5-氮杂胞苷抑制血管紧张素Ⅱ诱导的心肌细胞肥大的作用研究

发布时间：2018-06-29 06:32

本文选题：心肌细胞肥大 + 5-氮杂胞苷　；参考：《广州医科大学》2017年硕士论文

【摘要】：【研究背景】据中国心血管病报告2011公布的资料显示,心血管疾病死亡率逐年上升,农村居民心血管病死亡率增加速度高于城市居民;中国人口的总死亡率中心血管病死亡占总死亡原因的41%,居各种死因的首位,全国每年350万人死于心血管病,我国10省市20个城乡调查发现,35~74岁人群的慢性心衰发生率为0.9%,且发病年龄趋于年轻化,对社会经济建设造成巨大影响。高血压、冠心病、动脉粥样硬化、心脏瓣膜疾病等众多心血管疾病的代偿性病理生理表现。而钙水平紊乱在心肌肥大的发生机制中起极为重要的作用。本研究是基于我们相关前期研究,前期研究结果表明,心肌细胞内钙调控相关基因的异常表达可导致细胞内钙稳态失衡,继而激活钙敏感钙调素依赖钙调神经磷酸酶(CaN)和钙调素依赖蛋白激酶(CaMKs)途径,导致心肌细胞肥大的发生。心肌细胞肥大时钙调控和钙信号途径相关基因的调控机制目前尚未完全明了,而相关基因启动子区甲基化水平变化可调节基因的异常表达,导致了疾病发生。在前期工作基础上,本研究拟分析甲基化酶抑制剂5-氮杂胞苷在体外诱导的心肌细胞肥大中,对细胞钙调控相关基因SERCA2a、CaMKⅡ、p-CaMKⅡ表达水平和相关基因表达的异常改变以及细胞内钙水平的变化,并进一步分析钙信号途径相关基因变化与心肌细胞肥大的相互关系,以期探索钙调控和钙信号途径相关基因表达水平改变、调控机理及其在心肌细胞肥大发生的作用。该研究将为进一步探讨心肌细胞肥大的发生机制以及临床治疗奠定基础,具有较大的社会和经济效益。【研究目的】探讨甲基化酶抑制剂5-氮杂胞苷(5-AZA-2’-d C)抑制血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大的作用。【研究方法】1、原代乳鼠心肌细胞分离、纯化、培养,免疫荧光进行原代鉴定。2、建立心肌细胞肥大模型、干预药物浓度选择及实验分组:采用不同浓度血管紧张素Ⅱ诱导心肌细胞肥大;不同浓度5-氮杂胞苷处理,cck8检测细胞存活率。根据处理方法,分为5组,分别为对照组、血管紧张素Ⅱ组、5-氮杂胞苷组、血管紧张素Ⅱ与5-氮杂胞苷同时处理组和5-氮杂胞苷预处理组。3、测定心肌细胞ANP mRNA及蛋白表达、细胞面积等心肌细胞肥大的指标;4、采用Western blot法检测各组心肌细胞ANP、SERCA2a、CaMKⅡ、p-CaMKⅡ的蛋白质表达的水平;5、钙离子探针孵育细胞,激光共聚焦检测胞内钙变化情况。【研究结果】1、血管紧张素Ⅱ体外诱导原代乳鼠心肌细胞肥大呈浓度依赖性;5-氮杂胞苷对心肌细胞毒性呈浓度依赖性。2、血管紧张素Ⅱ(10-6 mol/L)处理心肌细胞48 h,可使心房利钠肽(ANP)增加,而该作用可被5-氮杂胞苷(10-6 mol/L)抑制。与对照组相比,血管紧张素Ⅱ组心肌的ANP、p-CaMKⅡ蛋白表达明显增加;而5-氮杂胞苷同时加药组及预处理组较之血管紧张素Ⅱ组则下降。3、与对照组相比,血管紧张素Ⅱ组心肌的SERCA2a蛋白质表达下降,5-氮杂胞苷同时加药组及预处理组较血管紧张素Ⅱ组SERCA2a表达量上升。4、钙离子变化情况,与对照组相比,血管紧张素Ⅱ组胞内钙离子峰值上升时间和下降时间均延长,与5-氮杂胞苷同时加药组及预处理组较之肥大组则稍缩短。【研究结论】5-氮杂胞苷可增加SERCA2a蛋白表达,从而缩短胞浆钙再摄取入肌浆网的时间,有利于维持胞内钙稳态,抑制血管紧张素Ⅱ诱导的心肌细胞肥大。
[Abstract]:[background] according to the data published in China's cardiovascular disease report 2011, the mortality rate of cardiovascular disease is rising year by year, and the rate of cardiovascular disease mortality in rural residents is higher than that of urban residents; the death rate of total mortality center of China's population accounts for 41% of the total cause of death, ranking first in all kinds of causes of death, and 3 million 500 thousand people in the country die every year. Cardiovascular disease, 20 urban and rural surveys in 10 provinces and cities in China found that the incidence of chronic heart failure in 35~74 years old is 0.9%, and the age of the disease tends to be younger and has a great influence on the social and economic construction. Hypertension, coronary heart disease, atherosclerosis, heart valve disease, and so on, the compensatory pathophysiology of many cardiovascular diseases, and the disorder of calcium level It plays an important role in the pathogenesis of cardiac hypertrophy. This study is based on our previous studies. Earlier results showed that abnormal calcium regulation related genes in cardiac myocytes could lead to intracellular calcium homeostasis, and then activated calcium sensitive calmodulin dependent calcineurin (CaN) and calmodulin dependent protein. The pathway of kinase (CaMKs) leads to the occurrence of hypertrophy of cardiac myocytes. The regulation mechanism of calcium regulation and calcium signal pathway related genes is not fully understood at present. The change of the level of methylation in the related gene promoter region can regulate the abnormal expression of genes and lead to the occurrence of disease. Based on the earlier work, this study is to be analyzed. The methylation inhibitor 5- aza cytidine was used to induce cardiac myocyte fertilizer in vitro. The abnormal changes in the expression of SERCA2a, CaMK II, p-CaMK II and related gene expression and the intracellular calcium level were changed in vitro, and the relationship between calcium signal pathway related gene changes and cardiac myocyte hypertrophy was further analyzed. In order to explore the changes of calcium regulation and calcium signaling pathway related gene expression level, regulation mechanism and its role in the occurrence of cardiomyocyte hypertrophy, this study will lay a foundation for further exploring the mechanism of cardiac myocyte hypertrophy and clinical treatment, and has great social and economic benefits. [Objective] to explore the inhibition of methylation enzyme. 5- nitrogen heterocytidine (5-AZA-2 '-d C) inhibits the role of angiotensin II (Ang II) induced cardiomyocyte hypertrophy. [method] 1, primary neonatal rat cardiomyocytes were isolated, purified, cultured, and immunofluorescent was used to identify.2, establish cardiomyocyte hypertrophy model, dry predrug concentration selection and experimental grouping: different concentrations of blood vessels were used. Zhang Su induced hypertrophy of cardiac myocytes; different concentrations of 5- aza cytidine treatment and CCK8 detection of cell survival rate were divided into 5 groups: control group, angiotensin II group, 5- heterocytidine group, angiotensin II and 5- aza cytidine simultaneous treatment group and 5- aza cytidine preconditioning group.3, determination of ANP mRNA and protein in cardiac myocytes Expression, cell area and other cardiac myocyte hypertrophy index; 4, Western blot method was used to detect the level of protein expression of ANP, SERCA2a, CaMK II, p-CaMK II in all groups; 5, calcium ion probe incubated cells, laser confocal detection of intracellular calcium changes. [results] 1, angiotensin II in vitro induced myocardial fine myocardium in vitro. The cytotoxicity of 5- was concentration dependent.2, and angiotensin II (10-6 mol/L) treated cardiomyocytes 48 h, which could increase the concentration of atrial natriuretic peptide (ANP), and this effect could be inhibited by 5- nitrogen heterocytidine (10-6 mol/L). Compared with the control group, the expression of ANP and p-CaMK II protein in the myocardium of angiotensin II group was clearly expressed. Compared with the control group, the expression of SERCA2a protein in the angiotensin II group decreased by.3. Compared with the control group, the expression of SERCA2a protein in the angiotensin II group was lower than that in the control group. The expression of SERCA2a in the group of 5- nitrocytidin and the pretreatment group was higher than that of the angiotensin II group.4, the change of calcium ion and the control group were compared with the control group. Compared with the group of angiotensin II, the peak time and decrease time of intracellular calcium ion in the angiotensin II Group extended, and the 5- nitrocytidin group and the preconditioning group were slightly shorter than those in the hypertrophic group. [Conclusion] 5- aza cytidine can increase the expression of SERCA2a protein, thus shortening the time of cytoplasmic calcium reuptake into the sarcoplasmic reticulum, which is beneficial to the maintenance of intracellular time. Calcium homeostasis inhibits angiotensin II induced cardiomyocyte hypertrophy.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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