软脂酸对人脐静脉血内皮祖细胞增殖、凋亡及Bcl-2表达的影响

Effect of Palmitate on Proliferation,Apoptosis,and Expression of Bcl-2 of Endothelial Progenitor Cells from Human Umbilical Cord Blood in vitro

目的探讨软脂酸(PA)对人脐静脉血内皮祖细胞(EPC)增殖、凋亡及Bcl-2表达的影响。方法密度梯度离心法获取人脐静脉血单个核细胞,培养7d后,收集贴壁细胞并加入不同浓度软脂酸(分别为200mmol/L、400mmol/L、600mmol/L)干预48h。MTT法检测PA对EPC增殖能力的影响;流式细胞仪检测PA对细胞凋亡率的影响;提取细胞RNA,采用逆转录一聚台酶链式反应(RT-PCR)技术检测Bcl-2mRNA表达;采用免疫组织化学法检测Bcl-2蛋白表达水平。结果PA呈浓度依赖性抑制EPC增殖,诱导EPC凋亡(P<0.05或P<0.01);基础状态下EPC表达Bcl-2mRNA及蛋白,PA处理能抑制其表达,并存在量效关系(P<0.05或P<0.01)。结论PA通过下调Bcl-2表达诱导EPC凋亡、抑制EPC增殖,这可能影响血管内皮的修复及新生血管的形成。

Objective To investigate the effect of palmitate on proliferation,apoptosis,and expression of bcl - 2 of endothelial progenitor cells （EPC） from human umbilical cord blood in vitro. Methods Total mononuclear cells were isolated from human umbilical cord blood in vitro by Ficoll density gradient eentrifugation and then the cells were plated on fibronectin - coated culture dishes. After 7 days of culture,attached cells were stimulated with different concentrations of PA for 48 hours. EPC were identified, by demonstra ring the expression of CD34,VEGFR- 2 and CD133 under a laser scanning confocal microscope. MTT assay was used to detect the effect of PA on the multiplication ability of EPC. Flow cytometry was used to detect the apoptosis caused by PA. The expression of Bcl - 2 genes mRNA and protein were detected respectively by RT - PCR and immunohistoehemistry technology. Result After exposure to PA,the proliferation of EPC was lower than that in control group,apoptosis rate was higher than that in control group （P〈 0.01 ） ,and it was dose - dependent in experiment range. Expression of Bcl - 2 genes mRNA and protein were down - regulated （P〈 0.05）. Conclusion PA could inhibit the proliferation of EPC and promote the apoptosis of the cells by down - regulation of bcl - 2 ex pression, which might contribute to vasculogenesis and reparation of blood vessel endothelium.