稳定表达人骨形态发生蛋白2基因骨组织工程种子细胞的构建

Construction of seed cells with the stable expression of human bone morphogenetic protein 2 gene for bone tissue engineering

背景:由于人与动物之间蛋白基因的非完全同源性,人源基因构建组织工程人工骨促进动物成骨或脊柱融合影响了实验验证效果。目的:构建可稳定表达分泌人骨形态发生蛋白2的组织工程种子细胞系。方法:以巢式RT-PCR方法从人肌肉组织中克隆人骨形态发生蛋白2基因全长基因,纯化后连接构建pc DNA3-h BMP2真核表达载体系统,实验组利用脂质体介导转染修饰人骨髓间充质干细胞,置于G418培养液中筛选培养,设定空载质粒转染细胞(阳性对照组)及正常培养细胞(空白对照组)作为对照,观察检测转染后细胞的生长增殖生物学性状;在培养48 h和3周后,行人骨形态发生蛋白2 m RNA基因表达的RT-PCR测定,人骨形态发生蛋白2蛋白表达和分泌的免疫组织化学和免疫酶斑点测定,以及转染1周后细胞成骨潜能碱性磷酸酶比活性的检测。结果与结论:(1)转染人骨形态发生蛋白2基因的人骨髓间充质干细胞成簇样生长聚集,传代培养生长增殖良好,G418筛选培养可得到抗性细胞克隆,有类似钙结节样形成,细胞增殖与两对照组无差异;(2)RT-PCR、免疫酶斑点及免疫组织化学检测结果检测,实验组转染48 h和3周时均能转录和表达人骨形态发生蛋白2,转染48 h的表达较3周时要高,两对照组未见有明显蛋白转录表达;(3)正常培养或G418筛选培养时,实验组碱性磷酸酶比活性显著高于阳性对照组、空白对照组(P<0.01);(4)结果表明,人骨形态发生蛋白2基因转染修饰的人骨髓间充质干细胞生长增殖分化等生物学活性良好,可稳定表达分泌外源转染基因人骨形态发生蛋白2。

BACKGROUND： Because of the non-homology of protein and gene between human and animals, to promote osteogenesis or spinal fusion of animals by construction of tissue-engineered bone with the human gene has influenced the experimental validation.OBJECTIVE： To construct the seed cell line for bone tissue engineering with stable expression of human bone morphogenetic protein 2 （hBMP2）.METHODS： The full-length hBMP2 gene was cloned from human muscle tissues by nested RT-PCR and transfected to human bone marrow mesenchymal stem cells （hBMSCs） with lipidosome. The transfected hBMSCs were cultured with G418 in vitro to screen and purify the cells. A series of analyses such as RT-PCR, dot-ELISA, immunohistochemstry and alkaline phosphatase activity analysis were performed to evaluate the situation of hBMP2 expression and secretion at 48 hours and 3 weeks after the transduction. hBMSCs transduced with empty plasmid and the normal hBMSCs served as positive control and blank control groups, respectively, which were used for observation of cell growth, proliferation and biological characteristics of transfected cells. RESULTS AND CONCLUSION： The transfected hBMSCs appeared in small groups or clusters, and had a good proliferation after subculture in vitro. Some G418-resistance cell clones and calcium nodules were found when cultured with G418 in vitro. No significant difference was noted in the cell proliferation between the hBMP2 transfection group and two control groups. The ALP activity in the hBMP2 transfection group remained significantly higher than that in the two control groups （P 〈 0.01）. At 48 hours and 3 weeks after transduction, hBMSCs could express actively hBMP2 by RT-PCR monitoring, and had a positive reaction of dot-ELISA and immunohistochemical analysis. The expression of hBMP2 gene in the experiment group at 48 hours was significantly higher than that at 3 weeks after transduction while there was no expression of hBMP2 gene in the two control groups. The above results show that the hBMSCs transfected by hBMP2 gene not only have potentials of normal proliferation and osteogenic differentiation, but also can stably express hBMP2.