人外周血内皮祖细胞的培养与鉴定

Culture and identification of endothelial progenitor cells from human peripheral blood

目的:探讨人外周血内皮祖细胞体外诱导分化及鉴定的方法,为自体外周血内皮祖细胞移植的临床应用打基础。方法:实验于2004-09/2005-05在解放军第四军医大学西京医院肝胆外科实验室完成。采集献血员抗凝外周血,经Ficoll密度梯度离心法获取单个核细胞,在含有体积分数为0.1胎牛血清、20μg/L的血管内皮细胞生长因子和4μg/L的碱性成纤维细胞生长因子的M199培养液中培养和扩增,分别在培养的第6和14天,流式细胞仪检测其KDR,CD133,CD34和vWF阳性率,并通过检测其对异硫氰酸荧光素标记的荆豆凝集素的吸附和内吞DiI-acLDL来进行细胞功能学的鉴定。结果:①人外周血单个核细胞经体外诱导分化,培养第6天的贴壁细胞表达KDR,CD133,CD34和vWF,流式细胞仪检测其阳性率分别为(46.4±9.3)%、(33.8±11.7)%、(73.5±6.3)%和(38.9±8.2)%,能特异性吸附异硫氰酸荧光素标记的荆豆凝集素并内吞DiI-acLDL。②培养第14天的贴壁细胞表达KDR,CD34和vWF,不表达CD133,流式细胞仪检测其阳性率分别为(81.5±7.6)%、(88.9±5.4)%和(78.3±13.6)%,能特异性吸附异硫氰酸荧光素标记的荆豆凝集素并内吞DiI-acLDL。结论:外周血来源的单个核细胞在一定的培养条件下可以分化为内皮祖细胞,为自体外周血内皮祖细胞移植的临床应用打下基础。

AIM： To examine whether endothelial progenitor cells （EPCs） can be induced and differentiated from human peripheral blood （PB） and identified in vitro, and expiore the background for clinical application of autologous PB EPCs transplantations. METHODS： The experiment was conducted at the Laboratory of Hepatobiliary Surgery, Xijing Hospital of the Fourth Military Medical University of Chinese PLA from September 2004 and May 2005. The mononuclear cells of PB from blood volunteers obtained by Ficoll densitygradient centrifugation were cultured in medium 199 （M199） supplemented with 10% fetal bovine serum, 20μg/L vascular endothelial growth factor （VEGF） and 4μg/L basic fibreblast growth factor （bFGF） in vitro. The expressions of specific antigens （KDR, CD133, CD34 and vWF） on cell surface were analyzed by flow cytometer at the 6th and 14th days during culture respectively. The biological functions of endothelial cells Were examined by the adsorption of ulex eurepaeus-agglutinin （UEA） labeled by fluorescein isothiacyanate （F1TC） and DiI-Ac-LDL internalization. RESULTS： ①After in vitro induction and differentiation of mononuclear cells in human PB, the adhesive cells expressed KDR, CDI33,CD34 and vWF at the 6th day during culture, and the positive ratio were （46.4±9.3）%, （33.8±11.7）%, （73.5±6.3）% and （38.9±8.2）% respectively.②At the 14th day during culture, the adhesive cells expressed KDR, CD34 and vWF except CD133, and the positive ratio were （81.5±7.6）%, （88.9±5.4）% and （78.3±13.6）% respectively. The experiment of FITClabeled UEA adsorption and DiI-Ac-LDL internalization were both positive at the 6th and 14th days during culture. CONCLUSION： The mononuclear ceils from human PB can be differentiated into EPCs under certain condition, which provides the basement for clinical application of autologous PB EPCs transplantations.