【摘要】：目的:不同濃度氟化鈉(Na F)對H9c2心肌細(xì)胞進(jìn)行染毒培養(yǎng),研究AMPK參與線粒體通路介導(dǎo)的氟對H9c2心肌細(xì)胞毒性損傷的機理。方法:H9c2心肌細(xì)胞經(jīng)不同濃度Na F作用48h和72h后,采用MTT法對細(xì)胞增殖進(jìn)行研究;運用HE染色法觀察心肌細(xì)胞的形態(tài)變化;采用流式細(xì)胞術(shù)觀測氟對細(xì)胞線粒體膜電位(ΔΨm)和細(xì)胞早期凋亡率的影響;實時熒光定量PCR法檢測凋亡途徑(線粒體途徑)中的重要基因(caspase-3、caspase-9和Cyt c)以及AMPKα的m RNA表達(dá)量;Western blot法檢測AMPKα蛋白磷酸化表達(dá)水平。結(jié)果:實驗結(jié)果顯示,低劑量的氟對細(xì)胞的增殖沒有明顯影響,隨Na F濃度的逐漸增大,氟對細(xì)胞增殖的抑制作用逐漸增強,細(xì)胞形態(tài)呈現(xiàn)較明顯的變化,細(xì)胞間的相互連接逐漸消失,細(xì)胞膜變得模糊不清。在20 mg/L Na F作用下,細(xì)胞核開始發(fā)生碎裂,胞核中染色質(zhì)聚集。在40mg/L Na F作用下,細(xì)胞間連接幾乎完全喪失,細(xì)胞外觀形態(tài)扭曲,輪廓模糊;經(jīng)Na F作用后,心肌細(xì)胞JC-1探針綠色熒光呈上升趨勢,說明氟會導(dǎo)致心肌細(xì)胞ΔΨm下降;細(xì)胞早期凋亡率隨Na F濃度的增大也呈上升趨勢,與ΔΨm的變化共同說明氟會導(dǎo)致H9c2心肌細(xì)胞發(fā)生凋亡;隨Na F劑量的增大,各實驗組caspase-3、caspase-9和Cyt c m RNA表達(dá)量與正常對照組比較均呈現(xiàn)上升趨勢;且心肌細(xì)胞AMPKαm RNA表達(dá)量逐漸增多;對照組心肌細(xì)胞p-AMPKα與AMPKα灰度值差別不是很大,隨Na F劑量的逐漸增大,p-AMPKα與AMPKα灰度值比值總體呈現(xiàn)上升趨勢。結(jié)論:細(xì)胞凋亡參與了氟誘導(dǎo)H9c2心肌細(xì)胞毒性的損傷過程;氟誘導(dǎo)H9c2心肌細(xì)胞凋亡的作用機制與激活線粒體通路有關(guān);AMPK參與了氟致H9c2心肌細(xì)胞的損傷過程。
[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 偽 were detected by real-time fluorescence quantitative PCR method. The phosphorylation of AMPK 偽 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 偽 m RNA in myocardial cells increased gradually, while the gray values of p-AMPK 偽 and AMPK 偽 in the control group were not significantly different. With the increasing of the dose of Na F, the ratio of p-AMPK 偽 to AMPK 偽 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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R54

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