摘要
目的探究醋酸铅对人绒毛外滋养层细胞(HTR-8/SVneo)增殖以及迁移与侵袭能力的影响,并从铅影响滋养层细胞凋亡与氧化应激的角度探讨铅导致流产的可能机制。方法用不同浓度的醋酸铅处理HTR-8/SVneo细胞,采用CCK-8法检测细胞增殖活性,最终选取100、200及400μmol/L三个浓度进行后续实验。使用细胞划痕实验及Transwell实验检测细胞的迁移侵袭能力;Western Blot分别检测细胞中侵袭迁移相关分子MMP-2、MMP-9的表达水平,凋亡相关分子Bax、Blc-2的表达水平以及氧化应激相关分子NQO1、HO1的表达水平;流式细胞术检测细胞凋亡水平及细胞内活性氧(ROS)含量;MDA试剂盒法检测细胞中丙二醛(MDA)的表达量。结果与对照组(0μmol/L醋酸铅)比较,25μmol/L以上醋酸铅对HTR-8/SVneo细胞增殖活性产生抑制作用(P<0.05),且抑制效果呈剂量依赖性。与对照组比较,各浓度醋酸铅(100、200及400μmol/L)处理后的HTR-8/SVneo细胞迁移距离和穿膜细胞数均显著减少(P<0.05),细胞迁移标志分子MMP-2、MMP-9蛋白的相对表达量显著下降(P<0.01),并呈剂量依赖性。与对照组比较,各浓度组醋酸铅(100、200及400μmol/L)处理后,HTR-8/SVneo细胞中的Bax蛋白相对表达量显著增加,而Bcl-2蛋白相对表达量显著降低(P<0.05);200μmol/L以上醋酸铅处理后的细胞中的抗氧化应激相关分子NQO1及HO1的蛋白表达量显著下降(P<0.01)。流式细胞仪检测结果显示,与对照组比较,各浓度醋酸铅处理后的HTR-8/SVneo细胞凋亡率均显著升高(P<0.05)、细胞内ROS及MDA水平均显著升高(P<0.01),并呈剂量依赖性。结论醋酸铅可导致HTR-8/SVneo细胞生长活力降低,侵袭迁移能力下降,凋亡水平增加,并导致氧化应激损伤,这些作用可能参与了流产的发病机制。
Objective:To explore the effect of lead acetate on the proliferation,migration and invasion ability of human extravillous trophoblast cells(HTR-8/SVneo),and to explore the possible mechanism of lead induced abortion from the perspective of the effect of lead on trophoblast cell apoptosis and oxidative stress level.Methods:HTR-8/SVneo cells were treated with different concentrations of lead acetate,and the cell proliferation activity was detected by cell counting kit-8(CCK-8)method.Finally,three concentrations of 100,200 and 400μmol/L were selected for subsequent experiments.The cell scratch test and Transwell assay were used to detect the migration and invasion ability of cells.The expression levels of the migration and invasion related molecules including matrix metalloproteinase(MMP)-2 and MMP-9,the protein expression levels of apoptosis related molecules regarding Bax and blc-2,and the expression levels of oxidative stress related molecules including the NAD(P)H:quinone oxidoreductase 1(NQO1)and heme oxygenase 1(HO1)were detected by Western blot.The levels of apoptosis and the concentration of intracellular reactive oxygen species(ROS)were detected by flow cytometry.The expression of malondialdehyde(MDA)in cells was detected with the MDA kit.Results:Compared with the control group with 0μmol/L of lead acetate,25μmol/L of lead acetate could inhibit the proliferation activity of HTR-8/SVneo(P<0.05),and the inhibitory effect increased significantly with the increase of concentration.Compared with the control group,both the migration distance and the number of transmembrane cells of HTR-8/SVneo cells were significantly reduced(P<0.05),and the relative expression levels of cell migration marker molecules regarding MMP-2 and MMP-9 proteins significantly decreased in the treatment groups with 100,200 and 400μmol/L of lead acetate(P<0.01),showing dose dependence.The relative expression levels of Bax protein were significantly higher and the levels of Bcl-2 protein were significantly lower in the treatment groups with 100,200 and 400μmol/L of lead acetate than those in the control group(P<0.05).After the treatment with 200μmol/L of lead acetate,the expression levels of anti-oxidative stress related molecules including NQO1 and HO1 proteins significantly decreased(P<0.01).The results of flow cytometry showed that the apoptosis rates of HTR-8/SVneo cells were significantly higher(P<0.05)and the levels of intracellular ROS and MDA were significantly lower(P<0.01)in all of the treatment groups than those in the control group,showing dose dependence.Conclusions:Lead acetate can reduce the growth viability,invasion and migration ability of HTR-8/SVneo cells,increase the level of apoptosis,and lead to oxidative stress injury.These effects may be involved in the pathogenesis of abortion.
作者
欧向阳
周梦琪
杨哲
张琪璇
吴庚香
OU Xiang-yang;ZHOU Meng-qi;YANG Zhe;ZHANG Qi-xuan;WU Geng-xiang(The Reproductive Medicine Center,Renmin Hospital of Wuhan University,Wuhan 430060)
出处
《生殖医学杂志》
2025年第7期961-968,共8页
Journal of Reproductive Medicine
基金
国家自然科学基金项目(82101749)。
关键词
醋酸铅
滋养层细胞
凋亡
氧化应激
流产
Lead acetate
Trophoblast cells
Apoptosis
Oxidative stress
Abortion
作者简介
欧向阳,男,湖南怀化人,硕士研究生,生殖医学专业;通讯作者:吴庚香。
