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

发布时间：2018-02-26 13:37

本文关键词： 干细胞骨形态发生蛋白质类基因疗法组织工程分化骨髓间充质干细胞骨形态发生蛋白骨组织工程基因治疗质粒转染成骨分化　出处：《中国组织工程研究》2017年17期 　论文类型：期刊论文

【摘要】：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(P0.01);(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。
[Abstract]:Background: due to the incomplete homology of human and animal protein genes, Human gene construction of tissue engineering artificial bone promotes animal osteogenesis or spinal fusion. Objective: to construct a tissue engineering seed cell line which can stably express and secrete human bone morphogenetic protein-2. Methods: to construct a tissue engineering seed cell line which can secrete human bone morphogenetic protein-2. Human bone morphogenetic protein-2 gene was cloned from human muscle tissue by nested RT-PCR. After purification, the eukaryotic expression vector system of PC DNA3-h BMP2 was constructed. The experimental group was transfected with liposome to modify human bone marrow mesenchymal stem cells and cultured in G418 medium. The biological characters of growth and proliferation of transfected cells were observed after 48 h and 3 weeks of culture. The expression of human bone morphogenetic protein 2 m RNA gene was detected by RT-PCR, and the expression and secretion of human bone morphogenetic protein 2 protein were detected by immunohistochemistry and immunoenzyme spots. Results and conclusion Human bone morphogenetic protein-2 gene transfected human bone marrow mesenchymal stem cells (BMSCs) grew and aggregated in clusters after one week of transfection of osteoblast potential alkaline phosphatase (ALP) activity. The resistant cell clones were obtained by G418 screening, which were similar to calcium nodule formation. The proliferation of cells was similar to that of the two control groups. The results of immunoenzyme spots and immunohistochemistry were detected. Human bone morphogenetic protein 2 was transcribed and expressed at 48 h and 3 weeks after transfection in the experimental group, and the expression at 48 h after transfection was higher than that at 3 weeks. The specific activity of alkaline phosphatase in the experimental group was significantly higher than that in the positive control group. The results showed that the human bone morphogenetic protein-2 gene transfection modified human bone marrow mesenchymal stem cells had good biological activities, such as growth, proliferation, differentiation and so on. Stable expression and secretion of exogenous gene transfected human bone morphogenetic protein 2.
【作者单位】：北京大学深圳医院脊柱外科;深圳市脊柱外科重点实验室;
【基金】：广东省医学科研基金(A2014654) 深圳市科技创新委员会基金(JCYJ20150403091443319)~~
【分类号】：R318.08;R68

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