内皮祖细胞对血管紧张素Ⅱ诱导的血管平滑肌细胞表型转化的影响
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Effect of Endothelial Progenitor Cell on the Phenotype Transformation of Cultured Rat Vascular Smooth Muscle Cells Induced by Angiotensin Ⅱ
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    目的观察内皮祖细胞对血管紧张素Ⅱ诱导的血管平滑肌细胞表型转化的影响。方法采用6%羟乙基淀粉沉降法和密度梯度离心法分离人脐血单个核细胞,EGM-2细胞培养基进行培养,诱导单个核细胞贴壁向内皮祖细胞分化。采用荧光显微镜双染色、流式细胞术鉴定内皮祖细胞,间接免疫荧光检测血管平滑肌细胞标志物平滑肌α-肌动蛋白、钙调节蛋白的表达,采用逆转录聚合酶链反应和免疫印迹检测早期内皮祖细胞条件培养液、晚期内皮祖细胞条件培养液以及人脐静脉内皮细胞条件培养液对血管紧张素Ⅱ诱导的血管平滑肌细胞收缩表型标志基因平滑肌α-肌动蛋白以及合成表型标志基因骨桥蛋白表达变化的影响。结果与对照组比较,血管紧张素Ⅱ(10~(-6) mmol/L)诱导血管平滑肌细胞增殖48 h后,平滑肌α-肌动蛋白mRNA和蛋白表达明显减少,而骨桥蛋白mRNA和蛋白表达明显增加,提示血管平滑肌细胞从收缩表型向合成表型转化;与血管紧张素Ⅱ组比较,早期内皮祖细胞条件培养液、晚期内皮祖细胞条件培养液以及人脐静脉内皮细胞条件培养液处理后均不同程度抑制血管紧张素Ⅱ诱导的平滑肌α-肌动蛋白表达减少和骨桥蛋白表达增加,其中以早期内皮祖细胞条件培养液的抑制效果最明显。结论内皮祖细胞能够抑制血管紧张素Ⅱ诱导的血管平滑肌细胞从收缩表型向合成表型转化。

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    Aim To explore the effect of endothelial progenitor cells(EPC) on the phenotype transformation of cultured rat vascular smooth muscle cells(VSMC) stimulated by angiotensinⅡ.Methods Mononuclear cells were isolated from fresh cord blood by 3%Gelatin and density gradient centrifugation.Isolated cells were cultured in EGM-2 medium supplemented with 20%FBS,VEGF,bFGF and other growth factors.Biological features of EPC were observed at different time points,and EPC was identified by morphology,fluorescence double-staining and flow cytometry.Indirect immunofluorescence was performed to analyze expression ofα-SM-actin,calponin of VSMC special antigen.RT-PCR and Western blotting were performed to analyze the effect of endothelial progenitor cell conditional medium(E-EPC-CM,LEPC -CM) and human umbilicus vein endothelial cell conditional medium(HUVEC-CM) on AngⅡ-induced expression ofα-SM-actin and osteopontin in VSMC.VSMC was divided into control group,AngⅡgroup,AngⅡ+EGM-2 group,AngⅡ+E-EPC-CM group,AngⅡ+L-EPC-CM group and AngⅡ+HUVEC-CM group.Results After stimulation with angiotensinⅡfor 48 h,the expression of theα-SM-actin mRNA and protein significantly decreased and the osteopontin mRNA and protein markerly increased compared with control group,suggesting that VSMC was changed from contractile to synthesize type by angiotensinⅡ.Treatments with E-EPC-CM,L-EPC-CM,HUVEC-CM,especially with E-FPC-CM, could inhibit significantly the down-regulation ofα-SM-actin expression and the up-regulation of osteopontin expression by AngⅡ.Conclusion EPC could inhibit the phenotype transformation of VSMC from contractile to synthesize type induced by angiotensinⅡ.

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方立,陈美芳,余国龙,肖智林,谢秀梅.内皮祖细胞对血管紧张素Ⅱ诱导的血管平滑肌细胞表型转化的影响[J].中国动脉硬化杂志,2009,17(6):459~464.

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  • 收稿日期:2009-02-23
  • 最后修改日期:2009-06-02
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