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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 active components of Guanxian Mixture on vascular endothelial cells damaged by TNF-a. Methods: Human umbilical vein endothelial cells were cultured, passaged and divided into control group, TNF-α group and drug active ingredient group. The control group received no drug for 24 h and incubated for 24 h. TNF -α cells were cultured for 18 h and then treated with 200 ng / mL of TNF-α for 6 h. The cells were washed with PBS and tested. The active ingredients were added to 80 μg / ml, 30 μg / ml and 20 μg / ml respectively Astragaloside, quercetin, isorhamnetin, and β-sitosterol at a concentration of 20 μg / ml (the concentration of which has been verified by the MTS method in the previous experiment) and cultured for 18 h, and then added with a concentration of 200 ng / m L After 6-h incubation of TNF-α, the cells were washed with PBS and tested. Quantitative detection was performed by TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay), and DNA Ladder (agarose gel electrophoresis) was used for qualitative detection. Results: In the TUNEL experiment, the structural integrity of the main active components of Guanxin Mixture was better than that of TNF-α group, and the number of apoptotic cells was significantly less than that of TNF-α group (P <0.001). Among the four drug-related active ingredients, Astragaloside had a significant protective effect on endothelial cells (P <0.001). Conclusion: The active ingredients of Guanxin Mixture can obviously protect the endothelial cells damaged by TNF-α. Of the four drug-related active ingredients, Astragaloside had the most obvious protective effect on endothelial cells (P <0.001). The experimental agarose gel electrophoresis did not appear in the apoptotic bands, consider the degree of apoptosis and apoptosis of asynchrony related.