内皮祖细胞对血管平滑肌细胞增殖的影响

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;Transwell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6,12,24,48,72 h后收集VSMCs,采用BrdU标记法、蛋白定量、流式细胞术分析VSMCs DNA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12,24,48,72 h后,VSMCs DNA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48 h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48 h最明显。结论：早期EPCs能够抑制VSMCs增殖。

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

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-culture 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 co-culture 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 control group(P＜0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G_1 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.