人胎盘滋养细胞HTR-8/SVneo胰岛素抵抗模型的建立

Establishment of placental trophoblastic cells model of insulin resistance

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 the placental trophoblastic cells model of insulin resistance by the method of insulin induced in vitro,and provide a reliable model in vitro for the study of insulin resistance in gestational diabetes. Methods：RPMI 1640 medium with different insulin concentrations was used to the placental trophoblastic cells,the medium glucose content was detected at different time points by the method of glucose-hexokinase and the placental trophoblastic cell viability of different insulin concentrations was detected by the method of CCK8,and to determine the optimal concentration and time of insulin resistance;then the changes of cell morphology was observed by the oil red O and HE staining;on the basis of the established model the method of glucose-hexokinase was used to detect the consumption of glucose at different time. Results：Placental trophoblastic cells consume the least glucose in 10^（-7）mol/L insulin concentration at 48h;after the establishment of the model,the proliferation rate is reduced and the lipid droplets of model cells was significantly increased compared with normal cells;the insulin resistance still exist in 60 h.Conclusion：By the method of insulin-induced in vitro,a stable placental trophoblastic cells model of insulin resistance can be established when the 10^（-7）mol/L insulin concentration act on cells at 48 h and the insulin resistance model can be stable in 60 h.