人脂肪组织来源内皮祖细胞的分离、培养及鉴定方法的实验研究

目的探讨人脂肪组织来源的内皮祖细胞的分离、培养及鉴定的方法。方法脂肪抽吸术获取人体脂肪组织,消化法得到的细胞接种在Fibronectin包被的培养皿内,用含2%胎牛血清的DMEM培养,传至第2代,分诱导组(EGM-2,2?S,VEGF,bFGF等)和非诱导组(DMEM,2?S)两组进行培养。免疫细胞荧光分别检测两组的CD34、vWF和PECAM-1表达;流式细胞仪分别检测两组的CD34、CD45、CD133和PECAM-1表达率;荧光显微镜观察细胞摄取DiI-ac-LDL的功能;诱导组细胞接种于甲基纤维素半固体培养基进行三维培养,观察血管样结构形成情况。结果诱导组细胞12d后呈现内皮细胞典型的铺路石样形态,未诱导组细胞未观察到这种变化;免疫细胞荧光显示诱导组vWF、PECAM-1表达阳性,未诱导组细胞CD34表达阳性;流式细胞仪检测显示诱导组PECAM-1阳性率为(67.41±13.35)%,明显高于非诱导组的(6.73±2.21)%(P<0.01),非诱导组CD34阳性率为(72.39±13.45)%,明显高于诱导组的(16.06±3.86)%(P<0.01);荧光显微镜观察显示诱导组细胞具有摄取DiI-ac-LDL的功能;诱导组细胞三维培养形成"树枝样"分叉结构。结论建立了一种从人脂肪组织分离、培养EPCs的方法,并对其表型进行鉴定,有望为血管组织工程提供新的种子细胞来源。

The isolation, subculture and identification of human adipose derived endothelial progenitor cells

OBJECTIVE: To investigate the methods of isolating and identifying human adipose derived EPCs. METHODS: The cells obtained from human lipoaspirates were plated on culture dishes coated with human fibronectin and were cultured in DMEM containing 2% FBS. Cells of passage 2 cultured in EGM-2 (2% FBS) served as the induced cells (experimental group), with cells cultured in DMEM (2% FBS) as the non-induced cells (control group) . Immunofluorescence was used to detect the expression of cell markers, including CD34, vWF and PECAM-1. FACS (fluorescence activated cell sorter) was used to quantitatively analyze the expression rate of cell markers (CD34, CD45, CD133 and PECAM-1). Fluorescence microscope was used to observe the function of taking up DiI-ac-LDL by the induced cells. To determine the ability of forming capillary-like structure in three-dimensional matrices, the induced cells were also cultured in methylcellulose. RESULTS: The induced cells of passage 2 exhibited cobblestone morphology, similar to that of the endothelial cells. In contrast, these morphological changes were not observed in non-induced cells. Immnofluorescence detected expression of vWF, PECAM-1 in induced cells and CD34 in non-induced cells. FACS analysis showed (67.41 +/- 13.35)% of the induced cells expressed PECAM-1 and (6.73 +/- 2.21)% of the non-induced cells expressed PECAM-1 (P < 0.01), while (72.39 +/- 13.45)% of the non-induced cells expressed CD34 and (16.06 +/- 3.86)% of the induced cells expressed CD34 (P < 0.01). Fluorescence microscopy observed the induced cells took up low-density lipoprotein (LDL). The formation of "branch-like" structure confirmed their functional activity. CONCLUSION: EPCs derived from human adipose may serve as another source of seeding cells for vascular tissue engineering.