心肌细胞肥大过程中心脏发育相关基因的时序性表达模式研究

发布时间：2018-03-03 20:38

  本文选题：心肌细胞肥大　切入点：心脏发育相关转录因子　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的观察在心肌细胞肥大过程中相关基因时序性表达规律,与心脏发育过程中相关规律进行对比,为心肌细胞肥大机制研究及治疗提供线索。方法选择出生1-3天的昆明乳鼠进行心肌细胞原代培养,用异丙肾上腺素(ISO)干预心肌细胞,用CCK-8、细胞计数仪、细胞免疫组化分别检测心肌细胞存活率、平均直径及表面积,筛选出构建模型的最佳ISO浓度。用RT-PCR、Western Blot分别检测相关基因mRNA及蛋白表达水平。结果用不同浓度ISO干预心肌细胞48h后细胞体积在随浓度浓度增加体积稍增大,到5μmol/L时达峰值(P0.05),后体积稍有减小(P0.05)。不同浓度ISO干预心肌细胞48h后,各组细胞存活率较正常对照组无明显变化(P0.05),但20μmol/L组细胞存活率明显低于5μmol/L及10μmol/L组(P0.05)。5μmol/L ISO干预心肌细胞48h细胞表面积较正常对照组明显增大(P0.05)。5μmol/L ISO干预后,GATA4、MEF2C、GATA5、BNP、ANP于24h,P300、α-MHC、TBX5于12h,SRF、β-MHC mRNA于6h开始升高(P0.05);其中GATA4、SRF、MEF2C、α-MHC、β-MHC、P300 mRNA表达呈先升后降的趋势,至干预72h GATA4、SRF、α-MHC、β-MHC、P300 mRNA表达仍高于正常(P0.05),而MEF2C降至正常(P0.05);GATA5、TBX5、ANP、BNP mRNA表达与正常对照组相比持续升高(P0.05),而NKX2.5 mRNA无明显变化(P0.05)。ISO干预48h后,GATA4、HEY2蛋白表达水平分别升高、降低(P0.05)。结论根据细胞体积、细胞存活率、细胞表面积及肥大基因的表达结果提示心肌细胞肥大模型构建成功。在该心肌细胞肥大模型中,MEF2C的时序表达规律与心脏发育过程相似,而GATA4、GATA5、SRF、TBX5的时序性表达规律与心脏发育过程不完全一致,提示其在心肌细胞肥大由代偿转为失代偿过程中的重要作用。同时GATA4、MEF2C、SRF与乙酰化酶P300的时序性表达规律相似,提示在心肌细胞肥大过程中其时序性表达可能受P300调控。
[Abstract]:Objective To observe the effect in the process of myocardial hypertrophy associated gene sequential expression rules and related rules and heart development comparison, provide clues for the study of mechanism of myocardial cell hypertrophy and treatment methods. Kunming rat was born 1-3 days of myocardial cells cultured with isoproterenol (ISO) on myocardial cells with CCK-8, cell count, cell immunohistochemistry were used to detect the myocardial cell survival rate, average diameter and surface area, screening the best concentration of ISO to construct the model. Using RT-PCR, Western Blot were used to detect related gene mRNA and protein expression level. The results with different concentrations of ISO on myocardial cells after 48h cells with the concentration increase in volume the volume increased slightly to 5, reached the peak at mol/L (P0.05), the volume is slightly decreased (P0.05). Different concentrations of ISO on myocardial cell 48h, cell survival rate compared with the normal control group. 鏃犳槑鏄惧彉鍖，

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