益肾蠲湿合剂对肾小球系膜细胞增殖及细胞外信号调节激酶激活影.docVIP

益肾蠲湿合剂对肾小球系膜细胞增殖及细胞外信号调节激酶激活影 　 作者：孙影， 张雪鹏， 张健， 杨方 【摘要】 目的观察益肾蠲湿合剂对肾小球系膜细胞(glomerular mesangial cell, GMC)增殖及细胞外信号调节激酶(extracellular signal-regulated kinases, ERK)磷酸化的抑制作用。方法将含低、中、高剂量益肾蠲湿合剂的药物血清作用于10%血清-DMEM培养液培养的GMC, 分别用MTT 和Western Blot 方法检测细胞增殖和磷酸化ERK1/2的表达。结果益肾蠲湿合剂对GMC增殖有明显的抑制作用，并随着剂量的增加作用也逐渐增强。细胞总ERK在各组间无明显差别。但不同浓度的益肾蠲湿合剂均显著地抑制血清所诱导的ERK1/2的磷酸化。结论益肾蠲湿合剂可通过抑制ERK1/2的磷酸化抑制GMC的增殖。 【关键词】 益肾蠲湿合剂；肾小球系膜细胞； 增殖； 细胞外信号调节激酶 　　Abstract：ObjectiveTo observe the effect of “Yishenjuanshi” on the proliferation of glomerular mesangial cells (GMCs) and the phosphorylation of extracellular signal-regulated kinases (ERK). MethodsAll herbs in “Yishenjuanshi” mixture were boiled in water and concentrated to different doses. After 7 days of “Yishenjuanshi” treatment, serum from rats eye blood was collected. GMCs were cultured in serum free medium for 48hs and then 10% FSC-DMEM with or without serum including “Yishenjuanshi” to detect GMCs proliferation and expression of phosphorylated ERK (p-ERK) by MTT and Western Blot respectively.ResultsLow, medium and high dose of Yishenjuanshi” treatment significantly inhibited GMC proliferation when GMCs were cultured for 24, 48 and 72 h; and the inhibition ratios increased as the dose increased. There was no difference in total ERK expression. However, “Yishenjuanshi” treatments with different dose all markedly inhibited p-ERK expression stimulated by FCS.Conclusion “Yishenjuanshi” treatment inhibits GMC proliferation via blocking ERK phosphorylation. 　　Key words：“Yishenjuanshi” mixture; Glomerular mesangial cell; Proliferation; ERK 　　复方益肾蠲湿合剂是华北煤炭医学院张健教授在多年临床和实验研究的基础上，拟定的治疗各型慢性肾炎的基本方药。本方直接针对慢性肾炎基本发病机理，重用黄芪，辅以枸杞、猪苓等药物，起到补气，滋肾阴、清热、解毒等作用。既往研究表明益肾蠲湿合剂在治疗临床狼疮性肾炎及实验性家兔系膜增生性肾炎取得了良好的效果［1，2］，不但明显缓解了慢性肾炎的临床症状，还全面改善了肾功能，从而延缓肾小球硬化和慢性肾炎的进展。导致慢性肾炎和肾小球硬化的重要原因之一是肾小球系膜细胞(glomerular mesangial cell, GMC)的过度增殖［3］，为此本研究将观察益肾蠲湿合剂对GMC增殖的抑制作用，并进一步探讨其作用机理，从细胞和分子生物学方面探讨其防治肾小球硬化的作用机制。 　　1 材料与仪器 　　1.1 益肾蠲湿合剂水煎液由华北煤炭医学院中西医结合医院提供。 　　1.2 细胞大鼠肾小球系膜细胞(HBZY1)由武汉大学中国典型培养物保藏中心