黄酒多酚对同型半胱氨酸诱导的血管平滑肌细胞增殖和迁移的影响及其机制研究  

Influence of Yellow Wine Polyphenol Compounds on Homocysteine Induced VSMCs Proliferation and Migration and Its Mechanism

作　　者：刘龙斌[1] 孟立平[1] 季政[1] 许富康[1] 池菊芳[1] 郭航远[1] 

机构地区：[1]浙江省绍兴市人民医院心内科浙江大学绍兴医院,312000

出　　处：《中国全科医学》2016年第14期1676-1683,共8页Chinese General Practice

基　　金：浙江省自然科学基金资助项目(LY14H020002);绍兴市科技局项目(2013B70072)

摘　　要：目的验证黄酒多酚（YWPC）是否具有抑制同型半胱氨酸（Hcy）诱导的大鼠血管平滑肌细胞（VSMCs）增殖和迁移的作用以及其产生作用的信号通路。方法采用组织贴块法培育SD大鼠胸主动脉VSMCs，取第4～7代细胞用于实验。细胞分为5组，分别为对照组，Hey组（500μmol／LHey），YWPC1mg／L组（500μmol／LHcy＋1mg／L YWPC），YWPC10mg／L组（500μmol／L Hey＋10mg／L YWPC），YWPC100mg／L组（500μmol／LHey＋100mg／L YWPC）。采用M1Tr法检测VSMCs增殖情况；细胞划痕实验和Transwell法检测VSMCs迁移和侵袭情况；Western blotting法检Np—AKT／AKT表达情况。为验证YWPC是否经Pi3K／AKT通路发挥抑制VSMCs的增殖和迁移，分别加入L Y-294002和胰岛素生长因子1（IGF-1）用来抑制和激活Pi3K／AKT通路，细胞被分为Hcy组，YWPC组，LY-294002＋Hey组，LY-294002＋YVCPC组；Hcy组，YWPC组，IGF-1＋Hcy组，IGF-1＋YWPC组，再分别采用上述方法检测-SMCs增殖、迁移和侵袭情况。结果培养大鼠胸主动脉VSMCs，2周左右细胞融合可以传代，经SN—actin细胞免疫荧光鉴定及DAPI核染，确定细胞纯度在99％以上。24、48h时，5组OD值比较，差异有统计学意义（F=52．575，P=0．016；F=83．756，P=0．014）；其中Hcy组OD值高于对照组和YWPC组，且与YWPC呈剂量依赖性（P〈0．05）。24、48、72、96h时，5组VSMCs迁移面积比较，差异均有统计学意义（P〈0．05）；其中Hcy组VSMCs迁移面积多于对照组（P〈0．05）；YWPC组VSMCs迁移面积低于Hcy组，且与YWPC呈剂量依赖性（P〈0．05）。48h时，5组迁移细胞数比较，差异有统计学意义（F=79．354，P=0．001）；其中Hcy组穿透Transwell膜细胞数高于对照组（P〈0．05）；YWPC组穿透Transwell膜细胞数低于Hcy组，且与YWPC呈剂量依赖性（P〈0．05）。5组P—AKT／AKT比较，差异有统计学意义（F=56．723，P=0．002）；其中Hey组p-AKT／AKT高于对�Objective To verify whether yellow wine polyphenol compounds（ YWPC） could inhibit homocysteine（ Hcy） induced rat aortic vascular smooth muscle cells（ VSMCs） proliferation and migration and its signaling pathway.Methods Tissue- sticking method was used to culture SD rat VSMCs,and 4-7 generations of cells were obtained. The cells were divided into 5 groups,which were control group,Hcy group（ 500 μmol / L Hcy）,YWPC 1 mg / L group（ 500 μmol / L Hcy ＋ 1 mg / L YWPC）, YWPC 10 mg / L group（ 500 μmol / L Hcy ＋ 10 mg / L YWPC） and YWPC 100 mg / L group（ 500μmol /L Hcy ＋ 100 mg /L YWPC）. MTT method was used to detect VSMCs proliferation; cell wound scratch assay and Transwell method were used to detect VSMCs migration and invasion; Western blotting method was used to detect p-AKT / AKT expression. In order to verify whether YWPC can inhibit VSMCs proliferation and migration through Pi3K / AKT pathway,LY-294002 and IGF-1 were added respectively to inhibit and activate Pi3K / AKT pathway. The cells added with LY-294002 were divided into Hcy group,YWPC group,LY-294002 ＋ Hcy group,and LY-294002 ＋ YWPC group; the cells added with IGF-1were divided into Hcy group,YWPC group,IGF-1 ＋ Hcy group,and IGF-1 ＋ YWPC group. The above methods were employed again to detect the proliferation,migration and invasion of VSMCs. Results Rat aortic VSMCs were cultured. Two weeks later,cell fusion and passage occurred,and cell purity was determined to be above 99% by SM- actin cellular immune fluorescent identification and DAPI nuclear staining dye. At 24 h and 48 h, the five groups were significantly different in OD value（ F= 52. 575,P = 0. 016; F = 83. 756,P = 0. 014）,and the OD value of Hcy group was higher than that of control group and YWPC group,and in a dose- dependent manner（ P〈0. 05）. At 24,48,72 and 96 h,the five groups were significantly different in VSMCs migration area（ P〈0. 05）; Hcy group was higher than control group in VSMCs migration area（ P〈0. 05）;YWPC group was

关 键 词：多酚类 肌 平滑 血管 PI3K/AKT 

分 类 号：R543.31[医药卫生—心血管疾病]