冠心病患者高密度脂蛋白抗氧化能力及其对内皮细胞增殖、迁移的影响研究

摘要

目的 探究冠心病(CHD)患者高密度脂蛋白(HDL)抗氧化能力及其对内皮细胞增殖、迁移的影响.方法 选取2015年6-12月就诊于陕西中医药大学附属医院心内科的CHD患者48例,另选取同期在陕西中医药大学附属医院体检健康者28例,采用密度梯度离心法提取CHD患者和体检健康者HDL,采用cell-free法检测HDL抗氧化能力,比较CHD患者和体检健康者HDL的相对荧光强度值(RFU).将4代人脐静脉内皮细胞(HUVECs)分为空白对照组(不做任何处理)、阳性对照组(加入血管内皮生长因子)、对照组(加入体检健康者HDL)、实验组(加入CHD患者HDL),采用CCK4细胞增殖检测试剂盒检测内皮细胞增殖情况,比较4组内皮细胞吸光度值;采用细胞划痕实验检测内皮细胞迁移能力,比较4组内皮细胞“水渠”宽度比值;采用Western bloting法检测HDL对内皮细胞磷脂酰肌醇3-激酶(H3K)、丝氨酸/苏氨酸激酶(AKT)、磷酸化磷脂酰肌醇3-激酶(P-H3K)、磷酸化丝氨酸/苏氨酸激酶(P-AKT)蛋白表达的影响,比较空白对照组、对照组和实验组内皮细胞PI3K/GAPDH、AKT/GAPDH、P-H3K/H3K、P-AKT/AKT蛋白灰度值比值.结果 CHD患者HDL的RFU高于体检健康者(P＜0.05).空白对照组和实验组内皮细胞吸光度值高于阳性对照组和对照组(P＜0.05).实验组内皮细胞“水渠”宽度比值低于阳性对照组和对照组(P＜0.05).空白对照组、对照组和实验组内皮细胞H3K/GAPDH和AKT/GAPDH蛋白灰度值比值比较,差异无统计学意义(P＞0.05);对照组内皮细胞P-PI3 K/PI3K和P-AKT/AKT蛋白灰度值比值高于空白对照组和实验组,实验组内皮细胞P-PI3K/PI3K和P-AKT/AKT蛋白灰度值比值低于空白对照组(P＜0.05).结论 CHD患者HDL抗氧化能力减弱,HDL可能通过抑制PI3K-AKT通路而影响内皮细胞增殖和迁移.%Objective To study the antioxidant ability of high density lipoprotein and the impact on proliferation and migration of endothelial cells in patients with coronary heart disease.Methods From June to December in 2015 in the Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,48 patients with coronary heart disease in the Department of Cardiology and 28 healthy people admitted to this hospital for physical examination were selected,density-gradient centrifugation method was used to extract high density lipoprotein,and cell-free method was used to detect the antioxidant ability of high density lipoprotein,then relative fluorescence unit (RFU) was compared between patients with coronary heart disease and healthy people.The fourth generation of HUVECs were divided into A group (without any intervention),B group (added with vascular endothelial growth factor),C group (added with high density lipoprotein of healthy people) and D group (added with high density lipoprotein of patients with coronary heart disease),CCK-8 Cell Proliferation Detection Kit was used to detect the proliferation of endothelial cells,and absorbance value was compared among the four groups;cell scratch-wound test was used to detect the migration of endothelial cells,and the ratio of " channel" width was compared among the four groups;Western bloting method was used to detect the impact of high density lipoprotein on protein expression of PI3 K,AKT,P-PI3 K and P-AKT of endothelial cells,and protein gray value ratio of PI3K/GAPDH,AKT/GAPDH,P-PI3K/PI3K and P-AKT/AKT was compared among A group,C group and D group.Results RFU of high density lipoprotein of patients with coronary heart disease was statistically significantly higher than that of healthy people (P ＜ 0.05).Absorbance value of endothelial cells of A group and D group was statistically significantly higher than that of B group and C group,respectively (P ＜ 0.05).Ratio of " channel" width of endothelial cells of D group was statistically significantly lower than that of B group and C group,respectively (P ＜0.05).No statistically significant differences of protein gray value ratio of PI3K/GAPDH or AKT/GAPDH of endothelial cells was found among A group,C group and D group (P ＞0.05);protein gray value ratio of P-PI3K/PI3K and P-AKT/AKT of endothelial cells of C group was statistically significantly higher than that of A group and D group,respectively,while protein gray value ratio of P-PI3K/PI3K and P-AKT/AKT of endothelial cells of D group was statistically significantly lower than that of A group (P ＜ 0.05).Conclusion Antioxidant ability of high density lipoprotein is significantly decreased in patients with coronary heart disease,high density lipoprotein may affect the proliferation and migration of endothelial cells through inhibiting PI3 K-AKT signal pathway.