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  本文關(guān)鍵詞：胰島素對高糖引起H9c2細胞損傷的保護作用及機制研究　出處：《廣西醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胰島素 內(nèi)質(zhì)網(wǎng)應(yīng)激 高糖 H9c2 PI3K/AKT

【摘要】：目的：建立心肌細胞的高糖損傷模型；觀察高糖環(huán)境下胰島素對心肌細胞凋亡的影響及內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)分子標(biāo)志物的表達；觀察PI3K/AKT信號通路在高糖引起心肌細胞損傷中的作用。方法：1.心肌細胞培養(yǎng)和高糖損傷模型大鼠心肌來源的H9c2細胞的常規(guī)培養(yǎng)使用5.5 mmol/L低糖DMEM,加入10%FBS。用35mmol/L高糖(HG, High Glucose) DMEM培養(yǎng)基孵育細胞不同時間(6h,12h,24h,48h,72h),建立心肌細胞高糖損傷模型。2.細胞活力和凋亡檢測將細胞分為對照組(低糖5.5 mmol/L)、高糖組(35 mmol/L)、胰島素組(INS,100nmol/L)、胰島素+高糖組。用MTT法檢測細胞活力,分別觀察0,24h,48h,72h和96h并記錄數(shù)據(jù)。根據(jù)細胞活力的數(shù)據(jù)分析選擇48h時間點做凋亡檢測,實驗分組同上,Hoechst染色檢測細胞凋亡。3. RT-PCR檢測內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)分子標(biāo)記物的表達高糖處理H9c2細胞不同時間后,提取細胞總mRNA,檢測高糖對GRP78mRNA表達的時間效應(yīng)。根據(jù)時間效應(yīng)的結(jié)果,以48h為時間點,細胞分為4組：對照組(control),高糖組(HG),胰島素+高糖組,LY294002+胰島素+高糖組(LY294002預(yù)處理30min后,加入高糖和胰島素處理),檢測各組GRP78mRNA的表達。LY294002是PI3K/AKT信號通路特異性的抑制劑。4. Westen-blot檢測蛋白表達實驗分組：對照組(control),高糖組(HG),胰島素組,LY294002組,胰島素+高糖組,LY294002+胰島素+高糖組,48h后Western blot檢測各組細胞中t-Akt和p-Akt的蛋白表達水平。LY294002是PI3K/AKT信號通路特異性的抑制劑。結(jié)果：與對照組相比,高糖處理48h后,細胞活力下降(P0.5)；與高糖組比較,胰島素處理可以顯著提高細胞活力,細胞凋亡數(shù)也顯著減少；且48h時Hochest染色顯示,高糖組細胞核固縮,染色質(zhì)向外周聚集,或者細胞核碎裂成凋亡小體；RT-PCR結(jié)果提示高糖處理24h時,GRP78 mRNA水平表達被上調(diào)(P0.01 vs. control);與高糖組相比,胰島素+高糖組p-Akt蛋白的表達顯著增加(P0.01),且LY294002能明顯抑制胰島素引起的p-Akt的表達上調(diào)(P0.01)。結(jié)論高糖可通過內(nèi)質(zhì)網(wǎng)應(yīng)激引起心肌細胞凋亡,胰島素通過PI3K/AKT信號通路的活化,保護心肌細胞因高糖刺激引起的內(nèi)質(zhì)網(wǎng)應(yīng)激損傷。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R587.2

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