内皮祖细胞对血管平滑肌细胞增殖的影响

Effect of endothelial progenitor cells on the proliferation of co-cultured vascular smooth muscle cells

目的:观察内皮祖细胞(endothelial progenitor cell,EPCs)对血管平滑肌细胞(vascular smooth muscle cell,VSMCs)增殖的影响。方法:采用6%羟乙基淀粉沉降红细胞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并向EPCs分化;采用荧光显微镜双染色、流式细胞术鉴定EPCs,间接免疫荧光检测VSMCs收缩表型标志物α-SM-actin和calponin的表达;Tr-answell培养板建立早期EPCs和大鼠VSMCs共培养模式,以20%胎牛血清刺激VSMCs增殖,分别共培养6,12,24,48,72h后收集VSMCs,采用BrdU标记法、蛋白定量、流式细胞术分析VSMCsDNA合成能力、细胞总蛋白含量以及细胞周期进程。结果:从脐血单个核细胞成功培养了EPCs。EPCs和大鼠VSMCs共培养12,24,48,72h后,VSMCsDNA合成能力、细胞总蛋白含量均较对照组明显降低(P<0.05),其中以48h最明显;流式细胞术显示,共培养组VSMCs细胞周期中S期细胞所占百分率较对照组显著降低(P<0.05),而细胞周期中G1期细胞所占百分率均相应高于对照组(P<0.05),其中均以48h最明显。结论:早期EPCs能够抑制VSMCs增殖。

Objective To explore the effect of endothelial progenitor cells （EPCs） on the proliferation of co-cultured rat vascular smooth muscle cells （ VSMCs ）. Methods Mononuclear cells were isolated from fresh cord blood by 6 % hydroxyethyl starch （HES） and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum （FBS）, VEGF, bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of α-SM- actin, calponin of VSMCs special antigen. Co-cuhure system of EPCs and VSMCs was established by transwell permeable support. FBS （20%） was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method, protein quantitation and flow cytometry after coculture for 6, 12, 24,48 and 72 h. Results After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the con- trol group （ P 〈 0.05 ）. Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/ EPCs co-cuhured group significantly decreased and the percentage of Gt phase increased markedly compared with the control group （ P 〈 0.05 ）. The maximal inhibitory effect was observed at 48 h. Conclusion Early EPCs could inhibit the proliferation of VSMCs.