低分子肝素及轴突导向因子-1对人胎盘微血管内皮细胞生物学功能的影响

目的 研究低分子肝素及轴突导向因子-1(Netrin-1)对人胎盘微血管内皮细胞(microvascular endothelial cells,MECs)生物学功能的影响.方法 选取正常早孕(8～10周)绒毛,通过Percoll梯度离心法提取人胎盘微血管内皮细胞进行原代培养,将细胞分为6组,其中5组培养液中加入不同浓度低分子肝素(low molecular weight heparin,LMWH)(终浓度为0.01 U/ml、0.1 U/ml、1U/ml、10 U/ml、100 U/ml),未加低分子肝素的作为对照组.通过实时荧光定量-PCR和Western-Blot分别检测不同组别Netrin-1基因mRNA及蛋白表达情况;CCK-8检测不同组别细胞增殖率的变化;筛选出细胞增殖率最高组别进行流式细胞检测凋亡水平及细胞周期的变化;利用Transwell细胞迁移实验检测细胞侵袭和迁移能力的变化.结果 培养24 h 0.1U/ml、1U/ml组Netrin-1 mRNA及蛋白表达高于对照组(P＜0.05);10 U/ml、100 U/ml组Netrin-1 mRNA表达与对照组比较差异无统计学意义(P＞0.05),但蛋白表达低对照组(P＜0.05);培养48 h 0.1U/ml、1 U/ml、10 U/ml组Netrin-1 mRNA及蛋白表达均高于对照组;培养24 h 0.1 U/ml组MECs增值率高于对照组,100U/ml组细胞增值低于对照组;培养48 h 0.1U/ml、1U/ml、10 U/ml组细胞增值率高于对照组;0.1U/ml组培养24、48hS期细胞比例及细胞迁移数目高于对照组;0.1U/ml组培养24 h晚期细胞凋亡低于对照组(P＜0.05),各组细胞侵袭实验比较差异无统计学意义(P＞0.05).结论 适当浓度的低分子肝素可以促进人胎盘MECs的增殖,降低凋亡,迁移能力增高,这一变化可能与低分子肝素促进Netrin-1基因表达有关.

Modulation of low molecular weight heparin and Netrin-1 on biological functions of human placental microvascular endothelial cells

Objective To investigate the effect of low molecular weight heparin (LMWH) and Netrin-1 on biological functions of human placental microvascular endothelial cells (MECs).Methods Human placental MECs were collected and cultured aseptically from villi tissue of normal pregnant women who accepted artificial abortion in first trimester.Six groups were set,including LMWH 0.01 U/ml,0.1 U/ml,1 U/ml,10 U/ml,100 U/ml and control group without LMWH.mRNA and protein expression of Netrin-1 were detected by real-time fluorescent quantitative PCR and Western-Blot.Cell proliferation was detected by CCK-8 cellular proliferation assay,cell cycle distribution and apoptosis were detected by flow cytometry,cell migration and invasion were detected by Transwell assay.Results Netrin-1 mRNA and protein expression increased in 0.1 U/ml and 1 U/ml groups,but Netrin-1 protein expression decreased in 10 U/ml and 100 U/ml groups after 24 hours.Netrin-1 mRNA and protein expression increased in 0.1 U/ml,1 U/ml,10 U/ml groups after 48 hours.Proliferation indexes of 0.1 U/ml and 100 U/ml groups after 24 hours were significantly different from those of control group,and proliferation indexes of 0.1 U/ml,1 U/ml,10 U/ml groups after 48 hours were higher than those of control group (P＜0.05).Cell cycle assay showed that S phase of 0.1 U/ml group increased and migration ability enhanced (P＜0.05).Conclusions Proliferation and migration ability of human placental MECs enhanced,apoptosis decreased under proper concentration of LMWH.These biological changes were probably related to Netrin-1 expression,which could be modulated by LMWH.