平肝潜阳方含药血清对血管紧张素Ⅱ诱导血管平滑肌细胞增殖、迁移及DNA甲基化水平的影响  

Effect of Pinggan Qianyang Recipe Containing Serum on Angiotensin Ⅱ Induced Vascular Smooth Muscle Cell Proliferation and Migration and DNA Methylation

作　　者：钟广伟[1,2] 万玲[1] 王东生[1] 方霞[2] 陈琼[2] 谢明萱[2] 唐涛[1] 

机构地区：[1]中南大学湘雅医院中西医结合研究所,长沙410008 [2]中南大学湘雅医院国际医疗部,长沙410008

出　　处：《中国中西医结合杂志》2016年第5期580-585,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金资助项目(No.30500644);中南大学贵重仪器设备开放共享基金资助项目(No.CSUZC2012014);湖南省中医药科研计划项目(No.201245)

摘　　要：目的观察平肝潜阳方对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖和迁移的抑制作用及DNA甲基化水平的影响。方法采用组织贴块法培养VSMCs,制备平肝潜阳方含药血清。将培养细胞分为正常组(原代培育的VSMCs)、模型组、叶酸组及平肝潜阳方组(下称中药组)。以AngⅡ作为诱导复制VSMCs增殖及迁移模型,在AngⅡ诱导VSMCs培育24 h后,叶酸组加入40μg/mL叶酸干预48 h,中药组加入5%中药血清干预48 h。采用CCK-8(Cell Counting Kit)试剂盒绘制VSMCs生长曲线,MTT法测定VSMCs增殖活性,体外细胞划痕实验检测细胞迁移情况,以抗5-甲基胞嘧啶(5-mC)抗体进行免疫荧光检测细胞核中胞嘧啶甲基化水平;Real-time q-PCR方法检测DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)mRNA表达水平。结果 AngⅡ在10-6mol/L浓度下诱导24 h后可促进VSMCs生长。与正常组比较,模型组细胞增殖活性及迁移数量明显增加,DNA甲基化水平明显降低(P<0.05,P<0.01);与模型组比较,叶酸组及中药组VSMCs生长、细胞增殖活性及迁移数量均明显降低,DNA甲基化水平升高(P<0.05,P<0.01);与正常组比较,模型组VSMCs DNMT1 mRNA表达水平降低(P<0.01);与模型组比较,叶酸组及中药组VSMCs DNMT1 mRNA表达明显增强(P<0.01)。结论平肝潜阳方可抑制AngⅡ诱导的VSMCs增殖和迁移,可导致基因组DNA高甲基化,其DNA甲基化水平改变可能与DNMT1表达有关。Objective To observe the effect of Pinggan Qianyang Recipe（PQR） on inhibiting angiotensinⅡ（AngⅡ） induced proliferation and migration of vascular smooth muscle cells（VSMCs） and changes of DNA methylation. Methods VSMCs were cultured using tissue explant method, and PQR containing serum was prepared. Primarily cultured VSMCs were divided into four groups, the normal group, the model group, the folate group（folic acid intervention）, and the PQR group. The proliferation and migration of VSMCs was duplicated by Ang Ⅱ.After 24-h AngⅡ induced culture, 40 μg/m L folic acid was added to the folate group for 48 h, while 5% PQR containing serum was added to the PQR group for 48 h. The cell growth curve of VSMCs was drawn by using Cell Counting Kit（CCK-8）. The proliferative activity of VSMC was determined by MTT assay. The migration of VSMCs was measured by Mil icell chamber. The general level of cytosine methylation in cell nucleus was detected via 5-m C antibodies immunofluorescence, and m RNA expression levels of DNA methyltransferase 1（DNMT1） weremeasured by Real-time q-polymerase chain reaction（q-PCR）. Results VSMCs were promoted by AngⅡ at 10- 6mol/L for 24 h. Compared with the normal group, the proliferative activity and migration quantity of VSMCs obvious-ly increased, and DNA methylation level obviously decreased（P〈0. 05, P〈0. 01）. Compared with the model group, the cell growth, proliferative activity and migration quantity of VSMCs obviously decreased and the general DNA methylation level increased in the folate group and the PQR group（P〈0. 05, P〈0. 01）. Compared with the normal group, the m RNA expression of DNMT1 decreased in the model group（P〈0. 01）. Compared with the model group, m RNA expression of DNMT1 in AngⅡ induced VSMCs was obviously enhanced in the folate group and the PQR group（P〈0. 01）. Conclusions PQR could inhibit Ang Ⅱ induced proliferation and migration of VSMCs, and cause high genomic DNA methylation level. Changes of DNA meth

关 键 词：血管平滑肌细胞 增殖与迁移 DNA甲基化 平肝潜阳方 DNA甲基转移酶1 

分 类 号：R285[医药卫生—中药学]