兔骨髓间充质干细胞的分离培养鉴定及BrdU标记的体外研究

目的：拟在体外建立一种分离纯化、培养扩增、标记兔骨髓间充质干细胞（BMSCs）的方法。方法：密度梯度离心和贴壁培养相结合,台盼蓝染色观察离心后细胞活性,绘制BMSCs生长曲线,流式细胞仪检测BMSCs表面标记。诱导传代细胞向成软骨细胞分化,2周后免疫组化检测Ⅱ型胶原的表达和阿尔新蓝染色检测蛋白多糖的表达。BrdU标记BMSCs,观察最佳标记率及标记时间,并予四甲基偶氮唑盐方法测定标记后细胞活性。结果：密度梯度离心结合贴壁法能分离培养出纯度较高的BMSCs,分离后细胞活性均大于99%,BMSCs表面标记CD90、CD45、CD34阳性细胞表达分别为99.24%、0.03%、1.37%,BMSCs的生长曲线呈S形。成软骨诱导2周后Ⅱ型胶原免疫组化阳性,阿尔新蓝染色细胞基质被蓝染。BrdU标记后,MSCs标记率〉95%,BrdU细胞标记最佳时间为48 h,最佳浓度为10 mg/L,标记后细胞活性高。结论：采用密度梯度离心和贴壁培养相结合,可获得纯度较高BMSCs;BrdU是一种简单、有效的标记BMSCs的方法,BrdU对细胞标记率高。

SEPARATION, CULTURE,IDENTIFICATION AND BrdU LABELING OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS IN VITRO

Objective：To establish rabbit bone mesenchymal stem cells（BMSC） isolation,purified,cultured,amplified,labeled methods in vitro.Methods：BMSCs were isolated by density gradient centrifugation and adherent culture,a Trypan blue demonstrated cytoactive after the density gradient centrifugation,the grow curve of the BMSCs was described through cell counting,cell phenotype was detected by flow cytometry.The passage BMSCs were inducted and differentiated into chondroblast,immunohistochemical assay was performed to test the secretion of collagen type Ⅱ and Al new blue staining detection cells proteoglycan expression.Observed the best mark rate and mark time after BMSCs was labeled by BrdU,at the same time,by the MTT observe the activity the cells labeled.Results：Density gradient centrifugation and adherent culture can be isolated with high purity BMSCs,Trypan blue exclusion demonstrated 99 % viability,the positive expression rates of cell phenotypes were various as followings respectively： CD90,99.24%;CD45,0.03%;CD34,1.37%.The growth curve of rabbit BMSCs was ＂S＂ shape.After 2 weeks,collagen type Ⅱ immunohistochemical positive and Al new blue staining cells were seen by aizen matrix.The rabbit BMSCs nuclei show green fluor under fluorescence microscope after labeled by the BrdU.It showed that no significant toxicity on cells after BrdU labeling.The most appropriate time for BrdU labeling rabbit BMSCs is 48 hours,and the most appropriate concentration is 10 mg/L.Conclusion：Using density gradient centrifugation and adherent culture combination,can obtain training BMSCs highly purity.Labeling rabbit BMSCs with BrdU is a simple efficient labeled method,and the labeling rate is high and the cytotoxicity is little.