Objective: To investigate the effect and mechanism of tumor necrosis factor α (TNF-α) and its inhibitor etanercept (ETA) on the invasion ability of extravillous trophoblast in patients with unexplained recurrent spontaneous abortion (URSA). Methods: (1) Patients were collected from March to June in 2019. They were divided into the URSA group (n=15) and the normal control group (n=15), according to whether diagnosed with URSA or not. The mRNA expression levels of TNF-α in villi tissue of patients in the two groups were detected by quantitative real-time PCR (qRT-PCR). (2) The mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2), Slug and CXC chemokine rceptor 4 (CXCR4) in HTR-8/SVneo cells were detected by qRT-PCR or western blot after being stimulated by exogenous TNF-α (0.2, 2, 20 ng/ml) alone or TNF-α along with ETA, or phosphate buffered saline (PBS) as control. (3) The invasion ability of HTR-8/SVneo cells was investigated by transwell test after stimulating by TNF-α alone or TNF-α along with ETA. (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells, which were stimulated by TNF-α (2 ng/ml) alone after nuclear factor-κB (NF-κB) inhibitor, BAY 11-7028, preconditioning, were detected by qRT-PCR or western blot. Results: (1) The mRNA expression level of TNF-α in villi tissue of URSA group (4.10±0.49) was 4.1 times as much as the normal control group (t=10.51, P<0.05). (2) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells of TNF-α group were significantly lower than those in PBS control group (P<0.05) and those in TNF-α along with ETA group (P<0.05). (3) The invasion ability of HTR-8/SVneo cells in TNF-α group was significantly decreased than PBS group and TNF-α along with ETA group (78±14 vs 373±26 vs 227±44, P<0.05). (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells with BAY 11-7028 preconditioning (mRNA: 1.03±0.10, 1.03±0.06, 1.09±0.08; protein: 1.09±0.03, 1.49±0.03, 1.12±0.03) were significantly higher than without preconditioning after being stimulated by TNF-α (all P<0.05). Conclusions: The expression of TNF-α in the villi of URSA patients is much higher than normal early pregnant women. TNF-α could decrease the capacity of invasion by suppressing the expression of MMP-2, Slug and CXCR4 through NF-κB signaling pathway in extravillous trophoblast cells. While ETA could improve the invasiveness capability of extravillous trophoblast cells through inhibiting the negative effect of TNF-α.
目的： 探讨肿瘤坏死因子α（TNF-α）及其抑制剂依那西普（ETA）对原因不明复发性流产（URSA）患者绒毛外滋养细胞侵袭力的调控作用及机制。 方法： （1）选取2019年3—6月就诊于北京大学第一医院的URSA患者（n=15）为URSA组，同期正常早孕期人工流产妇女（n=15）为对照组，用实时荧光定量PCR（qRT-PCR）技术检测两组绒毛组织中TNF-α mRNA的表达水平。（2）体外培养绒毛外滋养细胞系HTR-8/SVneo，使用TNF-α（0.2、2、20 ng/ml）单独刺激HTR-8/SVneo细胞以及TNF-α（2 ng/ml）联合ETA（3 μg/ml）共同刺激HTR-8/SVneo细胞，以磷酸盐缓冲液（PBS）为对照，通过qRT-PCR技术和蛋白印迹法（western blot）检测侵袭因子基质金属蛋白酶2（MMP-2）、锌指转录因子Slug和CXC型趋化因子受体4（CXCR4）mRNA和蛋白的表达水平。（3）通过细胞侵袭实验检测TNF-α及其抑制剂ETA对HTR-8/SVneo细胞侵袭力的影响。（4）核因子κB（NF-κB）抑制剂BAY 11-7082预处理HTR-8/SVneo细胞后再加入TNF-α（2 ng/ml），通过qRT-PCR技术和western blot检测MMP-2、Slug和CXCR4 mRNA和蛋白的表达水平。 结果： （1）URSA组患者绒毛组织中TNF-α mRNA的表达水平（4.10±0.49）显著升高，是对照组的4.1倍，两组比较，差异有统计学意义（t=10.51，P<0.05）。（2）TNF-α（0.2、2、20 ng/ml）单独刺激HTR-8/SVneo细胞，MMP-2、Slug和CXCR4 mRNA和蛋白的表达水平较PBS对照细胞显著下降（P均<0.05）。TNF-α+ETA共同刺激HTR-8/SVneo细胞时，MMP-2、Slug和CXCR4 mRNA和蛋白的表达水平较TNF-α单独刺激时显著升高（P均<0.05）。（3）TNF-α刺激HTR-8/SVneo细胞的侵袭细胞数［（78±14）个］较PBS对照［（373±26）个］显著下降（P<0.05），而TNF-α+ETA共同刺激HTR-8/SVneo细胞的侵袭细胞数［（227±44）个］显著高于TNF-α单独刺激时（P<0.05）。（4）加入NF-κB抑制剂BAY 11-7082预处理HTR-8/SVneo细胞后再以TNF-α刺激，MMP-2、Slug、CXCR4 mRNA和蛋白的表达水平（mRNA：1.03±0.10、1.03±0.06、1.09±0.08，蛋白：1.09±0.03、1.49±0.03、1.12±0.03）均较TNF-α单独刺激时显著升高（P均<0.05）。 结论： URSA患者绒毛组织中TNF-α的表达水平较正常早孕期人工流产妇女明显升高，TNF-α可以通过NF-κB信号通路抑制侵袭因子MMP-2、Slug和趋化因子受体CXCR4的表达，降低绒毛外滋养细胞的侵袭力；而ETA可以通过抑制TNF-α对MMP-2、Slug和CXCR4的降调作用来改善TNF-α对于绒毛外滋养细胞侵袭力的抑制作用。.