EGCG上調(diào)老年相關(guān)SERCA2a低表達(dá)的表觀遺傳機(jī)理研究
發(fā)布時(shí)間：2019-06-19 01:53
【摘要】：目的:心臟舒張功能障礙是老年人群中常見(jiàn)的心血管疾病,而SERCA2a在老年心臟中表達(dá)降低,與其密切相關(guān)。課題組前期研究發(fā)現(xiàn),ATP2a2基因的表達(dá)受到組蛋白乙�；揎椀恼{(diào)控,而EGCG(表沒(méi)食子兒茶素沒(méi)食子酸酯)能顯著影響組蛋白乙�；�,調(diào)控基因的轉(zhuǎn)錄和表達(dá)。因此,本研究采用EGCG作為干預(yù)方式,探討EGCG對(duì)老年小鼠心臟SERCA2a的表達(dá)及其對(duì)心臟組織形態(tài)學(xué)的影響,并探索其內(nèi)在機(jī)理。方法:16月齡雄性老年小鼠隨機(jī)分為EGCG+老年組、DMSO+老年組及未處理的老年組,3月齡雄性成年小鼠為青年組;EGCG+老年組小鼠腹腔注射EGCG(50mg/kg/d),DMSO+老年組小鼠腹腔注射與溶解EGCG等劑量的DMSO,干預(yù)8周,收集心臟組織。并做如下檢測(cè):1、RT-PCR技術(shù)檢測(cè)各組小鼠心臟ATP2a2、HDAC1等基因m RNA的表達(dá)水平;2、Western blot技術(shù)檢測(cè)各組小鼠心臟SERCA2a蛋白的表達(dá)水平;3、Ch IP-Q-PCR技術(shù)檢測(cè)ATP2a2啟動(dòng)子區(qū)域的組蛋白H3及H3K9、H3K4、H3K27乙�；�,組蛋白去乙酰化酶HDAC1的結(jié)合水平,以及核心轉(zhuǎn)錄因子GATA4、Mef2c與ATP2a2啟動(dòng)子區(qū)域的結(jié)合水平;4、取EGCG+老年組及老年組小鼠心臟組織,利用透射電鏡觀察心臟的超微結(jié)構(gòu),并采用Masson染色觀察心肌的纖維化程度,TUNEL法檢測(cè)心肌細(xì)胞的凋亡水平。結(jié)果:1、SERCA2a在轉(zhuǎn)錄和蛋白水平,EGCG+老年組及青年組顯著高于DMSO+老年組及老年組(p0.05);2、與DMSO+老年組及老年組比較,EGCG+老年組HDAC1m RNA表達(dá)水平及其與ATP2a2基因啟動(dòng)子區(qū)的結(jié)合量顯著降低(p0.05);3、EGCG+老年組ATP2a2基因啟動(dòng)子區(qū)域的組蛋白H3及H3K9乙�；斤@著高于DMSO+老年組及老年組(p0.05);而H3K4乙酰化水平在各組間無(wú)顯著性差異(p0.05);青年組啟動(dòng)子區(qū)H3K27的乙�；礁哂谄溆嗳M(p0.05),而這三組之間差異無(wú)統(tǒng)計(jì)學(xué)意義(p0.05);4、GATA4、Mef2c與ATP2a2基因啟動(dòng)子區(qū)域的結(jié)合量在EGCG+老年組及青年組高于DMSO+老年組及老年組(p0.05);5、老年組小鼠心肌組織出現(xiàn)肌絲溶解破壞,心肌細(xì)胞間膠原纖維增多,心肌細(xì)胞凋亡數(shù)量增加,而EGCG+老年組小鼠心肌組織肌絲溶解破壞、膠原纖維以及心肌細(xì)胞凋亡數(shù)量減少。結(jié)論:1、EGCG可能通過(guò)抑制心臟組蛋白去乙酰化酶HDAC1的表達(dá),降低其與ATP2a2基因啟動(dòng)子區(qū)域的結(jié)合水平,升高老年小鼠心臟ATP2a2基因組蛋白H3K9的乙�；�,促進(jìn)轉(zhuǎn)錄因子GATA4、MEF2c的結(jié)合,上調(diào)老年相關(guān)SECA2a的低表達(dá);2、老年小鼠心肌的超微結(jié)構(gòu)出現(xiàn)破壞,心肌纖維化及心肌細(xì)胞凋亡增加,EGCG可能在一定程度上可以改善這一系列衰老變化。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R54
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