血小板反应蛋白-1在PCOS患者中对卵巢血流调控作用及其对人卵巢微血管内皮细胞增殖能力的影响

Effect of thrombospondin-1 on ovarian blood flow regulation in PCOS patients and proliferation of human ovarian microvascular endothelial cells

目的探讨PCOS患者血清中血小板反应蛋白-1(TSP-1)表达水平与卵巢间质血供之间的关系,以及TSP-1对体外培养的人卵巢微血管内皮细胞(HOMEC)增殖能力的影响。方法选取2018年10月至2019年10月在我院生殖中心就诊的女性,50例诊断为PCOS的患者纳入PCOS组,50例因男方因素不孕的健康女性纳入对照组。ELISA法用于测定所有入组患者血清中的TSP-1水平。在患者处于卵泡早期时,经阴道超声测量双卵巢间质血供,计算血流动力学参数搏动指数(PI)和阻力指数(RI)。体外培养HOMEC,在不同浓度TSP-1(10^(-9) mmol/L、10^(-10) mmol/L和10^(-11) mmol/L)处理细胞12 h、24 h、48 h后,用CCK-8检测细胞增殖情况。结果PCOS组血清LH、T水平及LH/FSH均显著高于对照组(P<0.05)。与对照组比较,PCOS组血清TSP-1水平显著降低(P<0.05),卵巢间质血流动力学参数RI、PI值均显著降低(P<0.05)。Pearson相关性分析发现血清TSP-1水平与卵巢间质血流RI、PI均呈显著正相关(分别为r=0.83,P<0.05;r=0.85,P<0.05)。CCK-8实验显示,与对照组比较,不同浓度(10^(-11) mmol/L、10^(-10) mmol/L、10^(-9) mmol/L)TSP-1作用12 h、24 h、48 h后,对HOMEC增殖的抑制率均显著升高(P<0.05)。在TSP-1各浓度组,TSP-1作用24 h、48 h对HOMEC的抑制率均较作用12 h显著升高(P<0.05),且24 h时达到峰值。结论PCOS患者血清中TSP-1表达降低,卵巢间质血流动力学参数RI、PI值显著降低,卵巢间质血流量丰富,且TSP-1与RI、PI值呈正相关。TSP-1抑制HOMEC增殖,可能参与负调控血管生成。

Objective:To investigate the relationship between the expression level of serum thrombospondin-1(TSP-1)and ovarian stromal blood flow in patients with PCOS,and the effect of TSP-1 on the proliferation of human ovarian microvascular endothelial cell(HOMEC)in vitro.Methods:The female patients were selected in the Reproductive Center of the Second Affiliated Hospital of Harbin Medical University from October 2018 to October 2019.Fifty patients diagnosed with PCOS were included in the PCOS group,and 50 healthy women with infertility due to male factors were included in the control group.ELISA method was used to determine the serum level of TSP-1 in all patients of the groups.When the patients were in the early follicular phase,the stromal blood supply of both ovaries was measured by transvaginal ultrasound,and the hemodynamic parameters pulsatility index(PI)and resistance index(RI)were calculated.Human ovarian microvascular endothelial cells(HOMECs)were cultured in vitro and treated with different concentrations of TSP-1(10^(-9),10^(-10) and 10^(-11) mmol/L)for 12,24 and 48 hours,the cell proliferation was detected by CCK-8.Results:The levels of serum LH&testosterone and LH/FSH in PCOS group were significantly higher than those in control group(P<0.05).Compared with the control group,the expression level of TSP-1 in serum of PCOS group was significantly lower(P<0.05).The RI and PI values of ovarian stroma in PCOS group were significantly lower than those in control group(P<0.05).Pearson correlation analysis showed that serum TSP-1 were positively correlated with RI(r=0.83,P<0.05)and PI(r=0.85,P<0.05)in PCOS patients.CCK-8 assay showed that the inhibitory rates of different concentrations(10^(-11),10^(-10),10^(-9) mmol/L)of TSP-1 on the proliferation of HOMECs increased significantly after 12,24 and 48 hours compared with the control group(P<0.05).In each TSP-1 concentration group,the inhibitory rate of TSP-1 on HOMECs at 24 hours and 48 hours was significantly higher than that at 12 hours(P<0.05),and reached the peak at 24 hours.Conclusions:The expression of TSP-1 in serum of patients with PCOS is decreased,and the values of RI and PI are significantly decreased.The ovarian stromal blood flow is abundant,and TSP-1 is positively correlated with RI and PI.TSP-1 inhibits the proliferation of HOMECs and may be involved in negative regulation of angiogenesis.