本文關(guān)鍵詞： 硫化氫 同型半胱氨酸 過(guò)氧化物酶體增殖激活受體 吡格列酮 胱硫醚-γ-裂解酶　出處：《川北醫(yī)學(xué)院》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：探討硫氫化鈉和吡格列酮對(duì)體外培養(yǎng)的人臍靜脈內(nèi)皮細(xì)胞（HUVEC）內(nèi)胱硫醚-γ-裂解酶（CSE）的相關(guān)研究。 方法:(1) CCK-8法檢測(cè)不同處理組HUVEC的細(xì)胞活性。(2)實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)法（real-time polymerase chain reaction，RT-PCR）檢測(cè)不同處理組HUVEC細(xì)胞中的CSE mRNA表達(dá)，(3) Western-blot法檢測(cè)不同處理組HUVEC細(xì)胞中CSE蛋白的表達(dá)。(4)敏感硫電極法檢測(cè)不同處理組HUVEC細(xì)胞的H2S含量，以此計(jì)算CSE酶的活性。 結(jié)果:(1)隨著濃度的增加（10μM～1000μM），Hcy濃度依賴(lài)性降低HUVEC細(xì)胞活性，并在1000μM表現(xiàn)出顯著的毒性作用（P0.01）；Hcy濃度依賴(lài)性減少HUVEC對(duì)CSE mRNA的表達(dá)和蛋白的表達(dá)；并且Hcy濃度依賴(lài)性降低HUVEC CSE酶活性（P0.05）。(2)在濃度范圍（10μM～30μM）時(shí)，隨著濃度增加，NaHS對(duì)細(xì)胞活性有提高作用。在濃度范圍（50μM～1000μM）時(shí)，，隨著濃度增加，NaHS對(duì)細(xì)胞活性有降低作用（P0.05）。(3)當(dāng)30μM NaHS與1000μM Hcy組共同作用時(shí)，HUVEC細(xì)胞活性表現(xiàn)出不受1000μM Hcy影響，反之，細(xì)胞活性增加、細(xì)胞內(nèi)CSEmRNA的表達(dá)和蛋白的表達(dá)增加，同時(shí)，CSE酶活性增加（P0.05）。(4)10μM吡格列酮組有提高細(xì)胞活性的作用。而其他濃度組（1μM、100μM）沒(méi)有此作用。當(dāng)10μM吡格列酮與1000μM Hcy組共同作用時(shí)，HUVEC細(xì)胞表現(xiàn)出不受1000μM Hcy的影響反而提高了細(xì)胞活性，同時(shí)也提高了細(xì)胞內(nèi)CSEmRNA和蛋白的表達(dá)水平，并且提高了CSE酶活性（P0.05）。 結(jié)論:①高Hcy（高濃度同型半胱氨酸）可致HUVEC損傷。②一定濃度的NaHS或PPARγ激動(dòng)劑吡格列酮能降低高Hcy所致的HUVEC損傷。③NaHS和吡格列酮降低高Hcy所致的HUVEC的損傷與提高內(nèi)源性H2S的產(chǎn)生有關(guān)。④NaHS和吡格列酮通過(guò)增加細(xì)胞內(nèi)CSE的表達(dá)水平與活性來(lái)提高內(nèi)源性H2S的產(chǎn)生。
[Abstract]:Aim: to investigate the effects of sodium sulphide and pioglitazone on cystathithione- 緯 -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 緯 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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96

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