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[目的]将血管内皮生长因子121(vascular endothelial growth factor 121,VEGF121)基因转染兔骨髓基质细胞(rabbit bone marrow stroma cells,rMSCs)后,研究转染后细胞的成骨功能变化,为进一步利用经基因转染的rMSCs构建组织工程骨血管化打下基础。[方法]体外培养、扩增rMSCs,将其分为转染组和未转染组,脂质体介导下PCDI-VEGF121真核表达质粒转染rMSCs后,G-418筛选阳性细胞克隆。通过RT-PCR法检测VEGF mRNA水平,通过碱性磷酸酶(alkaline phosphatase,ALP)、I型胶原检测来评价两组细胞的成骨能力。将阳性克隆细胞和未转染的细胞植入裸鼠股部肌袋内。分别于植入后4、8周处死动物,观察异位成骨情况。[结果]转染组和未转染组的ALP表达随着时间逐渐增强,转染组细胞内ALP水平显著增高,Ⅰ型胶原在转染组细胞内高表达,转染组的rMSCs植入肌袋后,随着植入时间的延长,出现骨髓腔样结构及大量骨小梁,表现出较好的异位成骨能力。[结论]应用VEGF121基因转染rMSCs后,有利于发挥VEGF121的血管化作用,促进rMSCs的成骨能力,将会成为组织工程骨血管化探索的方向。
[Objective] To investigate the osteoblast function of transfected cells after transfection of VEGF121 gene into rabbit bone marrow stroma cells (rMSCs) for further utilization Transgenic rMSCs lay the foundation for the construction of tissue engineered bone vascularization. [Methods] The rMSCs were cultured and expanded in vitro. The rMSCs were divided into transfection group and untransfected group. The positive clones were screened by G-418 after transfection of rMSCs with liposome-mediated PCDI-VEGF121 eukaryotic expression plasmid. The mRNA level of VEGF was detected by RT-PCR. The osteogenic ability of the two groups of cells was evaluated by alkaline phosphatase (ALP) and type I collagen. Positive clonal cells and untransfected cells were implanted into the muscle bags of nude mice. Animals were sacrificed 4 and 8 weeks after implantation to observe the ectopic osteogenesis. [Results] The expression of ALP in transfection group and non-transfection group increased gradually with time, and the level of ALP in transfection group was significantly increased. Type Ⅰ collagen was highly expressed in the transfected group. Transfected group rMSCs implanted muscle After the bag, with the extension of implantation time, cavity-like structures and a large number of trabecular bone appeared, showing good ability of ectopic osteogenesis. [Conclusion] The VEGF121 gene transfection with rMSCs is beneficial to vascularization of VEGF121 and promote osteogenic ability of rMSCs, which will be the direction of tissue engineering vascular vascularization.