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目的采用胰岛素体外诱导法建立胎盘滋养层细胞胰岛素抵抗模型为研究妊娠期糖尿病中的胰岛素抵抗提供一种可靠的细胞模型。方法采用含不同浓度的胰岛素的RPMI1640培养基作用于胎盘滋养层细胞,用葡萄糖-己糖激酶法检测上清中葡萄糖含量,并用CCK8检测不同时间、不同胰岛素浓度细胞生存率,结合葡萄糖消耗量及细胞生存率确定产生胰岛素抵抗最佳胰岛素作用浓度及作用时间;并以油红及HE染色观察细胞形态变化;模型建立后再次用用葡萄糖-己糖激酶法检测不同作用时间上清中葡萄糖含量,以确定胰岛素抵抗模型持续时间。结果细胞在胰岛素浓度为10~(-7)mol/L,作用时间为48h,葡萄糖消耗量最小;模型细胞与正常细胞相比增殖减少,脂滴明显增多;模型建立后在60h内,胰岛素抵抗仍然存在。结论细胞产生胰岛素抵抗的最佳作用浓度为10~(-7)mol/L,最佳作用时间为48h,此模型于60h内稳定。
Objective To establish a model of insulin resistance of trophoblast cells by insulin in vitro and to provide a reliable cell model for the study of insulin resistance in gestational diabetes mellitus. Methods RPMI1640 medium containing different concentrations of insulin was used to act on placental trophoblast cells. The glucose content in the supernatant was measured by glucose-hexokinase assay. The survival rates of cells at different time and different insulin concentrations were detected by CCK8. Combined with glucose consumption and Cell survival rate to determine the best insulin concentration insulin resistance and the role of time; and oil red and HE staining observed cell morphological changes; model was established again with glucose - hexokinase method to detect the role of different time supernatant glucose content, To determine the duration of the insulin resistance model. Results The cells in the insulin concentration of 10 ~ (-7) mol / L, the role of time was 48h, the smallest amount of glucose consumption; model cells compared with normal cells decreased proliferation, lipid droplets increased significantly; model established within 60h, insulin resistance still exists. Conclusion The optimal concentration of insulin producing cells is 10 -7 mol / L and the optimal time is 48 h. The model is stable within 60 h.