转染碱性成纤维细胞生长因子骨髓间充质干细胞复合小肠黏膜下层构建的组织工程皮肤

背景：研究证实，小肠黏膜下层不存在免疫原性，不会引起异种移植中常见的排斥反应，是一种比较理想的人工真皮替代物。 目的：通过基因转染的方法将碱性成纤维细胞生长因子基因转染至骨髓间充质干细胞，并复合猪小肠黏膜下层构建组织工程皮肤。 方法：日本大耳兔骨髓间充质干细胞进行体外培养扩增后，通过pCDNA3.1质粒将碱性成纤维细胞生长因子基因转染至生长状态良好的骨髓间充质干细胞，并将转染后的细胞接种于制备好的猪小肠黏膜下层上，进行体外联合培养，构建组织工程皮肤。观察细胞生长情况，流式细胞术检测骨髓间充质干细胞表型，WesternBlot检测pCDNA-bFGF真核表达载体转染兔骨髓间充质干细胞的结果，并观察组织工程皮肤的结构。 结果与结论：传代后骨髓间充质干细胞生长迅速，呈长梭形，形态良好。细胞的表面抗原CD90、CD44有阳性表达，而CD45为阴性。碱性成纤维细胞生长因子基因转染入兔骨髓基质干细胞并能稳定表达，转染后的骨髓间充质干细胞在猪小肠黏膜下层中生长良好，可体外构建组织工程皮肤。

Construction of tissue-engineered skin using basic fibroblast growth factor gene transfected bone marrow mesenchymal stem cells combined with small intestinal submucosa

BACKGROUND: Studies demonstrated that small intestinal submucosa (SIS) had no immunogenicity, which can not lead to rejection following transplantation, thus, this is an ideal skin substitutes for natural skin. OBJECTIVE: Basic fibroblast growth factor (bFGF) gene was transfected into bone marrow mesenchymal stem cells (BMSCs), and combined the cells with SIS to construct tissue-engineered skin. METHODS: BMSCs were obtained from Japanese big-eared rabbits, and in vitro cultured. Then the subcultured BMSCs were transfected by pCDNA3.1 plasmid, followed by incubation on swine SIS to construct the tissue-engineered skin. The growth of cells and phenotype of BMSCs were detected by flow cytometry. In addition, the result of transfecting BMSCs with pCDNA-bFGF vector was measured by Western blot, and the structure of tissue-engineered skin was observed. RESULTS AND CONCLUSION: After passaged, BMSCs were grown quickly with long-fusiform shape. The cells were positive expressed CD90 and CD44, but negative expressed CD45. bFGF had been transfected into BMSCs, and stable expressed. The transfected BMSCs grew well in SIS. By this method, tissue-engineered skin can be constructed in vitro.