摘要
目的观察黄酒能否抑制同型半胱氨酸(Hcy)诱导的大鼠血管内皮细胞(vascular endothelial cells,VECs)中基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达及探讨其可能机制。方法大鼠原代主动脉VECs经分离培养及纯化鉴定后,取第3～4代细胞用于实验。将Hcy(0、50、100、500、1 000μmol/L)与VECs共同孵育48h及100μmol/L Hcy与VECs共同培养24、48、72h,分别用荧光定量PCR(FQ-PCR)和免疫印迹法检测Hcy对VECs中MMP-2mRNA和蛋白表达的影响,确定Hcy最佳的刺激浓度和时间。每种酒(10、12、14、16、18、20mL/L)与100μmol/L Hcy共同孵育VECs 48h,MTT法检测酒类对细胞活性的影响,确定最佳干预浓度后处理分为正常组、Hcy组、Hcy+黄酒组、Hcy+红葡萄酒组、Hcy+酒精组5组。培养48h后收集样品,FQ-PCR检测VECs中MMP-2mRNA水平,免疫印迹法测定VECs中MMP-2的表达量。结果在50～1 000μmol/L浓度范围内与对照组相比Hcy能明显地增强MMP-2 mRNA和蛋白表达(P<0.05或P<0.01),至500μmol/L时开始达到最高。Hcy(100μmol/L)刺激24、48、72h后MMP-2mRNA和蛋白表达较对照组有明显的增强(均P<0.01),48h后开始达到最大值。实验组与Hcy组相比,黄酒组和红酒组MMP-2mRNA和蛋白表达均明显下降,差异有统计学意义(均P<0.01)。酒精组MMP-2mRNA和蛋白表达下降,差异均无统计学意义(P>0.05)。与酒精组相比,黄酒组和红酒组VECs中MMP-2mRNA和蛋白表达均明显下降,差异有统计学意义(均P<0.01),黄酒组和红酒组之间差异无统计学意义(P>0.05)。结论 Hcy能增强VECs中MMP-2mRNA和蛋白的表达,这可能在动脉粥样硬化的发病机制中发挥重要作用。黄酒和红葡萄酒能抑制Hcy对MMP-2mRNA和蛋白表达的增强,这可能是它们抗动脉粥样硬化和心血管保护作用的机制之一。
Objective To study whether rice wine can inhibit the expression of matrix metalloproteinase-2 （MMP-2） in cultured rat vascular endothelial cells（VECs） induced by homocysteine （Hcy） and its mechanisms. Methods We carried out isolation, cultivation, purification and identification of VECs in rat aorta in vitro. Cell passages 3 and 4 were used for all studies. Either cells were incubated with different doses of Hcy （0, 50, 100, 500 and 1 000 μmol/L） for 48 h, or with 100/μmol/L Hcy for 24, 48 and 72 h to observe the effect of Hcy on mRNA and protein expressions of MMP-2 using fluorescent quantitative PCR （FQ-PCR） and Western blotting. The optimal concentration and time of Hcy were selected. The VECs were incubated with one type of wine （10, 12, 14, 16, 18, 20 mL/L） and 100 μmol/L Hcy for 48 hl then the optimal concentration of each type of wine was selected. In another experiment, cells were divided into 5 groups： control, Hcy, Hcy＋ rice wine, Hcy＋ red wine, Hcy＋ ethanol groups and then incubated for 48 h. The FQ-PCR was used to test MMP-2 expression at the mRNA level and the Western blotting was applied to detect MMP-2 expression at the protein level. Results The results showed that within the concentration range of 50 and 1 000 μmol/L, Hcy could significantly increase the mRNA and protein expressions of MMP-2 compared with those in the control group （P〈0.05 and P〈0.01）, and could reach the maximum at 500 μmol/L. At 24 h, 48 h and 72 h after stimulation by Hcy at the conccntration of 100 μmol/L, MMP-2 expression at the mRNA and protein levels was significantly increased compared with that in the control group （P〈0.01）, and could reach the maximum at 48 h. Compared with those in Hcy group, MMP-2 expression at the mRNA and protein levels in rice wine and red wine groups was significantly decreased （both P〈0.01）. No significant difference was found between ethanol group and Hcy group （P〉0.05）. Compared with ethanol group, use of rice wine and red wine als
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2013年第3期313-319,共7页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
浙江省自然科学基金项目(Y2100535)
卫生部科学研究基金-浙江省医药卫生重大科技计划项目(WKJ2011-2-018)
绍兴市科技计划项目(2011A23011)
浙江省卫生高层次创新人才培养工程项目(No.2012241)~~
