巴马小型猪骨髓来源内皮祖细胞的培养鉴定及生物学特性分析简

为探索并建立巴马小型猪骨髓来源内皮祖细胞(EPCs)的体外培养及鉴定方法，本研究在健康猪髂后上棘取骨髓，采用密度梯度分离法分离单个核细胞，按2×10⁷个/孔的密度加入预先包被人纤维连结素（HFN）的6孔细胞培养板中，用含内皮细胞生长因子的选择性培养基诱导培养，逐日在倒置相差显微镜下观察，MTT法检测EPCs增殖情况；流式细胞术（FACs）检测EPCs中AC133和vWF的表达纯度；免疫荧光法双染检测EPCs表面相关抗原ac-LDL和UEA-1。结果发现，从巴马小型猪骨髓分离的EPCs具有增殖能力；MTT结果显示细胞于48 h换液二次贴壁后3～5 d增殖明显，7～10 d增殖加速并出现条索状结构；FACs检测结果显示，二次贴壁细胞vWF阳性细胞百分率74.91%，AC133阳性细胞百分率79.54%，而未经诱导的二次贴壁细胞vWF和AC133阳性率分别是33.65%和39.87%；细胞经免疫荧光双染后Dil-ac-LDL和FITC-UEA-1表达呈阳性，vWF和AC133免疫荧光染色阳性。由此可见，分离培养的细胞具有自我增殖和分化潜能，是内皮祖细胞。

Research of the Culture Condition and Biological Characteristics Endothelial Progenitor Cells from Bama Minipigs Marrow in vitro

To investigate and establish the methods of in vitro culturing and identification of endothelial progenitor cells (EPCs) of Bama Minipigs. The mononuclear cells were gained from Bama Minipigs bone marrow by density gradient centrifugation，inoculated （2×10⁷） in the culture plates，which were coated with HFN and induced differentiation to EPCs.The EPCs were observed by inverted microscope, compare the proliferation status of both derived EPCs by observing the growth curve，MTT method，and the express of EPCs-related antigen ac-LDL, UEA-1, AC133 and vWF by immunofluorescence staining.MTT results showed that the proliferation of the cells were obvious at 3 to 5 d.FACs test results showed that vWF adherent cells twice percentage of positive cells was 74.91%, AC133-positive cell percentage was 79.54%, while the secondary without adherent cells induced vWF and AC133-positive rate were 33.65% and 39.87%. The cells were identified by immunofluorescence expression of ac-LDL, UEA-1, AC133 and vWF were positive. Thus, the cells isolated and cultured from Bama Minipigs bone marrow were shown that strong ability of self-renewal proliferation and multipotency of differentiation which identified as the endothelial progenitor cells.