同型半胱氨酸影响内皮祖细胞机制的探讨  

Effects of homocysteine on endothelial progenitor cells

作　　者：朱军慧[1] 马彩艳[2] 陈君柱[3] 王兴祥[3] 张芙荣[3] 傅国胜[1] 

机构地区：[1]浙江大学医学院附属邵逸夫医院心内科,浙江杭州310016 [2]浙江省立同德医院心内科,浙江杭州310012 [3]浙江大学医学院附属第一医院心内科,浙江杭州310003

出　　处：《中国病理生理杂志》2009年第1期73-78,共6页Chinese Journal of Pathophysiology

基　　金：浙江省科学技术研究基金资助项目(No.2004C30012)

摘　　要：目的:探讨同型半胱氨酸(Hcy)影响内皮祖细胞(EPCs)数量和功能的可能机制。方法:采用密度梯度离心法从外周血获取单个核细胞,将其接种在人纤维连接蛋白包被的培养板,培养4 d后,收集贴壁细胞,加入不同浓度Hcy(10μmol/L、30μmol/L、100μmol/L和200μmol/L)干预,或先予以1μmol/L阿托伐他汀预处理1 h,然后再用200μmol/L Hcy干预。采用SA-β-半乳糖苷酶染色试剂盒检测衰老细胞,细胞增殖ELISA试剂盒和集落生成能力测定实验检测EPCs的增殖能力和集落形成能力,端粒重复序列扩增法(TRAP)-ELISA定量检测端粒酶活性,Western blotting检测EPCs Akt Ser473磷酸化水平。结果:Hcy呈浓度依赖性增加SA-β-半乳糖苷酶阳性细胞数量,200μmol/L最为显著,较对照增加了2倍(15.2±9.8vs51.9±13.5,P<0.01),而阿托伐他汀能显著减少Hcy诱导的衰老细胞的数量。此外,Hcy干预后伴随EPCs增殖和集落形成能力的显著损害。Hcy随着浓度的增加而显著降低EPCs端粒酶活性。进一步研究发现Hcy显著减少Akt磷酸化。结论:Hcy加速EPCs衰老,伴随EPCs增殖和集落形成能力的损害,提示细胞衰老也许是Hcy损害EPCs的机制之一。Hcy加速EPCs衰老可能跟EPCs端粒酶活性下降以及Akt磷酸化水平的下降有关。阿托伐他汀可以预防Hcy对EPCs的损害效应。AIM： To investigate whether homocysteine （Hcy） accelerates the onset of endothelial progenitor cells （EPCs） senescence through telomerase inactivation, leading to cellular dysfunction. METHODS： Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin - coated culture dishes. After 4 d culture, attached cells were stimulated with either indicated doses of Hcy （ 10 μmol/L, 30 μmol/L, 100μmol/L, 200 μmol/L）, with or without pretreatment with atorvastatin （ 1 μmol/L） for 1 h. EPCs senescence was determined by acidic β - galactosidase staining. The proliferation of EPCs was assessed by BrdU incorporation assay and colony - forming capacity. Telomerase activity was measured by telomerase - PCR ELISA and the phosphorylation of Akt was determined by using Western blotting. RESULTS ： Hcy dose - dependently accelerated the onset of EPCs senescence in culture, with a maximal effect achieved at 200 μmol/L, approximately 2 - fold increase （ 15.2 ± 9. 8 vs 51.9 ± 13.5, P 〈0.01 ）. However, the increase in SA- β -gal- positive cells induced by Hcy was significantly reduced by atorvastatin. Moreover, Hcy decreased proliferation and colony -forming capacity of EPCs. In addition, Hcy dose -dependently diminished telomerase activity and Akt phosphorylation. CONCLUSION： The results of the present study demonstrate that Hcy accelerates the onset of EPCs senescence, leading to cellular dysfunction. The effect of Hcy might be dependent on telomerase inactivation, and Akt dephosphorylation also appears to play a major role. In addition, atorvastatin has a preventative effect against Hcy - induced EPCs senescence.

关 键 词：内皮祖细胞 高半胱氨酸 端粒 末端转移酶 衰老 

分 类 号：R540[医药卫生—心血管疾病]