跨膜蛋白16A对胚胎着床和子宫内膜容受性的影响

Effect of transmembrane protein 16A on embryo implantation and endometrial receptivity

目的研究跨膜蛋白16A(TMEM16A)对子宫内膜容受性和胚胎着床的影响。方法体外培养子宫内膜Ishikawa细胞系,分为空白对照组、E_2+P_4组、抑制剂组和E_2+P_4+抑制剂组。分别加药处理后,采用实时荧光定量(qRT-PCR)和免疫印记试验(Western blot)技术检测各组TMEM16A、白血病抑制因子(LIF)和整合素αvβ3mRNA和蛋白的表达水平。采用体外细胞粘附实验检测E_2+P_4组和E_2+P_4+T16Ainh-A01组Ishikawa细胞与人绒毛膜滋养细胞HTR8-SVneo之间的粘附作用。结果 qRT-PCR和Western blot结果显示,E_2+P_4组的TMEM16A、LIF和整合素αvβ3mRNA和蛋白的表达水平最高,在加入T16Ainh-A01后3个基因的表达水平均显著降低(P<0.01),但均显著高于空白对照组(P<0.01);各组TMEM16A、LIF和整合素αvβ3表达水平均与空白对照组存在显著差异(P<0.01)。细胞粘附实验结果显示,E_2+P_4+抑制剂组中Ishikawa细胞与HTR8-SVneo细胞的粘附作用显著小于E_2+P_4组(P<0.05)。结论抑制TMEM16A的表达降低着床期LIF和整合素αvβ3的表达,可能影响子宫内膜容受性;抑制TMEM16A可降低子宫内膜细胞与滋养层细胞之间的粘附作用,可能导致胚胎着床失败。

Objective:To investigate the effect of transmembrane protein 16A on embryo implantation and endometrial receptivity.Methods:Ishikawa cells were cultured in vitro and incubated with estrogen combined progesterone(E2+P4 group),T16Ainh-A0 1(inhibitor group),estrogen combined progesterone added T16AinhA0 1(inhibitor+E2+P4 group),and 0.1%DMSO(control group).The expressions of mRNA and the protein of TMEM16A,leukemia inhibitory factor(LIF)and Integrinαvβ3 in Ishikawa cells were detected by Q-PCR and western blot.The adhesion was detected by cell adhesion assay in vitro both in E2+P4+T16Ainh-A0 1 group and E2+P4 group in Ishikawa cell line and HTR8-SVneo cell line.Results:Q-PCR and western blot showed that the expression levels of TMEM16A,LIF and Integrinαvβ3 mRNA and protein after treated with E2+P4 were significantly higher than other groups(P<0.01),and the expression of the three genes were significantly reduced after adding the T16Ainh-A01(P<0.01),but they were significantly higher than the control group(P<0.01).The expression levels ofTMEM16A,LIF and integrinαvβ3 in each group were significantly different from those in the blank control group(P<0.01).The results of cell adhesion experiment showed that the adhesion of Ishikawa cells to HTR8-SVneo cells in the E2+P4+inhibitor group was significantly less than that of the E2+P4 group(P<0.05).Conclusions:Inhibiting the expression of TMEM16Acan reduce the expression of LIF and Integrinαvβ3 in the implantation period,which may affect the receptivity of endometrium.Inhibition of TMEM16A can reduce the adhesion between endometrial cells and trophoblast cells,which may lead to embryo implantation failure.