蛋白PTEN通过调节子宫内膜腔上皮细胞极性影响胚胎着床

PTEN affects embryo implantation by regulating the polarity of endometrial luminal epithelial cells

目的探讨人第10号染色体缺失的磷酸酶及张力蛋白同源基因(phosphatase and tensin homolog deleted on chromosome 10,PTEN)对子宫内膜腔上皮细胞极性的调控及对胚胎着床的影响。方法通过实时荧光定量PCR、Western blotting、细胞免疫荧光实验比较非容受态子宫内膜腔上皮细胞HEC-1A与容受态子宫内膜腔上皮细胞RL95-2之间PTEN的表达及定位差异;PTEN干扰质粒转染HEC-1A细胞,检测紧密连接相关蛋白质表达水平,透射电子显微镜检测紧密连接结构,Transwell实验检测细胞运动能力,与绒毛膜癌细胞JAR共培养检测HEC-1A细胞与JAR细胞之间的黏附水平;向体外培养的HEC-1A细胞分别加入二甲基亚砜、17β-雌二醇、孕酮、17β-雌二醇+孕酮,检测卵巢激素对PTEN表达的影响。结果相较HEC-1A细胞,RL95-2细胞PTEN基因及蛋白质表达水平均显著降低(P均=0.003);PTEN主要定位于RL95-2细胞核,而在HEC-1A细胞中,PTEN主要定位于细胞质;与质粒载体对照组相比,敲降PTEN基因后,HEC-1A细胞紧密连接相关蛋白ZO-1、Occludin和Claudin-4表达水平显著降低(P<0.001,P=0.038,P<0.001),细胞间紧密连接长度降低(P=0.046),迁移与侵袭能力增强(P均<0.001),与JAR细胞之间黏附率增强(P=0.016);与空白对照组(二甲基亚砜组)相比,17β-雌二醇组、孕酮组及17β-雌二醇+孕酮组的PTEN蛋白表达水平均显著降低(P均<0.001),17β-雌二醇+孕酮组的PTEN蛋白表达水平显著低于17β-雌二醇组和孕酮组(P均=0.001),孕酮组与雌二醇组的PTEN蛋白表达水平差异无统计学意义(P>0.05)。结论不同容受状态的子宫内膜细胞PTEN表达存在差异,雌二醇和孕酮可能通过抑制PTEN在子宫内膜的表达,进一步调控子宫内膜上皮细胞间紧密连接结构及细胞极性,从而增强子宫内膜容受性。

Objective To investigate the regulation of phosphoatase and tensin homolog deleted on chromosomes 10(PTEN)in the polarity of endometrial lumen epithelial cells and their effects on embryo implantation.Methods The differences in PTEN expression and localization between non-receptive endometrial epithelial cells(HEC-1A)and receptive endometrial epithelial cells(RL95-2)were compared by qRT-PCR,Western blotting and immunofluorescence.After the transfection of PTEN siRNA into HEC-1A cells,tight junction(TJ)related proteins,TJ structure,cell motility and adhesive capacity with choriocarcinoma cells(JAR)were detected respectively by Western blotting,transmission electron microscope(TEM),Transwell assay and adhesion assay.After dimethylsulfoxide(DMSO),17β-estradiol,progesterone,and 17β-estradiol+progesterone were respectively added into HEC-1A in vitro,the PTEN protein expression were detected by Western blotting to study the effect of ovarian hormone on PTEN.Results Compared with HEC-1A cells,the gene and protein expression levels of PTEN in RL95-2 cells were significantly reduced(both P=0.003),PTEN was mainly located in the nucleus of RL95-2 and cytoplasm of HEC-1A.Compared with the plasmid vector control group,the expression level of TJ related proteins(ZO-1,Occludin,Claudin-4)in HEC-1A cells was significantly reduced(P<0.001,P=0.038,P<0.001),the length of TJ between cells were reduced(P=0.046),the ability of migration and invasion were enhanced(both P<0.001),and the adhesion rate to JAR cells was enhanced after knockdown of PTEN in HEC-1A(P=0.016).Compared with the DMSO blank group,the expression level of PTEN protein in 17β-estradiol group,progesterone group and 17β-estradiol+progesterone group were significantly reduced(all P<0.001),the expression level of PTEN protein in 17β-estradiol+progesterone group was significantly lower than that in both 17β-estradiol group and progesterone group(both P=0.001),and there was no difference in the expression level of PTEN protein between progesterone group and 17β-estradiol group.Conclusion There are differences in the expression of PTEN in endometrial cells with different receptivity states.17β-estradiol and progesterone may regulate the TJ structure and cell polarity of endometrial epithelial cells by inhibiting the expression of PTEN in endometrial luminal epithelium,thereby enhancing the endometrial receptivity.