早发冠心病患者高密度脂蛋白抗HUVECs凋亡作用的机制研究

发布时间：2018-05-17 03:25

本文选题：高密度脂蛋白 + 氧化型低密度脂蛋白　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:通过细胞实验探究早发冠心病(PCAD)患者高密度脂蛋白(HDLPCAD)与健康人群HDL(HDLhealth)抗人脐静脉内皮细胞(HUVECs)凋亡作用是否有区别及其可能机制。方法:1.PCAD患者和与之相匹配的健康人血样的采集;2.使用Lipoprint脂蛋白分析仪分析HDLhealth和HDLPCAD亚组分(HDL1-HDL10)分布情况;3.PCAD患者和与之相匹配的健康人血样HDL的分离、提取、鉴定;4.分别用0μg/ml、50μg/ml、100μg/ml、150μg/ml、200μg/ml的氧化低密度脂蛋白(ox-LDL)处理HUVECs 24小时,MTT检测细胞存活度,明确ox-LDL引起HUVECs凋亡的合适浓度;5.首先分别用0μg/ml、50μg/ml、100μg/ml、150μg/ml、200μg/ml的HDLhealth预处理HUVECs 18小时,接着再用最适浓度的ox-LDL处理HUVECs 24小时,MTT检测细胞存活度,明确HDLhealth预处理HUVECs的最适浓度;6.首先用最适浓度HDLhealth分别预处理HUVECs 0小时、6小时、12小时、18小时、24小时,接着再用最适浓度的ox-LDL处理HUVECs24小时,MTT检测细胞存活度,明确HDLhealth预处理HUVECs的最适时间;7.用最适浓度HDLhealth、HDLPCAD对HUVECs进行最适作用时间的预处理,再用合适浓度的ox-LDL处理HUVECs 24小时,MTT检测细胞存活度;流式细胞仪检测细胞凋亡;Western blot检测caspase-3、caspase-9蛋白的表达;用试剂盒测定活性氧(ROS)活性。结果:1.HDLPCAD亚组分大颗粒含量(HDL1-HDL3)为(28.5±5.74),较HDLhealth(46.8±15.2)低,而小颗粒(HDL8-HDL10)含量为(21.4±7.75),较HDLhealth(10.9±5.38)高;2.100μg/ml ox-LDL处理HUVECs 24小时后细胞存活率为60.34%,存活率较空白处理组出现显著降低(P0.05);3.200μg/ml HDLhealth预处理HUVECs18h后,细胞存活率为82.01%,可以明显减弱ox-LDL对细胞的损伤;4.200μg/ml HDLhealth显著抑制100μg/ml ox-LDL诱导的HUVECs凋亡及caspase-3、caspase-9蛋白表达和ROS的产生;5.200μg/ml HDLPCAD预处理HUVECs 18小时后,细胞存活率为65.5%,其作用较HDLhealth(80.28%)减弱;6.200μg/ml HDLPCAD可抑制100μM ox-LDL诱导HUVECs凋亡、caspase-3、caspase-9蛋白表达及ROS产生,但作用较HDLhealth减弱。结论:HDLPCAD与HDLhealth比较,可能由于其亚组分大颗粒含量(HDL1-HDL3)较HDLhealth低,小颗粒较HDLhealth(HDL8-HDL10)高,导致其抗氧化功能减弱,抑制ox-LDL诱导内皮细胞凋亡的功能亦减弱,从而减弱或丧失抗As的作用。
[Abstract]:Aim: to investigate the difference between HDLPCAD (high density lipoprotein) (HDLPCAD) and HDLhealth (healthy) in patients with premature coronary heart disease (CHD) and healthy subjects (HDL) on human umbilical vein endothelial cells (HUVECs) apoptosis and its possible mechanism. Methods: 1. Blood samples from PCAD patients and matched healthy people were collected. The distribution of HDLhealth and HDLPCAD subfractions HDL1-HDL10 was analyzed by Lipoprint lipoprotein analyzer. 3. Isolation, extraction and identification of HDL from blood samples of PCAD patients and matched healthy people. HUVECs was treated with 0 渭 g / ml 50 渭 g / ml ~ (100 渭 g / ml) of oxidized low density lipoprotein 200 渭 g / ml for 24 hours, respectively. The cell viability was determined by MTT assay, and the appropriate concentration of ox-LDL induced HUVECs apoptosis was determined. HUVECs was pretreated with 0 渭 g / ml ~ 50 渭 g / ml ~ (50 渭 g / ml) ~ 100 渭 g / ml ~ (-1) HDLhealth for 渭 g / ml ~ (-1) g/ml for 18 hours, then HUVECs was treated with ox-LDL at the optimal concentration for 24 hours to determine the cell viability, and the optimal concentration of HUVECs pretreated by HDLhealth was 6 ~ (6). The optimal concentration of HDLhealth was used to pretreat HUVECs for 0 hours, 6 hours, 12 hours, 18 hours and 24 hours respectively. Then, the optimal concentration of ox-LDL was used to detect the viability of HUVECs24 cells, and the optimal time for HDLhealth pretreatment of HUVECs was determined. HUVECs was pretreated with the optimal concentration of HDLhealth HUVECs for 24 hours, then HUVECs was treated with appropriate concentration of ox-LDL for 24 hours to detect cell viability, flow cytometry was used to detect apoptosis and Western blot was used to detect the expression of caspase-3 and caspase-9 protein. The reactive oxygen species (Ros) activity was determined with the kit. Results 1. The content of HDL1-HDL3 in HDL1-HDL3 was lower than that of HDLhealth(46.8 卤15.2in HDL1-HDL3, while the content of HDL8-HDL10 in HDL8-HDL10 was 21.4 卤7.75, which was higher than that in HDLhealth(10.9 卤5.38. The survival rate of HUVECs treated with 2.100 渭 g/ml ox-LDL for 24 hours was 60.344.The survival rate of HDL1-HDL3 was significantly lower than that of control group, and the survival rate of HDL8-HDL10 was significantly lower than that of control group after pretreatment with HUVECs18h of 3.200 渭 g/ml HDLhealth. The cell survival rate was 82.01, which could significantly attenuate the damage induced by ox-LDL (4.200 渭 g/ml HDLhealth), inhibit the HUVECs apoptosis induced by 100 渭 g/ml ox-LDL, the expression of caspase-3 and caspase-9 protein, and the production of ROS (5.200 渭 g/ml HDLPCAD) for 18 hours after pretreatment with HUVECs. The cell survival rate was 65.5, the effect of which was lower than that of HDLhealth80.28) 6.200 渭 g/ml HDLPCAD could inhibit the expression of caspase-3 caspase-9 protein and the production of ROS in HUVECs induced by 100 渭 M ox-LDL, but the effect was weaker than that of HDLhealth. Conclusion compared with HDLhealth, it is possible that the content of large particles of HDLhealth is lower than that of HDLhealth, and the small particles are higher than HDL8-HDL10, which results in the decrease of antioxidant function and the inhibition of apoptosis induced by ox-LDL in endothelial cells, thus weakening or losing the anti-arsenic effect.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.4

【参考文献】