本文選題：心肌細胞肥大 + 5-氮雜胞苷��； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：【研究背景】據(jù)中國心血管病報告2011公布的資料顯示,心血管疾病死亡率逐年上升,農(nóng)村居民心血管病死亡率增加速度高于城市居民;中國人口的總死亡率中心血管病死亡占總死亡原因的41%,居各種死因的首位,全國每年350萬人死于心血管病,我國10省市20個城鄉(xiāng)調(diào)查發(fā)現(xiàn),35~74歲人群的慢性心衰發(fā)生率為0.9%,且發(fā)病年齡趨于年輕化,對社會經(jīng)濟建設(shè)造成巨大影響。高血壓、冠心病、動脈粥樣硬化、心臟瓣膜疾病等眾多心血管疾病的代償性病理生理表現(xiàn)。而鈣水平紊亂在心肌肥大的發(fā)生機制中起極為重要的作用。本研究是基于我們相關(guān)前期研究,前期研究結(jié)果表明,心肌細胞內(nèi)鈣調(diào)控相關(guān)基因的異常表達可導致細胞內(nèi)鈣穩(wěn)態(tài)失衡,繼而激活鈣敏感鈣調(diào)素依賴鈣調(diào)神經(jīng)磷酸酶(CaN)和鈣調(diào)素依賴蛋白激酶(CaMKs)途徑,導致心肌細胞肥大的發(fā)生。心肌細胞肥大時鈣調(diào)控和鈣信號途徑相關(guān)基因的調(diào)控機制目前尚未完全明了,而相關(guān)基因啟動子區(qū)甲基化水平變化可調(diào)節(jié)基因的異常表達,導致了疾病發(fā)生。在前期工作基礎(chǔ)上,本研究擬分析甲基化酶抑制劑5-氮雜胞苷在體外誘導的心肌細胞肥大中,對細胞鈣調(diào)控相關(guān)基因SERCA2a、CaMKⅡ、p-CaMKⅡ表達水平和相關(guān)基因表達的異常改變以及細胞內(nèi)鈣水平的變化,并進一步分析鈣信號途徑相關(guān)基因變化與心肌細胞肥大的相互關(guān)系,以期探索鈣調(diào)控和鈣信號途徑相關(guān)基因表達水平改變、調(diào)控機理及其在心肌細胞肥大發(fā)生的作用。該研究將為進一步探討心肌細胞肥大的發(fā)生機制以及臨床治療奠定基礎(chǔ),具有較大的社會和經(jīng)濟效益。【研究目的】探討甲基化酶抑制劑5-氮雜胞苷(5-AZA-2’-d C)抑制血管緊張素Ⅱ(AngⅡ)誘導的心肌細胞肥大的作用�！狙芯糠椒ā�1、原代乳鼠心肌細胞分離、純化、培養(yǎng),免疫熒光進行原代鑒定。2、建立心肌細胞肥大模型、干預藥物濃度選擇及實驗分組:采用不同濃度血管緊張素Ⅱ誘導心肌細胞肥大;不同濃度5-氮雜胞苷處理,cck8檢測細胞存活率。根據(jù)處理方法,分為5組,分別為對照組、血管緊張素Ⅱ組、5-氮雜胞苷組、血管緊張素Ⅱ與5-氮雜胞苷同時處理組和5-氮雜胞苷預處理組。3、測定心肌細胞ANP mRNA及蛋白表達、細胞面積等心肌細胞肥大的指標;4、采用Western blot法檢測各組心肌細胞ANP、SERCA2a、CaMKⅡ、p-CaMKⅡ的蛋白質(zhì)表達的水平;5、鈣離子探針孵育細胞,激光共聚焦檢測胞內(nèi)鈣變化情況�！狙芯拷Y(jié)果】1、血管緊張素Ⅱ體外誘導原代乳鼠心肌細胞肥大呈濃度依賴性;5-氮雜胞苷對心肌細胞毒性呈濃度依賴性。2、血管緊張素Ⅱ(10-6 mol/L)處理心肌細胞48 h,可使心房利鈉肽(ANP)增加,而該作用可被5-氮雜胞苷(10-6 mol/L)抑制。與對照組相比,血管緊張素Ⅱ組心肌的ANP、p-CaMKⅡ蛋白表達明顯增加;而5-氮雜胞苷同時加藥組及預處理組較之血管緊張素Ⅱ組則下降。3、與對照組相比,血管緊張素Ⅱ組心肌的SERCA2a蛋白質(zhì)表達下降,5-氮雜胞苷同時加藥組及預處理組較血管緊張素Ⅱ組SERCA2a表達量上升。4、鈣離子變化情況,與對照組相比,血管緊張素Ⅱ組胞內(nèi)鈣離子峰值上升時間和下降時間均延長,與5-氮雜胞苷同時加藥組及預處理組較之肥大組則稍縮短。【研究結(jié)論】5-氮雜胞苷可增加SERCA2a蛋白表達,從而縮短胞漿鈣再攝取入肌漿網(wǎng)的時間,有利于維持胞內(nèi)鈣穩(wěn)態(tài),抑制血管緊張素Ⅱ誘導的心肌細胞肥大。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54

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