大鼠内皮祖细胞的分离、培养与定向诱导分化

目的探讨建立一种从大鼠骨髓中分离培养与定向诱导分化内皮祖细胞(EPCs)的稳定、高效方法。方法密度梯度离心法从大鼠骨髓中分离单个核细胞,经差速贴壁结合特殊培养基扩增并向内皮细胞定向诱导分化EPCs。应用免疫荧光和流式细胞技术鉴定内皮细胞系列标志:CD34、CD31、Flk-1和祖细胞标志CD133。并通过检测其对FITC标记的UEA-1的吸附和内吞DiI-ac-LDL来进行细胞功能学的鉴定。对分化细胞行vWF、CD31免疫组化染色鉴定,并与血管内皮细胞合成前列腺素能力进行比较。结果梯度密度离心和贴壁法选择的细胞表达内皮细胞特异性抗原CD34、CD31、Flk-1,部分表达CD133。分离所得细胞经EBM-2专用培养基培养后,第5天可见集落形成,培养第9天CD133阳染细胞减少,CD31阳染细胞增加,细胞可特异性吸附FITC标记的荆豆凝集素并内吞DiI-ac-LDL,约3周左右形成铺路石样内皮细胞特有形态。诱导后的内皮祖细胞的前列腺素合成能力与血管内皮细胞之间无统计学差异(P>0.05)。结论该方法可以同时培养扩增和定向诱导分化内皮祖细胞,效率高,稳定性和重复性好。

Isolation,culture and directed differentiation of mesenchymai stem cells and endothelial progenitor cells from SD rat bone marrow

Objective It is to establish an efficient and stable method for simultaneous isolation,culture and directed differentiation of endothelial progenitor cells(EPCs) from SD rat bone marrow.Methods The mononuclear cells were isolated from SD rat bone marrow by density gradient after differential centrifugation.Then the resulted cells were cultured and differentiated to endothelial cells(ECs) in EBM-2.The expressions of specific antigens(CD(133),CD(34),Flk-1 and CD(31)) were analyzed by immunofluorescence and flow cytometer.The biological functions of endothelial cells were examined by the adsorp tion of ulex europaeus agglutinin(UEA) labeled by fluorescein isothiacyanate(FITC) and DiI-ac-LDL internalization.The expression of vWF and CD(31) of ECs differentiated from EPCs was also assessed by immunohistochemistry.Results The gained cells from mouse bone marrow by density gradient expressed CD(31),CD(34) and Flk-1,portion expressed CD(133).The adhesive cells formed clusters at the 5th day.At the 9th day,the experiment of FITC labeled UEA adsorption and DiI-ac-LDL internalization were positive,the expression of CD(133) in adhesive cells decreased,and CD(31) Increased.After 3 weeks they differentiated into mature ECs forming cobblestone monolayers.There was no difference in secretion PGI2 between ECs from EPCs and ECs from aorta of rat(P>0.05).Conclusion It is an efficient,stable and replicable method for simultaneous multiplication culture and directed differentiation of MPCs.