差速贴壁法分离兔骨髓源性间充质干细胞和内皮祖细胞及其生物学特性的研究

本研究探讨一种高效、稳定的从兔骨髓中同时分离培养间充质干细胞（Mesenchymal Stem Cells,MSC）和内皮祖细胞（Endothelial Progenitor Cells,EPC）的方法并观察其生物学特性。抽取兔骨髓,应用密度梯度分离单个核细胞,采用差速贴壁经纤连蛋白包被并结合EGM-2MV培养基分别扩增MSC和EPC。用台盼蓝法测定细胞传代成活率,通过绘制生长曲线法、MTT法、DNA周期检测MSC和EPC的增殖能力及诱导分化成骨细胞、成脂肪细胞能力,并结合流式细胞术（FCM）检测MSC免疫原型以鉴定MSC;细胞吞噬功能特异性地摄取Dil-ac-LDL及FITC-UEA-1,并结合CD133、VEGFR2/KDR、CD34免疫荧光鉴定EPC,并计算其纯度。结果表明,经密度梯度分离单个核细胞,在早期贴壁细胞24 h换液时即可见明显集落形成,8 d后达80%融合,细胞呈均匀一致的长梭形排列;2次贴壁细胞经EGM-2MV培养基培养,第3天开始伸展,约8 d可融合近80%,细胞呈多角形,出现条索状结构;2种细胞台盼蓝法测定细胞传代成活率均在90%以上,传至第2代后,生长曲线均近似＂S＂形;MTT法检测显示,细胞生长d 3至d 5时光密度值变化较明显;MSC G0-G1期为（93.32±1.65）%、EPC G0-G1为（93.05±1.95）%,2种细胞DNA周期无明显差异;早期贴壁细胞FCM检测CD90、CD44阳性率为（99.7±1.12）%、（99.1±2.33）%;CD14、CD45、CD79a阳性率分别为（4.8±0.38）%、（6.8±0.49）%及（0.4±0.08）%,经体外诱导能够向成骨细胞及脂肪细胞分化,鉴定为MSC;2次贴壁细胞传至第2代经Dil-ac-LDL、FITC-UEA-1双荧光染色阳性率（82.1±3.4）%,CD133、VEGFR2/KDR、CD34免疫荧光染色阳性率分别为（74.2±3.2）%、（64.7±4.3）%及（43.5±1.5）%,鉴定为EPC。结论：应用密度梯度分离法结合差速贴壁筛选法可培养出高纯度的MSC,2次贴壁细胞经纤连蛋白预包被并结合EGM-2MV培养基体外诱导可培养出增殖能力较强的EPC,为组织工程学研究提供种子细胞。

Mesenchymal Stem Cells and Endothelial Progenitor Cells Obtained from Rabbit Bone Marrow with Differential Adhesion Methods and their Biological Characteristics

This study was aimed to evaluate an effective and stable method for obtaining mesenchymal stem cells （MSC） and endothelial progenitor cells （El＇C） from the rabbit bone marrow and to investigate the biological characteris-tics of MSC and EPC. Mononuclear cells were obtained from rabbit bone marrrow using density gradient method, and were differentially adhered to the cell culture plate enclosed with fibronectin. Then, MSC and EPC were amplified with EGB-2MV medium. Trypan blue method was used to test the passage survival rate. Growth curve, MTr and DNA cycle were used to evaluate the proliferation ability of MSC and EPC. MSC ffere identified with induced differentiation into the osteoblasts and adipocytes, and their immune phenotype was detected by flow cytometry （FCM）. EPC were character- ized by the special digestion of Dil-ac-LDL, FITC-UEA-I, md the conjunction with CD133, VEGFR2/KDR andCD34, their purity was also calculated. The results indicated that the colony was obviously formed when the mononucle ar cells were cultured for 24 hours and,80% of the cells became long spindle and integrated at d 8. Cells, which were adhered for twice, were cultured with EGM-2MV medium, began to extend at d 3, and became strip-shaped and integrated for about 80% at d 8. Passage survial rates were more than 90% for both ceils, and after passage 2 the growth curve was like ＂S＂. Optical density was changed obviously when the cells were cultured for 3 - 5 d, but there were no significant difference of cell cycles between MSC and EPC, which GO - G1 was （ 93.32± 1.65 ） % and （ 93.05 ± 1.95 ） % respectively. Positive rates were （99.7 ± 1.12） %, （ 99.1 ± 2.33 ） %, （4.8 ± 0.38 ） %, （ 6.8 ± 0.49） % and （ 0.4± 0.08 ） % for CD90, CD44, CD 14, CD45 and CD79a respectively. MSC were identified by induced differentiation into osteoblasts and adipocytes. Positive rates of the EPC, which were adhered for twice and passaged 2, were （82.1 ± 3.4） % for fluorescent staining of Dil-ac-LDL and FITC-UEA-I, and （ 74.2 ± 3.2） %, （ 64.7 ± 4.3 ） % and （43.5 ± 1.5） % for CD133 ,VEGFR2/KDR and CD34 respectively. It is concluded that high-purity MSC can be obtained with density gradient and differential adhesion method, and high-proliferation EPC can be cultured with EGM-2MV medium in cell plates enclosed with fibronectin, so they may become the optimal seed cells for tissue engineering study.