摘要
目的:观察人参、三七、川芎提取物延缓自发性高血压大鼠(spontaneously hypertensive rat,SHR)血管平滑肌细胞(vascular smooth muscle cell,VSMC)衰老的作用及其可能的细胞周期调控机制。方法:取大鼠胸主动脉用于分离VMSC,采用原代细胞培养的方法建立SHR大鼠VMSC模型,实验分为Wistar-Kyoto(WKY)大鼠对照组、SHR模型组、缬沙坦组、中药低剂量组和中药高剂量组。采用衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)染色法观察细胞衰老情况,流式细胞仪分析细胞周期分布,逆转录聚合酶链式反应和蛋白质印迹法检测各组细胞p16、细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶4(cyclin-dependent kinase4,CDK4)和成视网膜母细胞瘤蛋白(retinoblastoma protein,Rb)的表达。结果:SA-β-gal染色显示,与WKY组比较,SHR组SA-β-gal阳性细胞数明显增多(P<0.01),人参、三七、川芎提取物可使SHR VSMC SA-β-gal阳性细胞数明显减少(P<0.01);流式细胞仪检测显示,与WKY组比较,SHR组VSMC在G1期明显减少,S期细胞明显增加,中药干预后SHR的VSMC G1期细胞明显增多,S期细胞明显减少(P<0.05);实时荧光定量聚合酶链式反应检测显示,与WKY组比较,SHR组VSMC的p16和Rb mRNA表达量明显减少,cyclin D1和CDK4mRNA表达增加;人参、三七、川芎提取物干预后SHR VSMC p16和Rb mRNA表达量增加,cyclin D1和CDK4mRNA减少。蛋白质印迹法显示,与WKY组比较,SHR组VSMC的p16蛋白表达量明显减少,cyclin D1、CDK4和磷酸化Rb蛋白表达量明显增加(P<0.05);中药干预后SHR VSMC p16蛋白表达增加,cyclin D1、CDK4和磷酸化Rb蛋白表达量减少(P<0.05)。结论:人参、三七、川芎提取物通过改变p16-cyclin D/CDK-Rb通路因子的表达而发挥延缓SHR VSMC衰老的作用。
OBJECTIVE： To observe the pathological changes of vascular aging in spontaneously hypertensive rats （SHRs）, and the effects of extracts from Panax ginseng, Panax notoginseng （Burk.） and Ligusticum chuanxiong on vascular aging. METHODS： Vascular smooth muscle cells （VSMCs） isolated from thoracic aorta of Wistar-Kyoto （WKY） rats and SHRs were primarily cultured and divided into WKY group, SHR group, valsartan group, low-dose extract group and high-dose extract group. Aging situation of VSMCs was detected by senescence-associated β-galactosidase （SA-β-gal） staining; the cell cycle was analyzed by flow cytometry; mRNA and protein expressions of p16, cyclin D1, cyclin-dependent kinase 4 （CDK4） and retinoblastoma protein （Rb） were detected by real-time fluorescence quantitative polymerase chain reaction （PCR） and Western blotting. RESULTS： SA-β-gal stain showed that compared with the WKY group, number of SA-β-gal-positive cells in the SHR group was increased significantly （P〈0.01）; extracts from Panax ginseng, Panax notoginseng （Burk.） and Ligusticum chuanxiong reduced the number of SA-β-gal-positive cells in the SHR group （P〈0.01）. Flow cytometry analysis showed that the number of VSMCs in G1 phase was reduced and in S phase was increased significantly （P〈0.05） in the SHR group compared with the WKY group, and the number of VSMCs in G1 phase was increased and in S phase was reduced significantly in the extracts groups （P〈0.05）. PCR result showed that compared with the WKY group, the mRNA expressions of p16 and Rb in the SHR group were reduced and those of cyclin D1 and CDK4 were increased significantly, the mRNA expressions of p16 and Rb in the extracts group were increased and mRNA expressions of cyclin D1 and CDK4 were reduced significantly. Western blotting result showed that compared with the WKY group, the protein expression of p16 in the SHR group was reduced and the protein expressions of cyclin D1, CDK4 and phospho-Rb were increased signifi
出处
《中西医结合学报》
CAS
2012年第1期76-84,共9页
Journal of Chinese Integrative Medicine
基金
国家自然科学基金资助项目(No.30973828)
国家重点基础研究发展计划(973计划)资助项目(No.2007CB507400)
中国中医科学院自主选题资助项目(No.Z02151)
