AMPK参与线粒体通路介导的氟致H9c2心肌细胞凋亡机制的研究

发布时间：2018-10-22 11:36

【摘要】：目的:不同浓度氟化钠(Na F)对H9c2心肌细胞进行染毒培养,研究AMPK参与线粒体通路介导的氟对H9c2心肌细胞毒性损伤的机理。方法:H9c2心肌细胞经不同浓度Na F作用48h和72h后,采用MTT法对细胞增殖进行研究;运用HE染色法观察心肌细胞的形态变化;采用流式细胞术观测氟对细胞线粒体膜电位(ΔΨm)和细胞早期凋亡率的影响;实时荧光定量PCR法检测凋亡途径(线粒体途径)中的重要基因(caspase-3、caspase-9和Cyt c)以及AMPKα的m RNA表达量;Western blot法检测AMPKα蛋白磷酸化表达水平。结果:实验结果显示,低剂量的氟对细胞的增殖没有明显影响,随Na F浓度的逐渐增大,氟对细胞增殖的抑制作用逐渐增强,细胞形态呈现较明显的变化,细胞间的相互连接逐渐消失,细胞膜变得模糊不清。在20 mg/L Na F作用下,细胞核开始发生碎裂,胞核中染色质聚集。在40mg/L Na F作用下,细胞间连接几乎完全丧失,细胞外观形态扭曲,轮廓模糊;经Na F作用后,心肌细胞JC-1探针绿色荧光呈上升趋势,说明氟会导致心肌细胞ΔΨm下降;细胞早期凋亡率随Na F浓度的增大也呈上升趋势,与ΔΨm的变化共同说明氟会导致H9c2心肌细胞发生凋亡;随Na F剂量的增大,各实验组caspase-3、caspase-9和Cyt c m RNA表达量与正常对照组比较均呈现上升趋势;且心肌细胞AMPKαm RNA表达量逐渐增多;对照组心肌细胞p-AMPKα与AMPKα灰度值差别不是很大,随Na F剂量的逐渐增大,p-AMPKα与AMPKα灰度值比值总体呈现上升趋势。结论:细胞凋亡参与了氟诱导H9c2心肌细胞毒性的损伤过程;氟诱导H9c2心肌细胞凋亡的作用机制与激活线粒体通路有关;AMPK参与了氟致H9c2心肌细胞的损伤过程。
[Abstract]:Aim: to study the mechanism of AMPK involved in mitochondrial pathway mediated toxicity injury of H9c2 cardiomyocytes induced by fluorine at different concentrations of sodium fluoride (Na F). Methods: H9c2 cardiomyocytes were treated with different concentrations of Na F for 48h and 72h, the proliferation was studied by MTT method, the morphological changes of cardiomyocytes were observed by HE staining. The effects of fluoride on mitochondrial membrane potential (螖蠄 m) and early apoptosis rate were observed by flow cytometry. The expression of caspase-3,caspase-9 and Cyt c) in apoptosis pathway (caspase-3,caspase-9 and Cyt c) and m RNA expression of AMPK 伪 were detected by real-time fluorescence quantitative PCR method. The phosphorylation of AMPK 伪 protein was detected by; Western blot method. Results: the results showed that low dose fluoride had no obvious effect on cell proliferation. With the increase of Na F concentration, the inhibitory effect of fluoride on cell proliferation was gradually enhanced, and the cell morphology showed obvious changes. The interconnectedness between cells gradually disappeared, and the cell membrane became blurred. Under the action of 20 mg/L Na F, the nuclei began to break apart and the chromatin accumulated in the nucleus. Under the action of 40mg/L Na F, the intercellular junctions were almost lost, the appearance of the cells was distorted and the outline was blurred, the green fluorescence of the JC-1 probe of cardiomyocytes increased after Na F treatment, which indicated that fluoride could lead to the decrease of 螖蠄 m in cardiomyocytes. The rate of early apoptosis increased with the increase of Na F concentration, and the changes of 螖蠄 m showed that fluoride could induce apoptosis of H9c2 cardiomyocytes, and with the increase of Na F dose, the apoptosis rate of H9c2 cardiomyocytes was increased with the increase of Na F dose. Compared with the normal control group, the expression of caspase-3,caspase-9 and Cyt c m RNA in each experimental group showed an upward trend, and the expression of AMPK 伪 m RNA in myocardial cells increased gradually, while the gray values of p-AMPK 伪 and AMPK 伪 in the control group were not significantly different. With the increasing of the dose of Na F, the ratio of p-AMPK 伪 to AMPK 伪 showed an increasing trend. Conclusion: apoptosis is involved in the damage of H9c2 induced by fluorine, the mechanism of fluoride induced apoptosis of H9c2 is related to the activation of mitochondrial pathway, and AMPK is involved in the process of fluorine-induced injury of H9c2 cardiomyocytes.
【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】：R54

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