【作者】 冯川；

【导师】 田杰；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 第一部分体外SAHA对MSCs向心肌细胞分化的诱导促进作用目的:探讨异羟肟酸(suberoylanilide hydroxamic acid, SAHA)体外诱导骨髓间充质干细胞(mesenchymal stem cells, MSCs)是否促进其向心肌细胞分化,以及SAHA与5氮杂胞苷(5-azacytidine, 5-aza)是否存在协同作用。方法:提取扩增大鼠MSCs,分别以SAHA、5-aza或SAHA+5-aza诱导其向心肌细胞方向分化。在分化过程中以定量聚合酶链反应(quantitative PCR, qPCR)、免疫荧光和Western blot检测心肌发育相关基因和心肌特异性蛋白的表达。结果:1. MSCs经诱导7天后心肌发育相关基因GATA4, NKx2.5和MEF2c的表达:(1)单纯SAHA诱导:GATA4, NKx2.5和MEF2c表达明显升高(p<0.05),呈剂量依赖性,在以1μM SAHA诱导组增加最高,与未诱导MSCs比较约增加9-15倍,2μM SAHA诱导组表达相对降低。(2)单纯5-aza诱导:GATA4, NKx2.5和MEF2c表达仅增高2-4倍(p<0.05)。(3)SAHA+5-aza诱导: GATA4, NKx2.5和MEF2c表达与单纯SAHA诱导相似(p>0.05)。2.MSCs经诱导4周后心肌特异蛋白cTnT的表达:(1)单纯SAHA诱导:免疫荧光检测可见细胞内较密集的绿色点状荧光;Western blot检测1μM SAHA诱导组cTnT增加最高(p<0.05),与未诱导MSCs比较约增加8倍,在诱导浓度为2μM时下降。(2)单纯5-aza诱导:免疫荧光检测可见细胞内稀疏绿色点状荧光,Western blot检测cTnT仅较未诱导MSCs升高1.3倍左右(p>0.05)。(3)SAHA+5-aza诱导:免疫荧光检测可见细胞内较密集的绿色点状荧光;Western blot检测cTnT升高与单纯SAHA诱导相似(p>0.05)。结论:1、SAHA可促进MSCs向心肌细胞分化,使MSCs中心肌发育相关基因和特异性蛋白表达明显升高。其诱导MSCs向心肌细胞分化效果强于单纯5-aza诱导。2、在MSCs向心肌细胞分化过程中未发现SAHA和5-aza的协同作用。第二部分体外SAHA促MSCs向心肌细胞分化的组蛋白乙酰化作用机制目的:探讨SAHA体外诱导MSCs是否通过升高心肌发育相关基因启动子DNA上结合组蛋白乙酰化水平来促进其表达,并探讨这些启动子区组蛋白高乙酰化是否与组蛋白去乙酰化酶4(histone deacetylase 4, HDAC4)抑制有关。方法:于1μM SAHA诱导MSCs 7天后收集细胞,以乙酰化H3和HDAC4抗体分别做染色质免疫共沉淀(Chromatin immunoprecipitation, ChIP),以心肌发育相关基因第一外显子前1000bp设计引物,qPCR检测沉淀后DNA中心肌发育相关基因启动子区DNA量的变化。结果: SAHA诱导后MSCs中以乙酰化组蛋白H3抗体ChIP沉淀的GATA4、MEF2c和NKx2.5启动子区DNA分别比未诱导MSCs增加约3倍、1.4倍和2倍(p<0.05),而以HDAC4抗体沉淀的GATA4、MEF2c和NKx2.5启动子区DNA分别比未诱导MSCs下降约0.41倍、0.31倍和0.43倍(p<0.05)。结论:1、SAHA诱导后MSCs的GATA4、MEF2c和NKx2.5表达增强可能与SAHA诱导后这些基因启动子区组蛋白乙酰化水平增加,而激活转录有关。2、GATA4、MEF2c和NKx2.5启动子区组蛋白乙酰化水平升高可能与HDAC4抑制有关。这可能也是SAHA诱导MSCs向心肌细胞特异性分化的乙酰化作用机制之一。第三部分SAHA对MSCs移植阿霉素心力衰竭大鼠心功能的影响目的:探讨SAHA诱导后MSCs移植治疗心力衰竭大鼠模型是否比单纯MSCs移植有更加明显的促进心功能改善的作用。方法:构建阿霉素心力衰竭大鼠模型,分别将MSCs、SAHA诱导7天的MSCs移植入大鼠心肌组织内,三周后心脏彩色超声心动图(ultrasonic cardiogram, UCG)检测其心功能变化,采用左室的射血分数(ejection fraction, EF)和短轴缩短率(fraction shortening, FS)指标评价移植后大鼠心功能改善情况。结果: MSCs组EF由移植前的65.92±2.98上升至移植后的73.25±3.58,FS由32.25±1.66上升至37.46±3.12。SAHA诱导MSCs组EF由移植前的66.04±2.02上升至移植后的75.13±2.05,FS由32.29±0.96上升至38.87±1.82。MSCs组和SAHA诱导MSCs组移植后与移植前心功能比较EF和FS均有明显改善(p<0.05),但MSCs组和SAHA诱导MSCs组之间移植后心功能比较EF和FS无明显差异(p>0.05)。结论:单纯MSCs和SAHA诱导MSCs移植阿霉素大鼠心力衰竭模型心肌组织内3周后检测,两组心功能均有改善,但两组间比较无明显差异。更多还原

【Abstract】 PART 1 SAHA promotes cardiomyocyte differentiation of mesenchymal stem cells in vitroObjective: This study was to investigate the effect of SAHA on cardiomyocyte differentiation of MSCs in vitro and the cooperative effect between SAHA and 5-aza.Methods: Rat MSCs were isolated and induced to differentiate into cardiomyocyte with SAHA or 5- aza or a combination of SAHA and 5-aza. The expression of cardiogenesis-related gene and cardiomyocyte-specific protein were detected by immunofluorescence staining, qPCR and Wstern-blot during the differentiation.Results:1.The mRNA levels of the transcriptional expression of GATA4, NKx2.5, and MEF2c after the 7 days induction on MSCs. (1)The induction with SAHA alone: the expressions of GATA4, NKx2.5, and MEF2c increased significantly (p<0.05), and were dose dependent. The expressions were increased highest when induction with 1uM SAHA, about 10-15 times higher than the group which with no induction, nevertheless, the expressions of which induction with 2uM SAHA were relative reduced. (2) The induction with 5-aza alone: the mRNA levels of GATA4, NKx2.5 and MEF2c increased only 2-4 times when induction with 10uM 5-aza(p<0.05). (3)The induction with SAHA + 5-aza:When MSCs were induced with both SAHA and 5-aza, the mRNA levels of GATA4, NKx2.5, and MEF2c were similar with those induced by SAHA alone(p>0.05).2.The expressin of cTnT after 4 weeks induction on MSCs:(1) The induction with SAHA alone: The immunofluorescence staining detect that green plaguliform fluorescences were dense in the cells. The Western blot detect that cTnT when induction with 1uM SAHA was increased about 8 times than the group with not induction(p<0.05),whereas the expression of the cTnT protein was decreased when induction with 2uM SAHA.(2) The induction with 5-aza alone: The immunofluorescence staining detect that green plaguliform fluorescences were sparseness in the cells.The Western blot detect that cTnT only increased about 1.3 times than the group with not induction ( p>0.05 ) . (3) The induction with SAHA + 5-aza: The immunofluorescence staining results and the Western blot results were similar with the group which induced by SAHA(p>0.05).Conclusion: 1.SAHA promoted cardiomyocyte differentiation of rat MSCs with a significant increase in the expression of cardiogenesis-related gene GATA4, NKx2.5, and MEF2c, and cardiomyocyte-specific protein cTnT in vitro. SAHA was much more effective on cardiomyocyte differentiation of MSCs than 5-aza.2.The cooperative effect between SAHA and 5- aza was not found during the differentiation of MSCs into cardiomyocyte.PART 2 The acetylation mechanism of cardiomyocyte differentiation of MSCs induced by SAHA in vitroObjective: This study was to investigate whether the increase of cardiogenesis-related genes transcription in MSCs was related to hyper-acetylation on their promoters DNA after induction by SAHA. Whether the repression of HDAC4 lead to the hyper-acetylation.Methods: Rat MSCs were collected after 7 days induced with 1μM SAHA. Then the DNA of these cells were precipitated by acetylated H3 antibody or HDAC4 antibody with ChIP . The primers were designed in the 1000bp region before the cardiogenesis-related genes first exon. The quantity of these genes promoter region DNA were evaluated using qPCR by the primers. Results: The DNA level of GATA4, MEF2c and NKx2.5 promotor region in MSCs after induction with SAHA were 3 times, 1.4times and 2 times higher than that in MSCs with not induction when acetylated H3 antibody was used with ChIP(p<0.05). The DNA level of GATA4, MEF2c and NKx2.5 in MSCs after induction with SAHA were decreased about 0.41 times, 0.31 times and 0.43 times than that in MSCs with not induction when HDAC4 antibody was used with ChIP(p<0.05).Conclusion:1.The increase of GATA4、MEF2c and NKx2.5 genes transcription may be related to the hyper-acetylation on their promoters DNA after induction with SAHA.2. The hyper-acetylation in GATA4、MEF2c and NKx2.5 promotor region probably related with the supression of HDAC4.And this is possible one of the reasons that differentiation of MSCs into cardiomyocyte induced by SAHA.PART 3 The effect of SAHA on cardiac function after MSCs transplantion into rat heart of doxorubicin heart failure model Objective: This study was to investigate the effect of SAHA on cardiac function after MSCs transplantion into rat heart of doxorubicin heart failure model.Methods: Doxorubicin heart failure model were constructed in rats. MSCs and MSCs which induced with 1μM SAHA by 7 days were transplanted into the myocardial tissue of the models. Cardiac function was evaluated with UCG 3 weeks after the transplantion, a nd the cardiac function on rats were assessed by EF and FS.Results: The EF was increased from 65.92±2.98 to 73.25±3.58 and the FS was increased from 32.25±1.66 to 37.46±3.12 in the MSCs transplantation rats. And in the rats which transplanted with MSCs induced by SAHA, the EF was increased from 66.04±2.02 to 75.13±2.05 and the FS was increased from32.29±0.96 to 38.87±1.82. In the two groups EF and FS were improved markedly after the transplantation than before(p<0.05).But there was no significant difference between the two groups after the transplantation(p>0.05).Conclusion: The cardiac function of doxorubicin heart failure rats was improved three weeks after the transplantion of MSCs which induced with SAHA or not. But there was no significant difference between the two groups.更多还原