卵巢细胞系糖脂代谢紊乱模型的构建及机制探索

Establishment of a Model of Glucolipid Metabolism Disorder in Ovarian Cell Line and Its Mechanism

目的通过在培养基中加入葡萄糖及油酸,模拟糖脂代谢紊乱时体内高糖高油酸微环境,建立中国仓鼠卵巢(CHO)细胞代谢紊乱模型并探究作用机制。方法培养卵巢细胞系CHO细胞,将细胞分为4组:对照组(C组)、单独高油酸模型组(O组)、单独高糖模型组(G组)、高糖高油酸联合模型组(GO组),采用25 mmol·L^(-1)的葡萄糖与62.5μmol·L^(-1)的油酸,分别诱导不同时长,选择最佳处理时间和药物处理方式,建立细胞模型,通过CCK-8检测细胞增殖水平,倒置显微镜观察模型细胞形态变化,Western blot、免疫荧光染色等方式检测半胱氨酸-天冬氨酸蛋白酶-3(Caspase-3)、增殖细胞核抗原(PCNA)蛋白表达,通过磷酸化腺苷酸活化蛋白激酶(p-AMPK)以及磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的蛋白表达,初步探索其机制。结果HE染色可见,与C组相比,O组、G组和GO组细胞数量均减少(P均<0.05),且细胞形态由长梭形皱缩变圆;CCK-8结果显示,高糖高油酸联合处理24 h细胞增殖水平变化最明显,且与C组相比,O组、G组和GO组在24 h时细胞增殖率均下降(P均<0.05);对细胞蛋白进行Western blot实验,与C组相比,O组、G组和GO组CHO细胞Caspase-3蛋白表达增加,G组及GO组PCNA蛋白表达减少(P均<0.05)。细胞免疫荧光染色结果亦显示,与C组相比,GO组细胞Caspase-3的表达增加(P<0.05),PCNA表达降低(P<0.05)。与C组相比,O组、G组及GO组CHO细胞中p-AMPK蛋白表达减少,G组p-mTOR蛋白表达增加(P均<0.05)。结论给予CHO细胞高油酸、高糖和高糖高油酸联合处理均可以导致细胞处于代谢紊乱微环境,并用作研究糖脂代谢紊乱对CHO细胞作用的体外模型。

Objective By adding glucose and oleic acid to the culture medium to simulate the high glucose and high oleic acid microenvironment during glucose and lipid metabolism disorder,the model of Chinese hamster ovary(CHO)cell metabolism disorder was established and the mechanism of action was explored.Methods Cultured ovarian cell line CHO cells,which were divided into four groups:control group(C group),separate high oleic acid model group(O group),separate high glucose model group(G group),high glucose and high oleic acid combined model group(GO group),then inducted by 25 mmol·L^(-1) of glucose and 62.5μmol·L^(-1) of oleic acid(oleic acid,OA)for 24 h.With varying induction,the optimal treatment time and drug treatment were selected to establish cell models.Cell proliferation levels were measured by CCK-8,and model cell morphology was observed by inverted microscopy.The expression of cysteine containing aspartate specific protease(Caspase-3)and proliferating cell nuclear antigen(PCNA)protein were detected by Western blot and immunofluorescence staining.The mechanism was initially explored by phosphorylated adenosine-activated protein kinase(p-AMPK)and phosphorylated mammalian rapamycin target(p-mTOR).Results HE staining showed that the numbers of cells were significantly decreased in groups O,G,and GO compared to group C(P all<0.05).And the cell morphology was rounded from the long spindle shape;CCK-8 showed that the cell viability change was most evident with high glucose and high oleic acid combination treatment for 24 h.Moreover,compared with group C,groups O,G and GO decreased at 24 h(P all<0.05);In Western blot experiments on cellular proteins,Caspase-3 protein expression was increased in groups O,G and GO compared with group C,and PCNA protein expression was decreased in groups G and GO compared with group C(P all<0.05).Cell immunofluorescence staining results also showed that,in comparison to group C,the expression of Caspase-3 increased significantly in the group GO(P<0.05).However,the PCNA expression was significantly reduced(P<0.05).Compared with group C,that p-mTOR protein expression was increased in G group,and the p-AMPK protein expression was decreased in the groups O,G and GO(P all<0.05).Conclusion Treatment of CHO cells with high oleic acid,high glucose,high glucose and high oleic acid combination can lead to cells in a metabolic disorder microenvironment and be used as an in vitro model to study the effect of glucose and lipid metabolism disorders on CHO cells.