体外培养基中加入细胞生长因子诱导大鼠骨髓单个核细胞向内皮细胞的分化  

In vitro differentiation of rat bone marrow monocytes into endothelial cells induced by adding cell growth factors in the medium

作　　者：陈文娜[1] 李大勇[2] 李曦明[1] 曲静[1] 马贤德[1] 孙宏伟[1] 

机构地区：[1]辽宁中医药大学,辽宁省沈阳市110032 [2]辽宁中医药大学附属第一医院,辽宁省沈阳市110032

出　　处：《中国组织工程研究与临床康复》2008年第21期4065-4068,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30600843):疏肝活血法促血管生成治疗肢体缺血性疾病的实验研究~~

摘　　要：背景:内皮祖细胞在骨髓中的含量高于外周血,但也仅占骨髓单个核细胞的1%。因此,在一般实验室培养过程中也需要解决提高内皮祖细胞体外扩增数量并保持其低分化状态的问题。目的:观察在含胎牛血清的M199培养基中加入血管内皮细胞生长因子和碱性成纤维细胞生长因子诱导内皮祖细胞的定向分化能力。设计、时间及地点:以细胞为观察对象的开放性实验,于2007-09/2008-02在辽宁中医药大学教学实验中心bsl-2级实验室完成。材料:体质量(200±20)g的健康成年Wistar大鼠。方法:无菌采集健康Wistar大鼠股骨骨髓,应用Ficoll密度梯度离心的方法获得单个核细胞,加入含胎牛血清+肝素+血管内皮细胞生长因子和碱性成纤维细胞生长因子的M199实验培养液和不含诱导生长因子与肝素的M199培养液为对照组。主要观察指标:诱导骨髓细胞向内皮样细胞生长过程中,应用表面抗原免疫细胞化学染色鉴定细胞,以Dil-ac-LDL和FITC-UEA-1双荧光实验鉴定培养细胞向内皮细胞分化的程度。结果:贴壁细胞培养诱导后具有内皮细胞的形态学特征,细胞呈上皮细胞样铺路石状贴壁生长,贴壁时间相对缓慢。加入M199实验培养液诱导后的第9天Ⅷ因子、CD31阳性表达的细胞分别为(55.1±7.2)%、(45.6±5.8)%,Dil-ac-LDL和FITC-UEA-1双荧光染色阳性率为(78.2±5.4)%,提示为正在分化的内皮祖细胞。对照组相关抗原表达阴性,提示为分化早期的骨髓细胞。结论:结果显示骨髓单个核细胞培养出可稳定扩增的内皮祖细胞,在体外特殊诱导环境下定向分化为内皮样细胞,且效果优于对照组。BACKGROUND： The endothelial progenitor cells （EPCs） in bone marrow are more excessive than those in peripheral blood monocytes, but EPCs only hold 1% of all the monocytes in bone marrow. So it is an urgent problem to amplify the quantity of EPCs in vitro and keep those cells in poorly differentiated status. OBJECTIVE： To investigate the oriented differentiation capacity of EPCs by the addition of vascular endothelial cell growth factor （VEGF） and basic fibroblast growth factor （bFGF） in M199 medium containing fetal bovine serum （FBS）. DESIGN, TIME AND SETTING： From September 2007 to February 2008. an open trial subjected as cells was carried out in the Center of Teaching and Researching （bsl-2 laboratory） of Liaoning University of Traditional Chinese Medicine （Shenyang, Liaoning China）. MATERIALS： Healthy adult Wistar rats weighing （200±20） g were adopted. METHODS： Femoral bone marrow of adult Wistar rats was gathered in germ free. Mononuclear cells in the middle layer were collected by Ficoll discontinuous gradient centrifugation, and the cells were suspended and cultured in M199 medium supplemented with VEGF and bFGF, while those in M199 without growth factor and heparin and were taken as control group. MAIN OUTCOME MEASURES： During the induction of bone marrow monocytes into endothelioid cells, surface antigen cytochemical staining was applied to identify various cells. The differentiation degree of the cultured cells into endothelial cells was calculated by Dil-ac-LDL and FITC-UEA-1 double immunofluorescent staining. RESULTS： After the culture and induction, the cells represented the morphology identical with the endothelial cells, they adhered gradually and grew in a shape of paving stone. At day 9 after the M 199 was added, the expression rates of factor Ⅷ and CD31 were （55.1±7.2）% and （45.6±5.8）%, respectively. EPCs were characterized as adherent cells （78.2±5.4）% positive for Dil-ac-LDL and FITC-UEA-1, which showed the differentiation p

关 键 词：骨髓单个核细胞 内皮祖细胞 分化 

分 类 号：R394.2[医药卫生—医学遗传学]