同步诱导培养兔骨髓源性内皮祖细胞和平滑肌祖细胞

Synchronous culture of rabbit bone narrow-derived endothelial progenitor cells and smooth muscle progenitor cells

目的采用兔骨髓来源的单个核细胞同步诱导分化为兔内皮祖细胞(EPCs)和平滑肌祖细胞(SPCs),研究其生物学特性,评估其作为组织工程化静脉瓣种子细胞的可能性。方法梯度密度离心法获取兔骨髓血单个核细胞沉淀,分别用含5%胎牛血清(FBS)的EGM-2完全培养液向EPCs方向诱导培养;用含20ng/mL血小板源性生长因子BB、5%FBS,不含血管内皮生长因子(VEGF)的EBM-2培养液向SPCs方向诱导培养。鉴定两种细胞的分化情况。结果诱导的EPCs培养至10d左右,细胞单层融合呈"铺路石"状;表达血管内皮细胞生长因子受体2(VEGFR-2)、血管性血友病因子(vWF)、CD133,不表达α-平滑肌肌动蛋白(α-SMA);透射电镜可见细胞质内特征性Weibel-Palade小体;细胞生物学功能检测可见EPCs在基质胶上呈现血管状生长。诱导的SPCs培养至14d左右呈现血管平滑肌细胞"峰-谷"样生长特性;表达CD34、α-SMA,不表达vWF和VEGFR-2;在透射电镜下可见细胞内含有与细胞纵轴平行排列的肌丝;在基质胶上不规则生长。结论兔骨髓血梯度密度离心得到的单个核细胞可同步诱导分化为EPCs和SPCs,为构建组织工程化静脉瓣提供了经济且可简便获取的种子细胞。

Objective To induce synchronous differentiation of rabbit bone marrow-derived mononuelear cells into endothelial progenitor cells （EPCs） and smooth muscle progenitor cells （SPCs）, and to study their biological properties and the possibility of them as seed cells for tissue-engineered venous valves. Methods Gradient density centrifugation was used to obtain bone marrow blood mononuclear cells, which were separately cultured with EGM-2 complete medium containing 5% FBS for differentiation of EPCs and with EBM-2 medium without vascular endothelial growth factor （VEGF） containing 5 % FBS and 20 ng/mL platelet-derived growth factor-BB （PDGF-BB） for differentiation of SPCs. The differentiation of EPCs and SPCs was identified by various methods. Results EPCs were cultured for 10 days and the cells fused into monolayer, showing a ＂stepping stone＂ appearance and expressing VEGF receptor-2 （VEGFR-2）, yon Willebrand factor （vWF） and CD133, but not α-smooth muscle actin （α-SMA） ; Weibel-Palade bodies were seen within the EPCs cytoplasm under the transmission electron microscope. Biological function tests showed visible EPCs growing on the matrigel in a blood vessel-like form. SPCs were cultured for 14 days and showed the specific features of the vascular smooth muscle growth, namely, the ＂peak-valley＂ growth way. SPCs expressed CD34 and α-SMA but not vWF and VEGFR-2. Myofilaments, paralleling with the cell longitudinal axis, were seen under the transmission electron microscope. SPCs could not form vessel-like structures on the matrigel. Conclusion Mononuclear cells can be obtained through gradient density centrifugation of the bone marrow blood, which can be synchronously induced into EPCs and SPCs, providing economical and easy seed cells for tissue-engineered venous valves.