摘要
目的体外诱导大鼠骨髓内皮祖细胞(EPCs)成内皮细胞,探索其作为血管组织工程内皮种子细胞的可行性。方法冲洗大鼠骨髓腔,通过密度梯度离心得到单个核细胞;按诱导(M199,20%FBS,VEGF10ng/mL,bFGF2ng/mL)和未诱导(M199,20%FBS)分组培养骨髓单个核细胞,体外培养至第二代;免疫细胞荧光分别检测诱导组和未诱导组细胞VWF、VEGFR-2、VE-cadherin和PECAM-1的表达;流式细胞仪分别检测骨髓单个核细胞、诱导组细胞和未诱导组细胞VEGFR-2、VE-cadherin和PECAM-1表达率;诱导组细胞接种于胶原凝胶中三维培养,观察血管生成能力。结果诱导组原代细胞呈小梭形,5～7d时出现典型的线样结构,P2、P3细胞逐渐成内皮细胞典型铺路石样结构,未诱导细胞未见这两种典型结构;免疫细胞荧光发现,诱导组细胞有VWF、VEGFR-2、VE-cadherin和PECAM-1表达,未诱导组未见明显表达;流式细胞仪检测显示三种标记(VEGFR-2、VE-cadherin和PECAM-1)在原代时的比例均不到总细胞数5%,经两代培养后,诱导组中阳性细胞率高于35%,未诱导组阳性细胞率明显低于诱导组(P<0.01);三维培养状态下诱导组细胞集落在胶原内有“出芽式”生长。结论本实验建立了一套骨髓EPCs分离、诱导培养和表型检测的方法;以VEGF和bFGF为主要诱导因子的诱导体系,对骨髓EPCs成内皮细胞?
Objective To investigate the feasibility of inducing rat bone marrow derived EPCs (endothelial progenitor cells) into endothelial cells in vitro. Methods EPCs harvested from bone marrow by flushing rat femur and tibia were isolated by Histopaque1083 density gradient centrifugation. The isolated cells that were induced in M199 medium containing 20% FBS, VEGF (10ng/mL) and bFGF (2ng/mL) served as the experimental group, and those not induced in M199 containing 20% FBS served as the control group. Immunofluorescence was used to detect the expression of endothelial cell markers, such as vWF, VEGFR 2, VE cadherin and PECAM 1 in the passage 2 cells. FACS (fluorescence activated cell sorter) was used to quantitatively analyze the expression rate of endothelial cell markers (VEGFR 2, VE cadherin and PECAM 1) of non induced and induced cells respectively. The cells were also cultured in rat tail type Ⅰ collagen gel to observe the EPCs’ ability of forming capillary structure in three dimensional matrices. Results The induced passage 0 EPCs exhibited small spindle shape and were able to line up in the typical cord like structure. At passages 2 and 3, the cells exhibited cobblestone morphology, similar to endothelial cells. In contrast, these morphological changes were not observed in non induced cells. Immunofluorescence detected expression of endothelial cell markers in induced cells, but not in non induced cells. FACS analysis also demonstrated that more than 35 percent of induced cells expressed specific markers, while the marker expression in non induced cells was less then 5 percent (P<0.01). Interestingly, capillary like structure with terminal branches was observed in the collagen gel containing induced cells, but not in non induced cells. Conclusion Bone marrow derived EPCs can be differentiated into endothelial cells with VEGF and bFGF induction. The differentiated EPCs have the potential to serve as seeding cells for endothelium construction in blood vessel tissue engineering.
出处
《中华创伤骨科杂志》
CAS
CSCD
2004年第10期1154-1159,共6页
Chinese Journal of Orthopaedic Trauma
基金
国家高技术研究发展计划(863计划)课题(2001AA216021)
国家高技术研究发展计划(863计划)课题(2002AA205011)
关键词
骨髓
内皮祖细胞
内皮细胞
组织工程
Bone marrow
Endothelial progenitor cells
Endothelial cell
Tissue engineering
