EGCG上调老年相关SERCA2a低表达的表观遗传机理研究
发布时间：2019-06-19 01:53
【摘要】：目的:心脏舒张功能障碍是老年人群中常见的心血管疾病,而SERCA2a在老年心脏中表达降低,与其密切相关。课题组前期研究发现,ATP2a2基因的表达受到组蛋白乙酰化修饰的调控,而EGCG(表没食子儿茶素没食子酸酯)能显著影响组蛋白乙酰化水平,调控基因的转录和表达。因此,本研究采用EGCG作为干预方式,探讨EGCG对老年小鼠心脏SERCA2a的表达及其对心脏组织形态学的影响,并探索其内在机理。方法:16月龄雄性老年小鼠随机分为EGCG+老年组、DMSO+老年组及未处理的老年组,3月龄雄性成年小鼠为青年组;EGCG+老年组小鼠腹腔注射EGCG(50mg/kg/d),DMSO+老年组小鼠腹腔注射与溶解EGCG等剂量的DMSO,干预8周,收集心脏组织。并做如下检测:1、RT-PCR技术检测各组小鼠心脏ATP2a2、HDAC1等基因m RNA的表达水平;2、Western blot技术检测各组小鼠心脏SERCA2a蛋白的表达水平;3、Ch IP-Q-PCR技术检测ATP2a2启动子区域的组蛋白H3及H3K9、H3K4、H3K27乙酰化水平,组蛋白去乙酰化酶HDAC1的结合水平,以及核心转录因子GATA4、Mef2c与ATP2a2启动子区域的结合水平;4、取EGCG+老年组及老年组小鼠心脏组织,利用透射电镜观察心脏的超微结构,并采用Masson染色观察心肌的纤维化程度,TUNEL法检测心肌细胞的凋亡水平。结果:1、SERCA2a在转录和蛋白水平,EGCG+老年组及青年组显著高于DMSO+老年组及老年组(p0.05);2、与DMSO+老年组及老年组比较,EGCG+老年组HDAC1m RNA表达水平及其与ATP2a2基因启动子区的结合量显著降低(p0.05);3、EGCG+老年组ATP2a2基因启动子区域的组蛋白H3及H3K9乙酰化水平显著高于DMSO+老年组及老年组(p0.05);而H3K4乙酰化水平在各组间无显著性差异(p0.05);青年组启动子区H3K27的乙酰化水平高于其余三组(p0.05),而这三组之间差异无统计学意义(p0.05);4、GATA4、Mef2c与ATP2a2基因启动子区域的结合量在EGCG+老年组及青年组高于DMSO+老年组及老年组(p0.05);5、老年组小鼠心肌组织出现肌丝溶解破坏,心肌细胞间胶原纤维增多,心肌细胞凋亡数量增加,而EGCG+老年组小鼠心肌组织肌丝溶解破坏、胶原纤维以及心肌细胞凋亡数量减少。结论:1、EGCG可能通过抑制心脏组蛋白去乙酰化酶HDAC1的表达,降低其与ATP2a2基因启动子区域的结合水平,升高老年小鼠心脏ATP2a2基因组蛋白H3K9的乙酰化水平,促进转录因子GATA4、MEF2c的结合,上调老年相关SECA2a的低表达;2、老年小鼠心肌的超微结构出现破坏,心肌纤维化及心肌细胞凋亡增加,EGCG可能在一定程度上可以改善这一系列衰老变化。
[Abstract]:Objective: cardiac diastolic dysfunction is a common cardiovascular disease in the elderly population, and the decreased expression of SERCA2a in the elderly heart is closely related to it. It was found that the expression of ATP2a2 gene was regulated by histone acetylation, while EGCG (epigallocatechin gallate) significantly affected the level of histone acetylation and regulated the transcription and expression of histone gene. Therefore, EGCG was used as an intervention method to investigate the effect of EGCG on the expression of SERCA2a in the heart of aged mice and its effect on cardiac histomorphology, and to explore its internal mechanism. Methods: 16-month-old male mice were randomly divided into EGCG elderly group, DMSO elderly group and untreated elderly group, 3-month-old male adult mice were young group, EGCG elderly mice were intraperitoneally injected with EGCG (50mg/kg/d), DMSO elderly group mice were intraabdominal injection and dissolved EGCG equal dose DMSO, intervention for 8 weeks, and cardiac tissue was collected. The results were as follows: (1) the expression of ATP2a2,HDAC1 and other genes m RNA in the heart of mice in each group was detected by RT PCR, and the expression of SERCA2a protein in the heart of mice in each group was detected by Western blot. 3. The histone H3 and H3K9, H3K4, H3K27 acetylated levels, histone deacetylase HDAC1 binding level, and the binding level of core transcription factor GATA4,Mef2c to ATP2a2 promoter region were detected by Ch IP-Q-PCR technique. 4. The ultrastructure of the heart was observed by transmission electron microscope, the degree of myocardial fibrosis was observed by Masson staining, and the apoptosis level of cardiomyocytes was detected by TUNEL assay. Results: 1the transcription and protein levels of EGCG in elderly group and youth group were significantly higher than those in DMSO elderly group and elderly group (p0.05). 2, compared with DMSO elderly group and elderly group, the expression of HDAC1m RNA and its binding to ATP2a2 gene promoter region in EGCG elderly group were significantly lower than those in ATP2a2 gene promoter group (p0.05). 3The histone H3 and H3K9 acetylation levels in the promoter region of ATP2a2 gene in the elderly group were significantly higher than those in the elderly group and the elderly group (p0.05), but there was no significant difference in the level of H3K4 acetylation among the three groups (p0.05). The acetylation level of H3K27 in the promoter region of the young group was higher than that in the other three groups (p0.05), but there was no significant difference among the three groups (p0.05). 4. The binding capacity of Mef2c to the promoter region of ATP2a2 gene in EGCG elderly group and young group was higher than that in DMSO elderly group and elderly group (p0.05). 5, myolysis and destruction of myocardial filaments, increase of collagen fibers and apoptosis of cardiomyocytes in aged group were higher than those in DMSO group (p0.05). In elderly group, the number of myocardial filaments dissolved and destroyed and the number of collagen fibers and cardiomyocytes in aged group decreased. Conclusion: 1ECG may inhibit the expression of cardiac histone deacetylase HDAC1, decrease its binding level to the promoter region of ATP2a2 gene, increase the acetylation level of ATP2a2 genomic protein H3K9 in aged mice, promote the binding of transcription factor GATA4,MEF2c, and up-regulate the low expression of SECA2a associated with the elderly. 2. The ultrastructure of myocardium was destroyed, myocardial fibrosis and cardiomyocyte apoptosis were increased in elderly mice. EGCG may improve this series of aging changes to a certain extent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
[Abstract]:Objective: cardiac diastolic dysfunction is a common cardiovascular disease in the elderly population, and the decreased expression of SERCA2a in the elderly heart is closely related to it. It was found that the expression of ATP2a2 gene was regulated by histone acetylation, while EGCG (epigallocatechin gallate) significantly affected the level of histone acetylation and regulated the transcription and expression of histone gene. Therefore, EGCG was used as an intervention method to investigate the effect of EGCG on the expression of SERCA2a in the heart of aged mice and its effect on cardiac histomorphology, and to explore its internal mechanism. Methods: 16-month-old male mice were randomly divided into EGCG elderly group, DMSO elderly group and untreated elderly group, 3-month-old male adult mice were young group, EGCG elderly mice were intraperitoneally injected with EGCG (50mg/kg/d), DMSO elderly group mice were intraabdominal injection and dissolved EGCG equal dose DMSO, intervention for 8 weeks, and cardiac tissue was collected. The results were as follows: (1) the expression of ATP2a2,HDAC1 and other genes m RNA in the heart of mice in each group was detected by RT PCR, and the expression of SERCA2a protein in the heart of mice in each group was detected by Western blot. 3. The histone H3 and H3K9, H3K4, H3K27 acetylated levels, histone deacetylase HDAC1 binding level, and the binding level of core transcription factor GATA4,Mef2c to ATP2a2 promoter region were detected by Ch IP-Q-PCR technique. 4. The ultrastructure of the heart was observed by transmission electron microscope, the degree of myocardial fibrosis was observed by Masson staining, and the apoptosis level of cardiomyocytes was detected by TUNEL assay. Results: 1the transcription and protein levels of EGCG in elderly group and youth group were significantly higher than those in DMSO elderly group and elderly group (p0.05). 2, compared with DMSO elderly group and elderly group, the expression of HDAC1m RNA and its binding to ATP2a2 gene promoter region in EGCG elderly group were significantly lower than those in ATP2a2 gene promoter group (p0.05). 3The histone H3 and H3K9 acetylation levels in the promoter region of ATP2a2 gene in the elderly group were significantly higher than those in the elderly group and the elderly group (p0.05), but there was no significant difference in the level of H3K4 acetylation among the three groups (p0.05). The acetylation level of H3K27 in the promoter region of the young group was higher than that in the other three groups (p0.05), but there was no significant difference among the three groups (p0.05). 4. The binding capacity of Mef2c to the promoter region of ATP2a2 gene in EGCG elderly group and young group was higher than that in DMSO elderly group and elderly group (p0.05). 5, myolysis and destruction of myocardial filaments, increase of collagen fibers and apoptosis of cardiomyocytes in aged group were higher than those in DMSO group (p0.05). In elderly group, the number of myocardial filaments dissolved and destroyed and the number of collagen fibers and cardiomyocytes in aged group decreased. Conclusion: 1ECG may inhibit the expression of cardiac histone deacetylase HDAC1, decrease its binding level to the promoter region of ATP2a2 gene, increase the acetylation level of ATP2a2 genomic protein H3K9 in aged mice, promote the binding of transcription factor GATA4,MEF2c, and up-regulate the low expression of SECA2a associated with the elderly. 2. The ultrastructure of myocardium was destroyed, myocardial fibrosis and cardiomyocyte apoptosis were increased in elderly mice. EGCG may improve this series of aging changes to a certain extent.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
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