脂联素对高糖环境下人心肌细胞凋亡及Bax、Bcl-2表达的影响

发布时间：2018-11-27 18:16

【摘要】：目的：通过培养人心肌细胞株，观察高糖环境下及加入外源脂联素干预后不同时间点，人心肌细胞的增殖、凋亡情况，及Bax\Bcl-2 mRNA的表达变化，从而揭示脂联素对高糖环境下心肌细胞的保护作用，为临床糖尿病心肌病的治疗提供新的思路。方法：①将培养的人心肌细胞（HCM）随机分为正常对照组（D-葡萄糖浓度：5.5mmol/l）、高糖组（D-葡萄糖浓度：30.0mmol/l）、高糖+脂联素组（D-葡萄糖浓度：30.0mmol/l+ADPN浓度：2.5μg/ml），按上述条件分别培养24h、48h、72h；②运用四甲基偶氮唑蓝（MTT）法测定不同时间点各组细胞的光吸收值（OD值），检测高糖及脂联素对人心肌细胞增殖的影响；③收集各个时间段培养的人心肌细胞，离心后加入脂结合蛋白Annexin V/碘化丙啶（PI）染色后，运用流式细胞仪（FCM）测定各组细胞在不同时间段的凋亡率；④在各个时间段提取各组人心肌细胞的RNA，逆转录为cDNA，然后加入引物，通过实时荧光定量PCR（RT-PCR）技术测定各组人心肌细胞Bax、Bcl-2的表达情况。结果：①与正常对照组相比，高糖的持续作用抑制人心肌细胞增殖，而脂联素组人心肌细胞的增殖明显增加，差异有统计学意义（P0.05）； ②高糖组与对照组相比，明显增加了人心肌细胞的凋亡率，加入脂联素干预后，人心肌细胞的凋亡率明显下降，且差异有统计学意义（P0.05）； ③高糖组人心肌细胞Bax的表达上调、Bcl-2的表达下降；与对照组相比，差异有统计学意义。脂联素组人心肌细胞Bax的表达减少、Bcl-2的表达增加，且该作用呈时间依赖性，与高糖组相比，差异有统计学意义（P0.05）。结论：高糖环境下，人心肌细胞的增殖减少，凋亡率增加，Bax的表达增加，Bcl-2表达降低；而加入脂联素干预后，通过下调促进凋亡因子Bax的表达、上调抑制凋亡因子Bcl-2的表达，增加细胞的增殖，减少细胞的凋亡，从而保护高糖环境下人心肌细胞的生长，，发挥对糖尿病心肌病的保护作用，为临床治疗糖尿病心肌病提供一条新的途径。
[Abstract]:Objective: to observe the proliferation, apoptosis and Bax\ Bcl-2 mRNA expression of human cardiomyocytes in high glucose environment and at different time points after exogenous adiponectin (adiponectin). Thus, the protective effect of adiponectin on cardiomyocytes in high glucose environment was revealed, which provided a new idea for the treatment of diabetic cardiomyopathy. Methods: (1) cultured human cardiomyocytes (HCM) were randomly divided into normal control group (D- glucose concentration: 5.5mmol/l) and high glucose group (D- glucose concentration: 30.0mmol/l). High glucose group (D- glucose concentration: 30.0mmol/l ADPN concentration: 2.5 渭 g/ml) was cultured for 24 h and 48 h for 72 h. (2) the light absorption value (OD) of the cells at different time points was measured by (MTT) method, and the effects of high glucose and adiponectin on the proliferation of human cardiomyocytes were detected. 3The cultured human cardiomyocytes were collected. After centrifugation, lipid binding protein (Annexin V) / pyridine iodide (PI) was added, and the apoptosis rate of each group was measured by flow cytometry (FCM) (FCM). 4Recombinant RNA, of human cardiomyocytes was extracted from each group at each time and then primers were added. The expression of Bax,Bcl-2 in human cardiomyocytes was detected by real-time quantitative PCR (RT-PCR) technique. Results: 1Compared with the normal control group, the sustained effect of high glucose inhibited the proliferation of human cardiomyocytes, while the proliferation of human cardiomyocytes in adiponectin group increased significantly (P0.05). 2 compared with the control group, the high glucose group significantly increased the apoptosis rate of human cardiomyocytes. After adiponectin intervention, the apoptosis rate of human cardiomyocytes decreased significantly (P0.05). (3) the expression of Bax was up-regulated and the expression of Bcl-2 was decreased in high glucose group, compared with the control group, the difference was statistically significant. In adiponectin group, the expression of Bax was decreased and the expression of Bcl-2 was increased in a time dependent manner. The difference was statistically significant compared with high glucose group (P0.05). Conclusion: under high glucose condition, the proliferation of human cardiomyocytes decreased, the apoptosis rate increased, the expression of Bax increased and the expression of Bcl-2 decreased. After adiponectin was added, the expression of apoptotic factor Bax was down-regulated, the expression of apoptotic factor Bcl-2 was up-regulated, the proliferation of cells was increased, and the apoptosis was reduced, thus protecting the growth of human cardiomyocytes under high glucose environment. It provides a new way for the clinical treatment of diabetic cardiomyopathy.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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