大鼠脂肪干细胞和骨髓间充质干细胞向内皮分化的能力比较

摘要

目的：研究脂肪干细胞（ASCs）向内皮分化的能力，并与骨髓间充质干细胞（BMSCs）对比。方法从SD大鼠中分离培养ASCs、BMSCs，选择生长良好的一定代数的细胞，流式细胞仪鉴定细胞表面标志物；用CCK-8法绘制细胞生长曲线；进行标准内皮诱导3周，用qPCR检测内皮细胞特异性标志物CD31、KDR、vWF mRNA的表达；免疫荧光染色观察表面抗原CD31的表达；用Dil标记的乙酰化低密度脂蛋白（Dil-Ac-LDL）检测诱导组细胞的摄取功能；在Matrigel上验证诱导组细胞的成管能力。结果成功分离培养大鼠ASCs和BMSCs，形态上，ASCs与BMSCs类似，均呈长梭形、纺锤状，类成纤维细胞样形态；流式检测结果显示BMSCs与ASCs均高表达间充质干细胞表面标志物CD29（100%与96.9%）、CD90（96.5%与99.2%），低表达造血系细胞表面标志物CD45（3.9%与0.8%），证实所提取的是间充质干细胞；CCK-8增殖实验结果表明， ASCs的增殖速度比BMSCs的增殖速度快（P<0.05）。标准内皮诱导后，qPCR显示诱导组BMSCs和ASCs表达CD31、KDR、vWF mRNA水平均高于未诱导组（P<0.05）；免疫荧光染色显示CD31抗原表达在诱导组的细胞膜表面；荧光显微镜下诱导组细胞摄取Dil-Ac-LDL（未诱导组不摄取）；诱导组细胞在Matrigel上均具备成管能力，证实诱导后的细胞为内皮细胞。结论表明BMSCs和ASCs均可诱导向内皮细胞分化，ASCs广泛分化为内皮细胞的时间更短且增殖能力更强，提示ASCs比BMSCs更具有应用前景。%Objective To compared the differentiation capacity of rat adipose-derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) into endothelial cells. Methods Rat BMSCs and ASCs were isolated, cultured and identified for cell surface markers using flow cytometry. The cell growth curves were drawn by CCK-8 assay, and the cells in active growth were induced for endothelial differentiation following standard protocols. On day 21 of induction, the cells were examined for mRNA expressions of endothelial cell specific markers CD31, KDR, and vWF using qPCR. Immunostaining was performed to observe the expression of CD31 on the cells. The induced cells were also tested for Dil-labeled acetylated low-density lipoprotein (ac-LDL) uptake ability. The tube-forming ability of the induced cells was verified on Matrigel. Results We successfully isolated rat ASCs and BMSCs. Morphologically, ASCs were similar with BMSCs, both having long spindle-shaped and fibroblast-like morphology. Flow cytometry showed that both BMSCs and ASCs had high expressions of mesenchymal markers CD29 and CD90 and a low expression of hematopoietic cell surface markers CD45. CCK-8 assay showed that ASCs proliferated more quickly than BMSCs. The cells with induced endothelial differentiation exhibited increased levels of CD31, KDR, and vWF mRNA expressions and immunofluorescent staining identified CD31 antigen expression on the cell membrane. Fluorescence microscopy revealed red fluorescence in the induced cells suggesting uptake of Dil-Ac-LDL by the cells. The induced cells were capable of forming tube on Matrigel, confirming their identity of endothelial cells. Conclusion Both rat BMSCs and ASCs can be induced to differentiate into endothelial cells, but ASCs differentiate more quickly into endothelial cells and possess a stronger proliferation ability, suggesting its greater potential than BMSCs in future applications.