胰岛素对高糖引起H9c2细胞损伤的保护作用及机制研究

发布时间：2017-12-27 21:11

本文关键词：胰岛素对高糖引起H9c2细胞损伤的保护作用及机制研究　出处：《广西医科大学》2016年硕士论文　论文类型：学位论文

更多相关文章： 胰岛素 内质网应激 高糖 H9c2 PI3K/AKT

【摘要】：目的：建立心肌细胞的高糖损伤模型；观察高糖环境下胰岛素对心肌细胞凋亡的影响及内质网应激相关分子标志物的表达；观察PI3K/AKT信号通路在高糖引起心肌细胞损伤中的作用。方法：1.心肌细胞培养和高糖损伤模型大鼠心肌来源的H9c2细胞的常规培养使用5.5 mmol/L低糖DMEM,加入10%FBS。用35mmol/L高糖(HG, High Glucose) DMEM培养基孵育细胞不同时间(6h,12h,24h,48h,72h),建立心肌细胞高糖损伤模型。2.细胞活力和凋亡检测将细胞分为对照组(低糖5.5 mmol/L)、高糖组(35 mmol/L)、胰岛素组(INS,100nmol/L)、胰岛素+高糖组。用MTT法检测细胞活力,分别观察0,24h,48h,72h和96h并记录数据。根据细胞活力的数据分析选择48h时间点做凋亡检测,实验分组同上,Hoechst染色检测细胞凋亡。3. RT-PCR检测内质网应激相关分子标记物的表达高糖处理H9c2细胞不同时间后,提取细胞总mRNA,检测高糖对GRP78mRNA表达的时间效应。根据时间效应的结果,以48h为时间点,细胞分为4组：对照组(control),高糖组(HG),胰岛素+高糖组,LY294002+胰岛素+高糖组(LY294002预处理30min后,加入高糖和胰岛素处理),检测各组GRP78mRNA的表达。LY294002是PI3K/AKT信号通路特异性的抑制剂。4. Westen-blot检测蛋白表达实验分组：对照组(control),高糖组(HG),胰岛素组,LY294002组,胰岛素+高糖组,LY294002+胰岛素+高糖组,48h后Western blot检测各组细胞中t-Akt和p-Akt的蛋白表达水平。LY294002是PI3K/AKT信号通路特异性的抑制剂。结果：与对照组相比,高糖处理48h后,细胞活力下降(P0.5)；与高糖组比较,胰岛素处理可以显著提高细胞活力,细胞凋亡数也显著减少；且48h时Hochest染色显示,高糖组细胞核固缩,染色质向外周聚集,或者细胞核碎裂成凋亡小体；RT-PCR结果提示高糖处理24h时,GRP78 mRNA水平表达被上调(P0.01 vs. control);与高糖组相比,胰岛素+高糖组p-Akt蛋白的表达显著增加(P0.01),且LY294002能明显抑制胰岛素引起的p-Akt的表达上调(P0.01)。结论高糖可通过内质网应激引起心肌细胞凋亡,胰岛素通过PI3K/AKT信号通路的活化,保护心肌细胞因高糖刺激引起的内质网应激损伤。
[Abstract]:Objective: to establish a high glucose injury model of myocardial cells, observe the effects of insulin on myocardial cell apoptosis and expression of endoplasmic reticulum stress related molecular markers in high glucose environment, and observe the role of PI3K/AKT signaling pathway in myocardial injury induced by high glucose. Methods: 1. H9c2 cells derived from myocardial cell culture and high glucose injury model rats were routinely cultured with 5.5 mmol/L low sugar DMEM and 10%FBS. 35mmol/L high glucose (HG, High Glucose) DMEM medium was used to incubate cells at different time (6h, 12h, 24h, 48h, 72h), and a high glucose damage model of cardiomyocytes was established. 2. cell viability and apoptosis were divided into control group (low sugar 5.5 mmol/L), high glucose group (35 mmol/L), insulin group (INS, 100nmol/L), insulin + high glucose group. The cell viability was detected by MTT, and 0,24h, 48h, 72h and 96h were observed and the data were recorded. According to the data analysis of cell vitality, 48h time point was selected to do apoptosis detection. The experimental group was same, and Hoechst staining was used to detect cell apoptosis. 3. RT-PCR was used to detect the expression of endoplasmic reticulum stress related molecular markers. After high glucose treatment, H9c2 cells were extracted for different time, and the total mRNA was extracted. The time effect of high glucose on GRP78mRNA expression was detected. According to the time effect, 48h was used as a time point. The cells were divided into 4 groups: control group (control), high glucose group (HG), insulin plus high glucose group, LY294002+ insulin plus high glucose group (LY294002 pretreatment 30min, high glucose and insulin treatment), and the expression of GRP78mRNA in each group was detected. LY294002 is a specific inhibitor of PI3K/AKT signaling pathway. 4., Westen-blot detection protein expression test group: control group (control), high glucose group (HG), insulin group, LY294002 group, insulin + high glucose group, LY294002+ insulin + high glucose group, 48h Western blot after detection of t-Akt and p-Akt protein expression level in each group. LY294002 is a specific inhibitor of PI3K/AKT signaling pathway. Results: compared with control group, high glucose 48h after treatment, cell viability decreased (P0.5); compared with high glucose group, insulin treatment can significantly improve the cell vitality, the number of apoptotic cells also decreased significantly; and 48h Hochest staining showed that high glucose group karyopyknosis, chromatin aggregation to peripheral, or nuclear fragmentation apoptosis corpuscle; Hg treatment results of 24h RT-PCR, GRP78 mRNA expression levels were upregulated (P0.01 vs. control); compared with the high glucose group, the expression of insulin + p-Akt protein in high glucose group increased significantly (P0.01), and LY294002 can increase significantly inhibited insulin induced the expression of p-Akt (P0.01). Conclusion high glucose can induce cardiomyocyte apoptosis through endoplasmic reticulum stress, and insulin protects the cardiomyocytes from endoplasmic reticulum stress injury induced by high glucose stimulation through activation of PI3K/AKT signaling pathway.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2

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