骨髓CD34＋干细胞与单核细胞应用于组织工程小血管内皮化的比较

A compatitive research on the application of bone marrow CD34＋ stem cells vs. bone marrow mononuclear cells in small vascular endothelialization tissue engineering

目的探讨骨髓CD34＋干细胞与骨髓单核细胞（MNCs）应用于组织工程小血管内皮化的效果，并作比较。方法采集犬骨髓，经免疫磁珠分离出CD34＋细胞，内皮细胞生长因子（VEGF）诱导分化为内皮细胞并扩增，行细胞免疫荧光、免疫组织化学和体外成血管实验鉴定；将培养后的细胞和未经分离的MNCs分别种植于人工小血管支架，扫描电镜观察，比较两组组织工程小血管的内皮化。结果经流式细胞仪测定，分离后的细胞中CD34＋细胞含量为（78．46±6．37）％。CD34＋细胞培养2周后细胞基本铺满培养瓶底面，细胞呈“铺路石”状排列，CD31和Ⅷ因子免疫细胞化学染色均为阳性，体外成血管实验显示6h后CD34＋细胞组出现较多毛细血管样网状结构。CD34＋细胞组人工血管内膜化面积为（68．4±2．3）％，明显高于MNCs组人工血管内膜化面积（41．3±3．4）％，差异有统计学意义（P〈0．01）。结论通过免疫磁珠方法可分离得到高纯度的骨髓CD34＋细胞，经体外培养VEGF诱导后可定向分化为内皮细胞，种植于人工血管内表面，较MNCs组内皮化效果理想。

Objective To compare the application of bone marrow CD34＋ stem cells vs. bone marrow mononuclear cells （MNCs） in vascular tissue prosthesis engineering. Methods We gathered dog bone marrow, segregated endothelia progenitor cells （EPCs） through immunomagnetic beads, and induced them to differentiate to endothelia cells （ECs） by endothelial cell growth fatter（ VEGF）. Then we identified ECs through inverted phase contrast microscope, immunocytochemistry and tube-like structure formed test. Then we take some unfraetioned MNCs, after routine cultivation implant them in the vascular prosthesis, and compare them under the microscope to the previous group. Results CD34 ＋ ceils being segregated were （78.46 ± 6. 37 ） % in content evaluated by flowcytometry, and were spread on the undersurface of culture flask 2 weeks after being cultured, and lined up as slabstones, being masccline for CD31 and Ⅷ factor in immunocytochemical staining. Under scanning eletron microscope, ECs tiled on the surface of vascular prosthesis ,with their pseudopodia stretching out to micropore of vascular internalsurface; in the MNCs group no reticular structure can be readily found. For the CD34 cell group, the vascular prosthesis endothe- lialization area is （68.4 ±2. 3）%, which is obviously more than that for the MNCs cell group （41.3 ±3.4） % ,P 〈 . 01. Conclusion Highly purified bone marrow CD34 ＋ cells can be obtained through immu- nomagnetic beads, cultured in vitro and differentiated to ECs directionally by VEGF. Better endothelializa- tion effect can be gained from cell plantation on internal surface of vascular prosthesis than MNCs.