氧化型低密度脂蛋白诱导大鼠血管平滑肌细胞增殖及周期素D1的表达

背景：氧化型低密度脂蛋白可通过Ras/Raf/丝裂原活化蛋白激酶激酶/丝裂原活化蛋白激酶途径诱导血管平滑肌细胞增殖及细胞周期蛋白的表达。目的：观察胞外信号调节激酶是否参与了氧化型低密度脂蛋白诱导的血管平滑肌细胞周期推进以及周期素D1的表达。设计、时间及地点：对照观察细胞学实验，于2008-02/10在广州医学院完成。材料：人血浆低密度脂蛋白来自健康志愿者血清。SD大鼠由广州医学院实验动物中心提供。方法：采用超速离心法分离人低密度脂蛋白进行氧化修饰及鉴定。以酶消化法培养大鼠血管平滑肌细胞，采用免疫组织化学染色法鉴定。主要观察指标：在静止的血管平滑肌细胞中加入MEK1/2选择性抑制剂10 μmol/L PD98059和/或50 mg/L氧化型低密度脂蛋白，以CCK-8和细胞计数法测定细胞增殖，流式细胞术观察细胞周期，蛋白印迹检测周期素D1蛋白表达及胞外信号调节激酶的活性。结果：CCK-8和细胞计数法显示，氧化型低密度脂蛋白能明显诱导血管平滑肌细胞增殖，PD98059抑制氧化型低密度脂蛋白诱导的血管平滑肌细胞增殖，并且呈时间依赖性。流式细胞术分析表明，经氧化型低密度脂蛋白处理24 h后，血管平滑肌细胞从G0/G1期进入S期，S期细胞百分比明显增高(P < 0.01)，加入PD98059处理24 h后，血管平滑肌细胞由G0/G1期向S期的推进受到抑制。蛋白印迹结果显示，氧化型低密度脂蛋白能明显诱导周期素D1蛋白表达及胞外信号调节激酶的磷酸化，PD98059抑制氧化型低密度脂蛋所诱导的周期素D1蛋白表达以及胞外信号调节激酶的磷酸化。结论：氧化型低密度脂蛋白能诱导血管平滑肌细胞增殖，促进细胞由G0/G1期向S期转换以及周期素D1表达，其作用部分是由胞外信号调节激酶所介导。

Oxidized low-density lipoprotein induces vascular smooth muscle cells proliferation and cyclin D1 expression in rats

BACKGROUND: It has been demonstrated that oxidized low-density lipoprotein (ox-LDL) stimulates vascular smooth muscle cells (VSMCs) proliferation through Ras/Raf/MEK/MAPK pathway and induces cell cycle proteins expression.OBJECTIVE: To investigate whether ox-LDL induced cell cycle progression and cyclin D1 expression are mediated by extracellular signal-regulated kinase (ERK1/2) in VSMCs. DESIGN, TIME AND SETTING: A control observational cytology study was performed at the Guangzhou Medical College between February and October of 2008.MATERIALS: Human low density lipoprotein (LDL) was obtained form healthy volunteers. Sprague Dawley rats were provided by experimental animal centre of Guangzhou Medical College.METHODS: LDL was isolated from human plasma by sequential ultracentrifugation and oxidized. VSMCs were isolated from rat aorta by enzymatic dispersion method, and identified by immunocytochemical staining. MAIN OUTCOME MEASURES: Quiescent rat VSMCs were treated with ox-LDL with or without PD98059. Then cell proliferation was measured by CCK-8 assay and cell counts, and cell cycle distribution was determined by flow cytometry. Finally, cyclin D1 protein expression and ERK1/2 activity were examined by western blot.RESULTS: CCK-8 assay and cell counts revealed that ox-LDL greatly induced the VSMCs proliferation, and PD98059 inhibited ox-LDL stimulated VSMCs proliferation, which showed a time-depended manner. Flow cytometry showed that VSMCs proceeded to the S phase after 24 hours of ox-LDL stimulation, S phase cell percentage was obvious increased (P < 0.01), and PD98059 prevented ox-LDL induced G0/G1 to S phase transition. Western blot found that PD98059 diminished ox-LDL induced cyclin D1 expression and ERK1/2 activation. CONCLUSION: ox-LDL stimulates VSMCs proliferation, accelerates cell cycle progression from G0/G1 phase to S phase, and the cyclin D1 expression, which is partly mediated by ERK1/2.