绵羊骨髓来源内皮祖细胞的培养与鉴定

Culture and identification of sheep bone marrow-derived endothelial progenitor cells

背景:当今,组织工程化静脉瓣的研究刚刚起步,种子细胞的选择是其关键,内皮祖细胞可以作为组织工程化静脉瓣体外构建理想的种子细胞。目的:通过体外培养与鉴定绵羊骨髓来源内皮祖细胞,探讨绵羊骨髓来源内皮祖细胞培养方法,为绵羊组织工程静脉瓣种子细胞选择提供实验基础。方法:绵羊骨髓经条件培养基进行选择培养获取骨髓单个核细胞,传代扩增后用磁珠分选出CD133+细胞再培养,流式细胞检测确认分前细胞CD133的表达情况;分选传代后绘制细胞生长曲线观察细胞生长能力,用免疫细胞化学检测细胞特异性分子CD133,D34,VWF的表达情况;FITC标记BS-1-lectin和DiI标记ac-LDL标记细胞检测细胞吞噬功能。结果与结论:绵羊骨髓单个核细胞原代培养2d细胞开始贴壁,7d细胞完全融合,传代后第2天进入对数生长期,传代3～5d形成为典型的铺路石样,传代后第7d细胞进入增殖平台期;细胞传代后磁珠分选率为12.6%,流式细胞仪检测显示CD133阳性率为12.64%;免疫细胞化学检测显示细胞呈CD133、CD34、血管性血友病因子阳性表达。细胞能同时吞噬FITC-labeledBS-1-lectin,DiI-ac-LDL阳性率达85.3%。证实实验成功地从绵羊骨髓单个核细胞中分离培养出内皮祖细胞。

BACKGROUND：Nowadays, the study of tissue-engineered venous valve has just started, the choice of seed cells is the key, endothelial progenitor cells are optimal seed cells for tissue-engineered venous valve construction in vitro.OBJECTIVE：To study the culture method of sheep bone marrow-derived endothelial progenitor cells （EPCs） and provide experimental basis for seed cells selection of sheep tissue engineering by culture and identification of sheep bone marrow-derived endothelial progenitor cells in vitro.METHODS：Sheep bone marrow mononuclear cells were obtained after whole bone marrow culturing selectively by using bone marrow condition medium.After primary cells were passaged, CD133-positive cells were sorted by microbeds instrument and cultured continually.Flow cytometry confirmed CD133 expression in cells before separation.The cell growth curve was drawn after separation and passage to observe cell growth capacity.Cell specific molecule CD133, D34, VWF expression were detected by immunocytochemistry.FITC labeled BS-1-lectin and DiI labeled ac-LDL were used to label cells and detect cell phagocytosis.RESULTS AND CONCLUSION：The sheep bone marrow mononuclear cells began to adhere on day 2 in primary culture and fully integrated on day 7.Cells started to went into the logarithmic growth phase on day 2 after passage and cell morphology formed typical paving stone-like on 3-5 days after passage; cells went into platform phase on day 7 after passage.Cells microbead sorting rate was 12.6% after passage.Flow cytometry showed CD133-positive cells rate was 12.64%.Immunocytochemistry showed that cells presented CD133, CD34, VWF-positive expression.FITC-labeled BS-1-lectin and DiI-ac-LDL double positive cells rate was 85.3%.This study successfully isolated and cultured EPCs from sheep bone marrow mononuclear cells.