兔外周血与骨髓源性内皮祖细胞培养鉴定及生物学特性的研究

目的:探索并建立兔内皮祖细胞(EPCs)的体外培养、鉴定方法。方法:采用密度梯度分离法分别从兔外周血和骨髓中分离出单个核细胞,接种于预包被明胶的培养瓶,并应用培养基(EBM-2)诱导培养,诱导分化为EPCs;在倒置显微镜下观察两种源性细胞生长过程;台盼蓝法测定细胞传代成活率;通过绘制生长曲线法、MTT法、DNA周期检测比较两种源性获得的EPCs增殖情况;采用免疫荧光染色观察EPCs相关抗原CD34、CD31、CD133及vWF因子的表达。结果:两种源性EPCs均于培养3～4d完全贴壁,呈克隆样生长,7～9d形成类似成熟血管内皮细胞形态,细胞数量逐渐增多,细胞成活率均为95%。两种源性获得的第2代细胞生长曲线近似"s"形、MTT法显示3～5d细胞增殖较明显,外周血源性EPCs G0-G1期和S+G2+M期所占比例分别为96.48%和3.52%,骨髓源性EPCs G0-G1期和S+G2+M期所占比例分别为97.11%和1.84%。第2代EPCs进行免疫荧光鉴定结果显示:两种源性内皮祖细胞均阳性表达CD34、CD133、vWF因子,CD31弱阳性表达。结论:两种源性的EPCs均可高效获得且在体外稳定培养。与传统的骨髓源性EPCs相比较,从外周血源获得的EPCs是一种可靠、简便的培养方法,为组织工程学提供理想的细胞来源。

Research of the culture condition and biological characteristics endothelial progenitor cells from rabbit marrow and peripheral blood in vitro

Objective:To investigate and establish the methods of in vitro culturing,differentiation and identification of endothelial progenitor cells(EPCs) of rabbit.Methods: The mononuclear cells were gained separately from rabbit bone marrow and peripheral blood by density gradient centrifugation,inoculated in the culture bottle that coated with gelatin and cultured in endothelial cell basel medium-2 then induced differentiation to EPCs.We observe the growth process of both derived EPCs by inverted microscope;measure the survival rate of passaging cells by typan blue exclusion;compare the proliferation status of both derived EPCs by observing the growth curve,MTT method,and testing the cell cycle;observe the express of EPCs-related antigen CD34,CD31,CD133,and von willebrand factor(vWF) by immunofluorescence staining.Results: Both bone marrow-derived EPCs and peripheral blood-derived EPCs are completely adherent after being cultured 3～4 d,both are clone-like growth,form a similar mature vascular endothelial cell morphology after 7～9 d,the number of cells increases gradually,the cell survival rates are 95%.The growth curves of the second generation of Both derived EPCs are S-shaped,MTT method shows that the proliferation of the cells are obvious at 3～5 d,the G0-G1 phase and S + G2 + M phase proportion of peripheral blood-derived EPCs are 96.48% and 3.52%,the G0-G1 phase and S + G2 + M phase proportion of bone marrow-derived EPCs are 97.11% and 1.84%.The result of the identification of the second generation of EPCs by immunofluorescence shows: two sources of EPCs are both positively expressed CD34、CD133 and vWF,but CD31 is weakly expressed.Conclusion: Both derived EPCs could be obtained efficiently stably cultured in vitro.And compared to the traditionally achieved EPCs from bone marrow,achieved EPCs from peripheral blood is reliable and more convenient method,providing the ideal source of cells for tissue engineering.