人骨髓间充质干细胞在兔膀胱脱细胞基质微环境中的生物学特性

Biological characteristics of human bone marrow mesenchymal stem cells in the microenvironment of rabbit bladder acellular matrix graft

目的利用兔膀胱脱细胞基质(bladder acellular matrix graft,BAMG)构建微环境,从细胞增殖能力、表面标记物及分子蛋白水平探讨其对人骨髓间充质干细胞(human bone marrow-derived stem cells,hBMSCs)的影响。方法制备BAMG浸泡液培养基,MTT法检测其对hBMSCs增殖的影响;流式细胞仪检测hBMSCs表面标志物CD44、CD45、CD73及PDGFRβ的表达;RT-PCR检测BAMG微环境对PPAR、OCN、α-SMA等基因表达的影响,Western blot检测不同处理方法 OCT-4蛋白的表达情况。结果 hBMSCs与BAMG有良好的相容性;实验组与对照组细胞曲线均呈"S"型增殖,于第3天进入快速增殖期,第7天趋于平稳;两组细胞CD44、CD45、CD73、PDGFRβ的表达量基本一致,差异不具有统计学意义;OCN、PPAR、α-SMA两组均表达目的基因;Western blot法检测亦显示OCT-4阳性表达。结论 hBMSCs在兔BAMG的微环境中仍能良好保持原有的生物学特性,将该种子细胞与基质材料结合可构建需要的组织材料,为泌尿系统组织工程修复提供可能。

Objective To explore the effects of the microenvironment of rabbit bladder acellular matrix graft(BAMG)on proliferation,cell surface markers,and molecular protein level of human bone marrow mesenchymal stem cells(hBMSCs).Methods We prepared BAMG immersion fluid medium and detected its effect on the proliferation of hBMSCs by MTT method.The expressions of CD44,CD45,CD73 and PDGFRβwere detected by flow cytometry.The expressions of PPAR,OCN andα-SMA were detected by RT-PCR,and the expression of OCT was detected by Western blot.Results hBMSCs had good compatibility with BAMG.The MTT method showed that BAMG and BAMG immersion medium did not affect the proliferation capacity of hBMSCs.The surface of hBMSCs cells cultured with immersion fluid still expressed CD44,CD73 and PDGFRβ,but not CD45.RT-PCR showed that OCN,PPAR,andα-SMA were all expressed.Western blot test also showed the positive expression of OCT-4.Conclusion hBMSCs can still keep their original biological characteristics in the microenvironment of rabbit BAMG.It can be the seed cells and combined substrate materials for urinary system tissue engineering.