犬骨髓内皮祖细胞生物学特性及诱导分化

目的:探讨犬骨髓内皮祖细胞(EPCs)随培养时间延长其生物学特性变化及向内皮细胞(ECs)分化能力。方法:免疫微珠分选方法纯化犬骨髓CD14 细胞,EGM-MV2条件培养基培养,于不同时间检测EPCs的生物学特性;将存活至8周的EPCs以分化培养基(M199 20%胎牛血清 VEGF)诱导培养,检测ECs表面标志的表达。结果:早期EPCs为分选后8代之前的EPC,体积略小,类圆形,60%融合时呈串珠样排列,表达CD133、CD14;晚期EPCs由表达VE-cadherin、KDR、CD14的早期EPCs分化而来,形态为多边形,分泌活动明显加强,表达CD133、VE-cadherin、CD14和KDR;诱导分化细胞呈鹅卵石样外观,表达Ⅷ因子、CD31。结论:犬骨髓EPCs随培养时间呈现不同生物学特性,早期EPCs不稳定,晚期EPCs显示出更稳定的生物学特性,能够分化为ECs,是血管组织工程学研究良好的种子细胞。

Characterization and differentiation of endothelial progenitor cells from canine bone marrow

Objective:To reveal the changes of biological characteristics of endothelial progenitor cells(EPCs) throughout the course of cell culture in vitro,and to demonstrate the differentiated potential of EPCs in different stages.Methods: The CD14 cells derived from canine bone marrow and purified by using magnetic cell sorting were cultured in EGM-MV2.The biological characteristics of EPCs were tested at different time points in vitro.Eight weeks later,the late EPCs were cultured in M199 supplemented with 20% fetal calf serum and vascular endothelial growth factor(VEGF). Meanwhile,the endothelial cells(ECs) surface markers were examined.Results: The early EPCs were small and polygon in shape initially,and changed to typical string-of-beads ultimately,while the spindle-shape cells were exhibited dominantly in the late ones appeared at 34 weeks.After induced by specific media and VEGF,the late EPCs became cobblestone in shape,which was typical phenotype of ECs,and reflected positively to factor VIII and CD31.The positive reactions of CD133 and CD14 were found in both the early EPCs and the late EPCs.The expressions of KDR and VE-cadherin in mRNA level were detected in the early and late EPCs,respectively.The nitrogen monoxidum(NO) produced by the late EPCs was more than that by the early EPCs,but less than that by ECs statistically.Conclusion:The late EPCs differ to the early EPCs in morphological characteristics,survival and proliferative capability,gene expression,as well as in secretary function,suggesting that they are suitable to create engineered cardiovascular tissue as a cell resource due to their unchangeable biological characteristics and committed differentiation into ECs.