冠心合剂主要活性成分对TNF-a损伤血管内皮细胞保护作用的实验研究  

Study of Protective Effect of Guanxin Decoction on Endothelial Cells Injuried by TNF-α

作　　者：祝光礼[1] 刘昭[1] 杨利利[1] 

机构地区：[1]浙江中医药大学附属广兴医院心内科,浙江杭州310007

出　　处：《中华中医药学刊》2016年第2期263-266,共4页Chinese Archives of Traditional Chinese Medicine

基　　金：浙江省自然科学基金项目(Y2080946)

摘　　要：目的:研究冠心合剂主要活性成分对由TNF-a损伤的血管内皮细胞的保护作用。方法:将人脐静脉内皮细胞进行常规细胞培养、传代,后分为对照组、TNF-α组、药物活性成分组;其中对照组不加任何药物,于培养箱培养24 h后待检;TNF-α组细胞先正常培养18 h,后加入200 ng/m L的TNF-α作用6 h后,PBS洗涤待测;药物活性成分组分别加入80μg/m L、30μg/m L、20μg/m L、20μg/m L浓度的黄芪甲苷、槲皮素、异鼠李素、β-谷甾醇(药物浓度已经前期实验MTS法验证),培养18 h,再加入浓度为200 ng/m L的TNF-α继续培养6 h后,PBS洗涤细胞待测。通过TUNEL(末端脱氧核苷酸转移酶介导的d UTP缺口末端标记测定法)进行定量检测、DNA Ladder(琼脂糖凝胶电泳)进行定性检测。结果:TUNEL实验中,光镜下可看到冠心合剂主要活性成分组细胞的结构完整性优于TNF-α组,且凋亡细胞数明显少于TNF-α组(P﹤0.001)。四种药物相关活性成分中以黄芪甲苷对内皮细胞保护作用明显(P﹤0.001)。结论:冠心合剂相关药物活性成分能明显保护由TNF-α损伤的内皮细胞。四种药物相关活性成分中以黄芪甲苷对内皮细胞的保护作用最明显(P﹤0.001)。本实验琼脂糖凝胶电泳结果中并未出现细胞凋亡条带,考虑与细胞凋亡程度及凋亡时间的非同步性有关。Objective： To study the protective effect of the main ingredients of Guanxin Decoctionon to the vascular endothelial cells injuried by TNF-α in order to further clarify the material basis and molecular pharmacological mechanism of Guanxin Decoction anti atherosclerosis and at the same time to prove the scientificalness of Guanxin Decoction anti atherosclerosis.Methods： Human umbilical vein endothelial cells according with the Cytological Guide were given the conventional culture and subculture and then were divided into negative control group,positive group and drug group. The negative group was without any drug culture for 24 h. The positive cells were cultured for 18 h,and then joining TNF-α 200 ng / m L for 6 h and PBS washing for test. The drug group added 80 μg / m L,30 μg / m L,20 μg / m L,20 μg / m L of astragaloside,quercetin,isorhamnetin,β-sitosterol（ validation of drug concentration was tested by the MTS method before）,cultured for 18 h,and then adding 200 ng / m L TNF-α cultured for 6 h after PBS washing the cells to be tested. Through this experiment,two kinds of classical ways to detection of apoptosis： one was TUNEL（ Td T-mediated d UTP nick end labeling） for the quantitative detection and the other was DNA Ladder（ agarose gel electrophoresis） for qualitative detection.Results： Confirmation of Guanxin Decoction had a protective effect on endothelial cells injured by TNF-α. The results of TUNEL showed the cell structure of drug groups was superior to that ofthe TNF-α group,also the apoptosis rate was far below that of the TNF-α group（ P〈0.001）. It was confirmed that there was difference between them. The astragaloside in all kinds of drugs active ingredients had the significant protective effect on the endothelial cells injured by TNF-α（ P〈0.001）. The result of DNA Ladder was all the groups showed no DNA ladder display,considering it was related to the degree of cell apoptosis and the cell apoptosis not at the same time. Conclusion： Active ingredients of

关 键 词：冠心合剂 内皮细胞 TNF-α 细胞凋亡 琼脂糖凝胶电泳 

分 类 号：R541.4[医药卫生—心血管疾病]