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物理方法构建肝细胞自噬模型
Hepatocyte autophagy model established by physical method
文章发布日期:2016年07月06日  来源:  作者:朱学敏,孟庆华  点击次数:1465次  下载次数:317次

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【摘要】:目的应用物理方法构建正常人肝细胞株7702细胞的低氧饥饿自噬模型,为研究细胞自噬对肝脏功能的影响机制奠定基础。方法选用7702细胞,使用完全DMEM培养基,在37 ℃和5%CO2培养箱中培养(正常对照组),使用Binder三气培养箱,设置温度为37 ℃,CO2浓度为5%、O2浓度为0.3%来提供缺氧环境,使用无血清DMEM培养细胞来诱导饥饿,将其分为低氧饥饿6、12、18和24 h 4个时间组。用Western Blot检测正常对照组和不同时间点实验组的Beclin-1、Atg5以及LC3蛋白表达水平的变化;通过实时荧光定量PCR法检测各组Beclin-1 mRNA、Atg5 mRNA的表达;转染LC3质粒后利用免疫荧光法观察各组细胞发生自噬的情况。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验;计数资料组间比较采用χ2检验。结果低氧饥饿处理6 h组的Beclin-1、Atg5及LC3的蛋白表达量高于正常对照组及其他处理组;低氧饥饿6 h组较其他各组的Beclin-1 mRNA和Atg5 mRNA的表达量显著增高,且其增高最显著,差异均有统计学意义(P值均<0.05);低氧饥饿组自噬小体数量均高于正常对照组,其中低氧饥饿6 h组的自噬小体数量最高,差异均有统计学意义(P值均<0.05)。结论通过物理方法构建的低氧饥饿可以诱导7702细胞发生自噬,并且在低氧饥饿6 h时,细胞发生自噬最为明显。
【Abstract】:ObjectiveTo establish the autophagy model of normal human liver cell line 7702 induced by hypoxia and starvation, and to lay a foundation for further studies on the influence of autophagy on liver function. MethodsThe 7702 cells were selected and incubated with 95% air and 5% CO2 at a temperature of 37 ℃(normal control group). The Binder three-gas incubator was used, with a temperature of 37 ℃, a CO2 concentration of 5%, and an O2 concentration of 0.3% to provide a hypoxic environment, and the serum-free DMEM was used to induce starvation. These cells were divided into 6-, 12-, 18-, and 24-hour hypoxia-starvation groups. Western blot was used to measure the protein expression of Beclin 1, Atg5, and LC3 in the normal control group and experimental groups, RT-qPCR was used to measure the mRNA expression of Beclin 1 and Atg5 in each group, and after transfection of LC3 plasmid, immunofluorescence assay was used to observe autophagy in each group. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for further comparison between any two groups; the chi-square test was used for comparison of categorical data between groups. ResultsThe 6-hour hypoxia-starvation groups had higher protein expression of Beclin 1, Atg5, and LC3 than the normal control group or other treated groups. Compared with all the other groups, the 6-hour hypoxia-starvation group showed significantly increased mRNA expression of Beclin 1 and Atg5, as well as significantly greater increases in the mRNA expression of Beclin 1 and Atg5 (all P<0.05). The hypoxia-starvation groups had significantly lower numbers of autophagosomes than the normal control group, and the 6-hour hypoxia-starvation group had the highest number of autophagosomes (all P<0.05). ConclusionHypoxia and starvation established by physical methods can successfully induce hepatocyte autophagy, which is the most remarkable at 6 hours of hypoxia and starvation.
【关键字】:肝疾病; 自噬; 模型, 生物学
【Key words】:liver diseases; autophagy; models, biological
【引证本文】:朱学敏, 孟庆华. 物理方法构建肝细胞自噬模型[J]. 临床肝胆病杂志, 2016, 32(8): 1566-1570.

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