摘要
目的：本实验通过研究不同浓度的同型半胱氨酸（homocysteine,HCY）对人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）增殖和细胞周期的影响,分析可能的细胞损伤机制,探讨 HCY对细胞增殖影响的相关信号通路。同时对叶酸（folic acid,FA）对 HCY 的拮抗作用进行论证,为临床治疗提供理论依据。方法正常人脐静脉内皮细胞体外培养,随机分成6组：HCY低剂量组（1μmol/L）,HCY 中剂量组（10μmol/L）,HCY 高剂量组（100μmol/L）,实验组（10μmol/L HCY＋100μmol/L叶酸）,叶酸组（100μmol/L）及阴性对照组。以 CCK-8法绘制相关组细胞生长曲线；用碘化丙啶单染法检测各组相关的细胞周期；Western-blotting检查周期相关蛋白表达。结果①CCK-8法绘制生长曲线显示,10μm HCY 吸光度明显抑制 HUVECs 的生长,而加入100μM FA后能改善这种抑制作用。②细胞周期分析显示：HCY 作用24 h 后,G0-G1期细胞百分比有明显增加,而 FA 能改善这种作用,表明 HCY 可能通过影响细胞周期的 R 点,使细胞停留在 G0-G1期；高浓度 HCY明显抑制了 S期,但没有明显规律；G2-M期随 HCY 浓度梯度增加,细胞百分比减少,而 FA能改善这种抑制作用。说明 HCY可能也影响了细胞的 M期的分裂过程。③周期相关蛋白表达结果显示 HCY 显著抑制 CDK 4/6的表达量,使细胞阻滞在 G0-G1期。结论 HCY 可通过对细胞周期的抑制作用来抑制 HUVECs的增殖,而叶酸对这种抑制作用具有拮抗作用。HCY 能破坏细胞DNA,使细胞被阻滞在 G1期,同时进入 S、M期的细胞减少。
Objective To study the possible injury mechanism of homocysteine （HCY）on hu-man umbilical vein endothelial cells （HUVECs）,explore the related signal pathways through which HCY affected HUVECs proliferation,and investigate the antagonistic effect of folic acid （FA）on HCY.Methods HUVECs were cultured in vitro and then randomized into six groups：low dose HCY （1μmol/L）,moderate dose HCY （10 μmol/L）,high dose HCY （100 μmol/L）and HCY＋ FA （HCY 10μmol/L,and FA 100μmol/L）,FA （100μmol/L）,and negative control group.We applied CCK-8 Law growth curve to analyze the effects of HCY on the cell cycle.The propidium iodide single staining was used to detect the related cell cycles.Western blotting was done to detect cell cycle relat-ed protein expression.Results （1 ）CCK-8 growth curve showed that the growth of HUVECs was significantly inhibited in the moderate HCY group,which was antagonized by FA （100μmol/L）;（2） Single propidium iodide staining of cell cycle analysis revealed that the percentage of cells in G0-G1 phase was increased significantly 24 h after HCY intervention,which was antagonized by FA,sugges-ting that HCY could arrest the cells in G0-G1 phase by affecting R point of the cell cycle.High concen-trations of HCY significantly inhibited the Sphase,but no regular rule was seen.The percentage of cells in G2-M phase was decreased as HCY concentration increased,which could be antagonized by FA,suggesting HCY might also affect the M phase of the cell division process.HCY significantly in-hibited the expression of CDK4/6 and arrested the cells in the G0-G1 phase and indirectly increased Cy-clinD1 content.Conclusions HCY injured HUVECs by influencing the cell cycle,and FA has a pro-tective effect against HCY.HCY injured cell DNA,arrested cells in the G1 phase and decreased the S and M phase cells.
出处
《临床肾脏病杂志》
2014年第11期686-690,共5页
Journal Of Clinical Nephrology
基金
深圳市福田区卫生公益性科研项目（NO.FTWS201203）
关键词
同型半胱氨酸
人脐静脉内皮细胞
细胞周期
叶酸
Homocysteine Human umbilical vein endothelial cells Cell cycle Folic acid
