【摘要】：目的建立缺血缺氧損傷細(xì)胞模型,探討永久性缺血缺氧致神經(jīng)細(xì)胞損傷機(jī)制及其對PC12細(xì)胞自噬的影響。方法采用經(jīng)典的神經(jīng)細(xì)胞模型PC12細(xì)胞作為研究對象,利用無糖的DMEM培養(yǎng)基和缺氧罐(95%N_2和5%CO_2)培養(yǎng)PC12細(xì)胞,模擬體內(nèi)神經(jīng)細(xì)胞缺血缺氧環(huán)境。細(xì)胞分為對照組(在正常條件下,使用完全培養(yǎng)基培養(yǎng)細(xì)胞)和糖氧剝奪(OGD)處理組(在缺氧條件下,使用OGD培養(yǎng)基培養(yǎng)細(xì)胞):0.5h(OGD+0.5h)、2h(OGD+2h)、6 h(OGD+6h)、12h(OGD+12h)和24h(OGD+24h)。利用MTT檢測細(xì)胞存活率,通過流式細(xì)胞儀檢測細(xì)胞凋亡率,比色法檢測細(xì)胞乳酸脫氫酶(LDH)釋放率,免疫熒光以及Western blotting法檢測低氧誘導(dǎo)因子-1α(HIF-1α)、環(huán)氧化酶-2(COX2)、微管相關(guān)蛋白輕鏈3(LC3)和Beclin-1的表達(dá),透射電子顯微術(shù)檢測自噬體的超微結(jié)構(gòu),探討不同永久性缺血缺氧時間對細(xì)胞損傷以及自噬的影響。結(jié)果與對照組相比,細(xì)胞存活率下降,呈OGD時間依賴性,且自O(shè)GD 6h顯著下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);細(xì)胞凋亡率和壞死率持續(xù)增高,與OGD時間呈正相關(guān),且自O(shè)GD 12h后顯著升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);HIF-1α和COX2蛋白表達(dá)隨OGD時間延長逐漸升高,OGD 12h后增高顯著,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。OGD組被熒光標(biāo)記的綠色的LC3蛋白相對于對照組,數(shù)量增多,綠色熒光增強(qiáng)。Beclin-1蛋白表達(dá)結(jié)果顯示,Beclin-1的表達(dá)與OGD時間呈正相關(guān),并由OGD6h開始明顯增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論缺血缺氧能夠誘導(dǎo)PC12細(xì)胞氧化應(yīng)激及自噬激活。
[Abstract]:Objective to establish a cell model of ischemia and hypoxia injury and to explore the mechanism of nerve cell injury induced by permanent ischemia and hypoxia and its effect on autophagy of PC12 cells. Methods the classical nerve cell model PC12 cells were used as the research object. PC12 cells were cultured in sugar-free DMEM medium and hypoxia tank (95%N_2 and 5%CO_2) to simulate the ischemic and anoxic environment of nerve cells in vivo. The cells were divided into two groups: control group (cultured in complete medium under normal conditions) and glucose-oxygen deprived (OGD) group (cultured in OGD medium under hypoxia): 0.5h (OGD 0.5h), 2h (OGD 2h), 6 h (OGD 6 h), 12 h (OGD 12 h) and 24 h (OGD 24 h). The cell survival rate was detected by MTT, the apoptosis rate was detected by flow cytometry, the release rate of lactic dehydrogenase (LDH) was detected by colorimetric assay, the expression of hypoxia inducible factor-1 偽 (HIF-1 偽), cyclooxygenase-2 (COX2), microtubule-associated protein light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence and Western blotting, and the ultrastructure of autophagy was detected by transmission electron microscopy. To investigate the effects of different permanent ischemia and hypoxia time on cell injury and autophagy. Results compared with the control group, the cell survival rate decreased in a OGD time-dependent manner, and decreased significantly since OGD 6 h, the difference was statistically significant (P 0.05), the apoptosis rate and death rate continued to increase, which was positively correlated with OGD time, and increased significantly after 12 h OGD, the difference was statistically significant (P 0.05). The expression of HIF-1 偽 and COX2 protein increased gradually with the prolongation of OGD time, and increased significantly after 12 hours of OGD, the difference was statistically significant (P 0.05). Compared with the control group, the number of green LC3 protein labeled with fluorescence increased and the green fluorescence increased. Beclin-1 protein expression showed that the expression of Beclin-1 was positively correlated with OGD time, and began to increase significantly by OGD6h, the difference was statistically significant (P 0.05). Conclusion ischemia and hypoxia can induce oxidative stress and autophagy activation in PC12 cells.
【作者單位】： 河南大學(xué)神經(jīng)生物學(xué)研究所;河南大學(xué)醫(yī)學(xué)院生物化學(xué)與分子生物學(xué)教研室;河南大學(xué)護(hù)理學(xué)院;
【基金】：國家自然科學(xué)基金(31070952,U1204311)
【分類號】：R743.3

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