吡格列酮和硫氢化钠对培养人脐静脉内皮细胞内胱硫醚-γ-裂解酶影响的相关性研究

发布时间：2018-02-04 20:12

本文关键词： 硫化氢同型半胱氨酸过氧化物酶体增殖激活受体吡格列酮胱硫醚-γ-裂解酶　出处：《川北医学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探讨硫氢化钠和吡格列酮对体外培养的人脐静脉内皮细胞（HUVEC）内胱硫醚-γ-裂解酶（CSE）的相关研究。方法:(1) CCK-8法检测不同处理组HUVEC的细胞活性。(2)实时荧光定量聚合酶链反应法（real-time polymerase chain reaction，RT-PCR）检测不同处理组HUVEC细胞中的CSE mRNA表达，(3) Western-blot法检测不同处理组HUVEC细胞中CSE蛋白的表达。(4)敏感硫电极法检测不同处理组HUVEC细胞的H2S含量，以此计算CSE酶的活性。结果:(1)随着浓度的增加（10μM～1000μM），Hcy浓度依赖性降低HUVEC细胞活性，并在1000μM表现出显著的毒性作用（P0.01）；Hcy浓度依赖性减少HUVEC对CSE mRNA的表达和蛋白的表达；并且Hcy浓度依赖性降低HUVEC CSE酶活性（P0.05）。(2)在浓度范围（10μM～30μM）时，随着浓度增加，NaHS对细胞活性有提高作用。在浓度范围（50μM～1000μM）时，，随着浓度增加，NaHS对细胞活性有降低作用（P0.05）。(3)当30μM NaHS与1000μM Hcy组共同作用时，HUVEC细胞活性表现出不受1000μM Hcy影响，反之，细胞活性增加、细胞内CSEmRNA的表达和蛋白的表达增加，同时，CSE酶活性增加（P0.05）。(4)10μM吡格列酮组有提高细胞活性的作用。而其他浓度组（1μM、100μM）没有此作用。当10μM吡格列酮与1000μM Hcy组共同作用时，HUVEC细胞表现出不受1000μM Hcy的影响反而提高了细胞活性，同时也提高了细胞内CSEmRNA和蛋白的表达水平，并且提高了CSE酶活性（P0.05）。结论:①高Hcy（高浓度同型半胱氨酸）可致HUVEC损伤。②一定浓度的NaHS或PPARγ激动剂吡格列酮能降低高Hcy所致的HUVEC损伤。③NaHS和吡格列酮降低高Hcy所致的HUVEC的损伤与提高内源性H2S的产生有关。④NaHS和吡格列酮通过增加细胞内CSE的表达水平与活性来提高内源性H2S的产生。
[Abstract]:Aim: to investigate the effects of sodium sulphide and pioglitazone on cystathithione- 纬 -lyase (CSE) in cultured human umbilical vein endothelial cells (HUVECs) in vitro. Methods CCK-8 assay was used to detect the cellular activity of HUVEC in different treatment groups. (1) Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the cell activity of HUVEC in different treatment groups. Real-time polymerase chain reaction. RT-PCR was used to detect the expression of CSE mRNA in HUVEC cells treated with different treatments. Detection of CSE protein expression in HUVEC cells by Western-blot. Sensitive sulfur electrode method was used to detect the content of H2S in HUVEC cells treated with different treatments. The activity of CSE enzyme was calculated. Results (1) the activity of HUVEC cells was decreased in a concentration-dependent manner with the increase of concentration of Hcy. At 1000 渭 M, it showed a significant toxic effect (P 0.01). Hcy decreased the expression of CSE mRNA and protein in a dose-dependent manner. Moreover, Hcy decreased the activity of HUVEC CSE in a dose-dependent manner (P 0.05 渭 M) at the concentration range of 10 渭 M ~ (30 渭 M), and increased with the concentration. NaHS could improve the cell activity. At the concentration range of 50 渭 m ~ (1 000 渭 m), the cell activity increased with the concentration of 50 渭 m ~ (-1) 渭 m ~ (-1). The effect of NaHS on cell activity was significantly decreased (P0.05. 3) when 30 渭 M NaHS was treated with 1 000 渭 M Hcy. The activity of HUVEC cells was not affected by 1000 渭 M Hcy. On the contrary, the cell activity increased, the expression of CSEmRNA and protein increased. The activity of CSE increased in 10 渭 M pioglitazone group and 1 渭 M in other concentration groups. When 10 渭 M pioglitazone was treated with 1 000 渭 M Hcy. HUVEC cells were not affected by 1000 渭 M Hcy, but increased the cell activity and the expression level of CSEmRNA and protein. And the activity of CSE was increased (P 0.05). Conclusion the high concentration of homocysteine (Hcy) is high in 1: 1. 2. 2 NaHS or PPAR 纬 agonist pioglitazone can reduce HUVEC damage induced by high Hcy. 3NaHS and pioglitazone reduce Hcy induced by high Hcy. The damage of HUVEC is related to the increase of endogenous H2S production. 4NaHS and pioglitazone increase the production of endogenous H2S by increasing the expression and activity of CSE.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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