探讨人绒毛膜促性腺激素(hCG)影响胚胎体外着床的分子调控机制。

选择人滋养层细胞株BeWo及人子宫内膜细胞株RL95-2为研究材料。将BeWo细胞与RL95-2细胞共培养，以制备体外胚胎着床模型。对该模型进行如下研究。①通过间接免疫荧光方法，观察整合素β₃及其配体骨桥蛋白(OPN)在BeWo细胞的定位表达；②采用逆转录聚合酶链反应(RT-PCR)技术，分析不同含量hCG作用于BeWo细胞后，对BeWo细胞整合素β₃及OPN的mRNA相对表达量影响；③通过BeWo球状体黏附实验和铺展实验，观察hCG作用于BeWo细胞后，对BeWo球状体在RL95-2单层细胞上黏附和植入的影响。采用单因素方差分析或t检验，对不同含量hCG作用于BeWo细胞后的BeWo细胞整合素β₃及骨桥蛋白mRNA相对表达量，以及hCG处理前、后BeWo球状体黏附率及铺展倍数进行统计学比较。

①整合素β₃及OPN均在BeWo细胞的细胞膜表面表达。②1 000 IU/mL hCG作用于BeWo细胞后，整合素β₃及OPN的mRNA相对表达量分别为(2.07±0.57)、(1.95±0.48)，均高于10 IU/mL hCG作用于BeWo细胞后的(1.12±0.31)、—(8.44±0.18)；1 000 IU/mL hCG作用于BeWo细胞后，整合素β₃的mRNA相对表达量，高于100 IU/mL hCG作用于BeWo细胞后的(1.24±0.19)，并且上述差异均有统计学意义(P＝0.007、0.009、0.014)。③采用1 000 IU/mL hCG处理BeWo细胞后，在BeWo球状体＋RL95-2单层细胞共培养0.5 h时的BeWo球状体黏附率为(89.51±6.47)%，显著高于无hCG处理的(50.24±7.73)%，并且差异有统计学意义(t＝90.900、P<0.001)。④采用1 000 IU/mL hCG处理BeWo球状体＋RL95-2单层细胞，共培养24 h时BeWo球状体铺展倍数为(2.28±0.52)，高于无hCG处理的铺展倍数(1.93±0.36)，并且差异有统计学意义(t＝6.864、P＝0.012)。

hCG可能通过上调滋养层细胞表面整合素β₃及OPN的表达水平，促进胚胎体外着床。

To explore the molecular regulatory mechanism of human chorionic gonadotropin (hCG) effect on embryo implantation in vitro.

Human trophoblast cell line BeWo and human endometrial cell line RL95-2 were selected as study cell lines. BeWo cells and RL95-2 cells were co-cultured to establish an in vitro embryo implantation model. Based on this model, the following research was carried out. ① The expression locations of integrin β₃ and its ligand osteopontin (OPN) in BeWo cells were observed by indirect immunofluorescence; ② The effects of different contents of hCG on the relative expressions of integrin β₃ and OPN in BeWo cells were analyzed through reverse transcription polymerase chain reaction (RT-PCR) technique; ③ The effects of hCG on the adhesion and implantation of BeWo spheroids to RL95-2 monolayer were observed by BeWo spheroids adhesion and outgrowth assays. The differences of relative expressions of integrin β₃ and OPN mRNA were compared among different contents of hCG treated to BeWo cell, also the adhesion rates and expansion times of BeWo spheroids were compared between before and after hCG treatment by one-way ANOVA or t test.

① Integrin β₃ and OPN were both expressed on the membrane surface of BeWo cells. ② The relative mRNA expression levels of integrin β₃ and OPN were (2.07±0.57) and (1.95±0.48), respectively, after BeWo cells being treated with 1 000 IU/mL hCG, which were both higher than those of (1.12±0.31) and —(8.44±0.18), respectively, after BeWo cells being treated with 10 IU/mL hCG; Also, the relative mRNA expression level of integrin β₃ after BeWo cells being treated with 1 000 IU/mL hCG was higher than that of (1.24±0.19) after BeWo cells being treated with 100 IU/mL hCG, and the differences above were statistically significant (P=0.007, 0.009, 0.014). ③ After BeWo cells being treated with 1 000 IU/mL hCG, the adhesion rate of BeWo globules co-cultrued with RL95-2 monolayers for 0.5 h was (89.51±6.47)%, which was significantly higher than that of (50.24±7.73)% of BeWo cells without being treated with hCG, and the difference was statistically significant (t=90.900, P<0.001). ④ After BeWo spheroids and RL95-2 monolayers being treated with 1 000 IU/mL hCG for 24 hours, the expansion times of BeWo spheroids was (2.28±0.52), which was higher than that of (1.93±0.36) without being treated with hCG, and the difference was statistically significant (t=6.864, P=0.012).

Being treated with hCG may promote embryo implantation in vitro by up-regulating the expression of integrin β₃ and OPN on trophoblast surface.