氧化型低密度脂蛋白对人脐静脉内皮祖细胞增殖、凋亡及bcl-2表达的影响

Effect of Oxidized Low Density Lipoprotein on Proliferation,Apoptosis,and Expression of bcl-2 of Endothelial Progenitor Cells from Human Umbilical Cord Blood in Vitro

目的探讨氧化型低密度脂蛋白对人脐静脉内皮祖细胞增殖、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7天后,收集贴壁细胞并加入不同浓度氧化型低密度脂蛋白(分别为5、10及20mg/L)干预48h。MTT法检测氧化型低密度脂蛋白对内皮祖细胞增殖能力的影响,流式细胞仪检测氧化型低密度脂蛋白对细胞凋亡率的影响,逆转录聚台酶链反应检测bcl-2mRNA的表达,免疫细胞化学法检测bcl-2蛋白的表达。结果氧化型低密度脂蛋白呈浓度依赖性抑制内皮祖细胞增殖,诱导内皮祖细胞凋亡(P<0.01);基础状态下内皮祖细胞表达bcl-2mRNA及蛋白,氧化型低密度脂蛋白处理能抑制其表达,并存在量效关系(P<0.05)。结论氧化型低密度脂蛋白通过下调bcl-2的表达诱导内皮祖细胞凋亡、抑制内皮祖细胞增殖,这可能影响血管内皮的修复及新生血管的形成。

Aim To investigate the effect of oxidized low density lipoprotein （ ox-LDL） 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 centrifugafion and then the cells were plated on fibronectin-coated culture dishes. After 7 d d culture, attached cells were stimulated with different cencentrations of ox-LDL （5,10 and 20 mg/L） for 48h. EPC were identified by demonstrating the expression of CD34, KDR and CD133 under a laser scanning confocal microscope. MTr assay was used to detect the effect of ox-LDL on the multiplication ability of EPC. Flow cytometry was used to detect the apoptosis caused by ox-LDL. The expression of bcl-2 genes mRNA and protein were detected respecfivdy by RT-PCR and immunohistochemistry technology. Results After exposure to ox-LDL, the proliferation of EPC was lower than that of control group, apoptosis rate was higher than that of control group （ P 〈 0.01 ）, and it was dose-dependent in experiment range; expression of bcl-2 mRNA and protein were down-regulated （P〈0.05）. Conclusion Ox-LDL can inhibit the proliferation of EPC and promote the apoptosis of the ceils by down-regulation of bcl-2 expression, which may contribute to vasculogenesis and reparation of blood vessel endothelitan.