs transfected by shRNA-ZJ3, HAT activity shows at a low-grade. The expression of Gcn5 and GATA-4 cross-linked to H3 peptide chain is obvious in the MSCs induced by 5-aza, and it is weak in the MSCs transfected by shRNA-ZJ3. Myofilament was detected in the cytoplasm of MSCs induced by 5-aza; and some apoptosis cells were found in the sample of MSCs transfected by shRNA-ZJ3; in the transfected MSCs, the percentage of heterochromatin is larger than in the normal MSCs and induced MSCs.4. In the myocardium transplanted by MSCs which transfected by shRNA-ZJ3, HAT activity drop off obviously; the expression of gene Gcn5 and GATA-4 depressed significantly; and the expression of protein MHC, Cx43 and Gcn5 expressed also at a low level.Conclusion:1. shRNA-ZJ3 is the effective plasmid targeted to Gcn5.2. Gcn5 blocked by shRNA-ZJ3 can obstruct the modification of histone, and break the balance of histone acetylation.3. The balance of histone acetylation plays an important role to regulate the process of MSCs differention into cardiomyocytes.4. In the myocardial microenvironment, the differentiation activity and priming of MSCs is closely related to the HAT activity.5. In the myocardial microenvironment, silenced MSCs resulted from the disbalance of acetylation could not differentiate into cardiomyocytes.6. The study established a technique platform for the research on histone acetylation mechanisams of regulating the gene expression in the process of MSCs differentiation successfully.更多还原