同型半胱氨酸、半胱氨酸在動(dòng)脈粥樣硬化發(fā)病機制中作用的對照研究
蘇娟 王生蘭 黃玉珊 姜怡鄧 王樹(shù)人1
四川大學(xué)華西基礎醫學(xué)和法醫學(xué)院病理生理學(xué)教研室,成都 610041
摘要:目的 以動(dòng)脈粥樣硬化(As) 病灶中的主要細胞類(lèi)型—血管平滑肌細胞為模型,對比研究同型半胱
氨酸(Hcy) 和半胱氨酸(Cys) 在致As 發(fā)病效應上的異同,以期為Hcy 作為As 獨立危驗因子的分子機制提供有比較的證據。方法 體外培養人臍靜脈血管平滑肌細胞(VSMCs) ,用不同濃度的Hcy 和Cys 作用于細胞24h 后: (1) 用分光光度比色法測定細胞中SOD 和MDA 的含量; (2) 流式細胞術(shù)檢測細胞凋亡率,半定量RT2PCR 方法檢測caspases23 mRNA 的表達; (3) MTT法測定細胞的增殖率; (4) 用1000μmolPL Hcy 和Cys 分別作用VSMCs 24、48 和72h 后,MS2PCR 法檢測ERα啟動(dòng)子區甲基化的狀態(tài),半定量RT2PCR 法檢測ERαmRNA 的表達。結果 (1) Hcy 可導致細胞內MDA、SOD 呈劑量依賴(lài)性增加,Cys 使MDA、SOD 增加的效應遠弱于Hcy ( P< 0101) 。(2) 流式細胞術(shù)、caspases23 mRNA 的表達和MTT試驗的結果顯示,Hcy、Cys 對血管平滑肌細胞凋亡率和增殖率的影響差異不太明顯。(3) Hcy 顯著(zhù)增加ERα基因啟動(dòng)區的甲基化修飾,并使ERαmRNA 的表達
進(jìn)行性減少。Cys 不影響ERα啟動(dòng)子區的甲基化狀態(tài),對ERαmRNA 表達的影響也遠小于Hcy。結論 氧化應激對DNA 甲基化修飾的影響可能在Hcy 致As 發(fā)病的機制中占據著(zhù)重要的地位,這也可能是Hcy 和Cys 在A(yíng)s 發(fā)病機制中作用不同的重要原因。
關(guān)鍵詞:同型半胱氨酸 半胱氨酸 動(dòng)脈粥樣硬化 血管平滑肌細胞
中圖分類(lèi)號:R54 R362 文獻標識碼:A
A comparative study on pathogenic effects of homocysteine and cysteine on atherosclerosis
SU Juan , WANG Shenglan , HUNAGYushan , JINAGYideng , et al.
Department of Pathophysiology , West China School of Preclinical Medical Sciences & Forensic Medicine ,Sichuan University , Chengdu 610041 , China
Abstract :Objective To investigate the similarities and differences of homocysteine and cysteine on pathogenesis of As in cultured human vascular smooth muscle cells (HVSMCs) . Methods Cultured HVSMCs were treated by different concentrations of Hcy and Cys for 24 h. (1) The SOD activity and MDA contents were measured by hromatometry. (2)The rates of apoptosis of HVSMCs were detected by flow cytometry. The expression of caspases23 mRNA in HVSMCs was detected by reverse2trascription polymerase chain reaction (RT2PCR) . (3) The proliferative activities of HVSMCs were examined by MTT assay. (4) HVSMCs were incubated with 1000μmolPL Hcy and Cys for 24 , 48 and 72h respectively ,then the DNA methylation status were assayed by nested methylation2specific polymerase chain reaction , and the expressions of ERαmRNA of HVSMCs were detected by RT2PCR. Results (1) Hcy significantly increased the MDA contents and SOD activities in a dose2dependent manner , but the increase of MDA contents and SOD activities were much weaker induced by Cys ( P < 0. 01) . (2) The results of flow cytometry , expression of caspases23 mRNA , and MTT test showed that the effects of Hcy and Cys on rate of apoptosis and proliferation of HVSMCs were not much different. (3) Hcy obviously induced de novo methylation in the promoter region of the ERα gene in HVSMCs , and the expressions of ERα mRNA were correspondingly down2regulated. The Cys had no effect on methylation modification of ERα gene , and very mild effect on ERαmRNA expression. Conclusion The oxidative stress and the methylative modification induced by Hcy may have the top position in its pathogenic mechanismof pro2atherosclerosis. The pro2apoptotic and proliferation2promoting effects of Hcy were similar with Cys , which suggested that these two effects might just play mild roles in its pro2 atheroscleroatic traits.
Key words :homocysteine , cysteine , atherosclerosis , pathogenic effect , human vascular smooth mscle cell
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基金項目:教育部博士點(diǎn)基金資助項目(No. 20050610050)
作者簡(jiǎn)介:蘇娟,女,工作單位云南大理學(xué)院,講師,碩士研究生,E2mail :616sj @1631com
1 通訊作者:王樹(shù)人,E2mail wangshuren1945 @yahoo. com. cn ,Tel :028285501268