Apelin抑制抵抗素诱导的H9c2心肌细胞肥大

发布时间：2018-05-10 20:22

本文选题：抵抗素 + Apelin　；参考：《山西医科大学》2015年硕士论文

【摘要】：目的:通过Apelin干预抵抗素诱导的H9c2大鼠心肌细胞肥大模型,检测心肌细胞表面积大小、单细胞总蛋白合成量、实时荧光定量PCR技术检测心肌细胞肥大标志物BNP和β-MHC的表达,研究其对抵抗素诱导的H9c2大鼠心肌细胞肥大的影响,探索有效防治心肌细胞肥大的新方法,为其应用于临床心力衰竭的防治提供理论依据。方法:H9c2大鼠心肌细胞是源于BD1X大鼠胚胎心脏组织的亚克隆细胞株,用含10%FBS的DMEM培养,每2-3 d换一次培养液,待细胞长到70%-80%时传代培养。在倒置显微镜下观察,H9c2大鼠心肌细胞呈圆形或椭圆形,未见心肌搏动。取长势良好的细胞用于实验。将1×105个H9c2大鼠心肌细胞分别接种于6孔板内,加入含10%FBS的DMEM 3 ml培养,待细胞贴壁后,换含0.1%FBS的DMEM 3 ml饥饿16 h。根据实验目的分为4组,C组(对照组):0.1%FBS的DMEM饥饿细胞后换含0.1%FBS的DMEM 3 ml培养48 h。R组(抵抗素组):0.1%FBS的DMEM饥饿细胞后换抵抗素浓度为50 ng/ml的含0.1%FBS的DMEM 3 ml培养48 h。A组(抵抗素+Apelin组):0.1%FBS的DMEM饥饿细胞后换Apelin浓度为10μmol/3 ml的含0.1%FBS的DMEM 3 ml培养2 h后,换抵抗素浓度为50 ng/ml的含0.1%FBS的DMEM 3 ml培养46 h。N组(抵抗素+Apelin+LKB1抑制剂组):0.1%FBS的DMEM饥饿细胞后换LKB1抑制剂(NHE)浓度为20μmol/3 ml的含0.1%FBS的DMEM 3 ml培养2 h后,换Apelin浓度为10μmol/3 ml的含0.1%FBS的DMEM 3 ml培养0.5 h后,换为抵抗素浓度为50 ng/ml的含0.1%FBS的DMEM 3 ml培养45.5 h。培养48 h后,细胞拍照,Image J软件分析细胞表面积;收集细胞计数,BCA法测总蛋白含量,计算单细胞总蛋白含量;实时荧光定量PCR检测心肌细胞肥大标志物BNP和β-MHC的相对表达量。结果:与C组相比,R组心肌细胞表面积、单细胞蛋白合成量以及心肌细胞肥大标志物的相对表达量明显增加,差异有统计学意义(P0.01),成功构建抵抗素诱导的H9c2大鼠心肌细胞肥大模型。与R组相比,A组心肌细胞表面积、单细胞蛋白合成量以及心肌细胞肥大标志物的相对表达量明显降低,差异有统计学意义(P0.01);与R组相比,N组心肌细胞表面积、单细胞蛋白合成量以及心肌细胞肥大标志物的相对表达量无明显减小,差异无统计学意义(P0.05)。结论:抵抗素可以诱导H9c2大鼠心肌细胞肥大;Apelin有抑制抵抗素诱导的H9c2大鼠心肌细胞肥大的作用;Apelin抑制抵抗素诱导的H9c2大鼠心肌细胞肥大的作用机制可能是通过激活LKB1/AMPK信号通路实现的。
[Abstract]:Aim: to investigate the effects of Apelin on cardiomyocyte hypertrophy induced by resistin in H9c2 rats, and to detect the surface area of cardiomyocytes, the total protein synthesis of single cell, and the expression of BNP and 尾 -MHC markers in cardiomyocyte hypertrophy by real-time fluorescence quantitative PCR. To study the effect of resistin on cardiomyocyte hypertrophy induced by resistin in H9c2 rats, to explore a new method to prevent and treat cardiomyocyte hypertrophy, and to provide a theoretical basis for its application in the prevention and treatment of clinical heart failure. Methods the rat cardiomyocytes were subcloned from the embryonic heart tissue of BD1X rats. They were cultured with DMEM containing 10s and cultured in different medium every 2-3 days. When the cells grew to 70% -80%, they were subcultured. The cardiomyocytes of H9c2 rats were round or oval under inverted microscope, and no myocardial pulsation was observed. The cells with good growth were used in the experiment. Myocardial cells of 1 脳 105 H9c2 rats were inoculated into 6-well plates and cultured with DMEM 3 ml containing 10s. After the cells were adhered to the cells, the cells were fed with 0.1 S DMEM 3 ml for 16 h. According to the objective of the experiment, four groups were divided into four groups (control group: 0.1 S DMEM starvation cells were cultured for 48h 路R with DMEM 3 ml containing 0.1 S) (resistin group: 0.1 S DMEM hunger cells were treated with resistin concentration of 50 ng/ml). DMEM 3 ml cultured for 48 h. A group (resistin Apelin group: 0. 1s DMEM starvation cells were cultured for 2 h with 10 渭 mol/3 ml of Apelin containing 0. 1 S DMEM 3 ml for 2 h). When the concentration of resistin was 50 ng/ml, 0.1 S DMEM 3 ml was cultured for 46 h. N group (resistin Apelin LKB1 inhibitor group: 0.1 S DMEM starvation cells were replaced with LKB1 inhibitor 3 ml) 20 渭 mol/3 ml, 0.1 S DMEM 3 ml was cultured for 2 h. When the concentration of Apelin was 10 渭 mol/3 / ml, the DMEM containing 0.1 S was incubated for 0.5 h, and the DMEM containing 0.1 S at the concentration of resistin was 50 ng/ml for 45.5 h. After 48 h culture, the cell surface area was analyzed by image-J software, the total protein content was measured by cell counting method, the total protein content of single cell was calculated, and the relative expressions of BNP and 尾 -MHC markers in cardiomyocyte hypertrophy were detected by real-time fluorescence quantitative PCR. Results: compared with group C, the surface area of cardiomyocytes, the amount of single cell protein synthesis and the relative expression of hypertrophic markers of cardiac myocytes in group R were significantly increased. The difference was statistically significant (P 0.01). Resistin induced cardiomyocyte hypertrophy model of H9c2 rats was successfully constructed. Compared with group R, the surface area of cardiomyocytes, the amount of single cell protein synthesis and the relative expression of hypertrophic markers of cardiomyocytes in group A were significantly lower than those in group R, the difference was statistically significant (P 0.01), and the surface area of cardiomyocytes in group N was significantly lower than that in group R. The amount of single cell protein synthesis and the relative expression of hypertrophic markers in cardiomyocytes were not significantly decreased, but the difference was not statistically significant (P 0.05). Conclusion: resistin can induce cardiomyocyte hypertrophy in H9c2 rats. Apelin can inhibit cardiomyocyte hypertrophy induced by resistin in H9c2 rats. The active LKB1/AMPK signal pathway is realized.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.6

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