构建外源性重组pcDNA3.1-hBMP-7真核表达载体转染兔骨髓基质干细胞

背景:在细胞获取、培养、移植等体外环境体系下,骨髓基质干细胞能否有效的应用于局部基因治疗尚不清楚.目的:课题创新性提出构建外源性hBMP-7基因真核表达载体,并期望可提高被转染的兔骨髓基质干细胞诱导成骨能力.设计、时间及地点:细胞-基因学体外观察,于2006-07/2007-07在哈尔滨医科大学附属第二医院科研实验中心完成.材料:人健康新鲜胎盘组织由哈尔滨医科大学附属二院妇产科提供,产妇知情同意.健康雄性新西兰大耳白兔1只,由哈尔滨医科大学动物中心提供.方法:从人胎盘组织中克隆出hBMP-7基因,与真核表达载体pcDNA3.1连接,构建重组pcDNA3.1-hBMP-7真核表达载体.从兔骨髓中分离培养骨髓基质干细胞,分为3组:pcDNA3.1-hBMP-7转染组、空载体pcDNA3.1转染组、未转染组.转染前1 d取第2代骨髓基质干细胞5×106个,接种到60 mm3含无抗生素培养基的培养皿中进行转染.主要观察指标:使用RT-PCR、免疫组织化学等方法检测hBMP-7在骨髓基质干细胞中的表达,检测各组细胞碱性磷酸酶、胶原、骨钙蛋白的合成情况.结果:转染72 h后,pcDNA3.1-hBMP-7转染组于1.3 kb处出现特异性条带,细胞胞浆有棕色的颗粒出现,另2组均呈阴性.pcDNA3.1-hBMP-7转染组骨髓基质干细胞碱性磷酸酶活性于转染后第2天显著增高,第8天达峰值,各时间点pcDNA3.1-hBMP-7转染组骨髓基质干细胞碱性磷酸酶活性、羟脯氨酸合成量、骨钙蛋白含量均明显高于其他2组(P<0.05或0.01).结论:实验成功构建了pcDNA3.1-hBMP-7真核表达载体.结局证明了外源性hBMP-7基因可在兔骨髓基质干细胞中充分、高效表达,并且这种外源性hBMP-7基因具备促进兔骨髓基质干细胞向成骨细胞转化的能力.

Construction of exogenous recombinant eukaryotic expression vector pcDNA3.1-hBMP-7 and transfection into rabbit bone marrow stromal cells

BACKGROUND:Under the in vitro conditions of cell harvesting, culture, and transplantation, whether bone marrow stromal cells (BMSCs) can be effectively applied in local gene therapy remains unclear.OBJECTIVE: To construct a recombinant eukaryotic expression plasmid carrying human bone morphogenetic protein-7 (hBMP-7) gene, and to expect to enhance osteoinductive properties of rabbit BMSCs transfected.DESIGN, TIME AND SETTING: A cell-genomics in vitro observation was performed at the Laboratory of Scientific Research, Second Affiliated Hospital of Harbin Medical University between July 2006 and July 2007.MATERIALS: Human healthy fresh placental tissue was provided by the Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from the women. One healthy male New Zealand rabbit was provided by the Laboratory Animal Center, Harbin Medical University.METHODS: hBMP-7 gene was cloned from human placental tissue to construct a recombinant eukaryotic expression plasmid carrying hBMP-7 gene by conjugating with eukaryotic expression vector pcDNA3.1. BMSCs were isolated from rabbit bone marrow and cultured in vitro. Then they were divided into 3 groups: pcDNA3.1-hBMP-7-transfected, pcDNA3.1 -transfected, and untransfected. 5×106 BMSCs were inoculated into a 60 mm3 flask containing antibiotic-free medium 1 day prior to transfection.MAIN OUTCOME MEASURES: RT-PCR and immunohistochemistry were employed to detect hBMP-7 expression in BMSCs, alkaline phosphatase activity, hydroxypreline content, and osteocalcin production in each group. RESULTS: After 72-hour transfection, a 1.3 kb fragment was seen in the pcDNA3.1-hBMP-7-transfected group, showing brown granules in the endochylema, but not seen in the pcDNA3.14ransfected and untransfected groups. ALP activity in the pcDNA3.1-hBMP-7-transfected group significantly increased at 2 days after transfection, peeked at 8 days, and still increased at 10 days. At each time point, alkaline phosphatase activity, hydroxyproline content, and osteocalcin production were significantly higher in the pcDNA3.1-hBMP-7-transfected group than in the pcDNA3.1 -transfected and untransfected groups (P<0.05 or P<0.01).CONCLUSION: Recombinant eukaryotic expression vector pcDNA3.1- BMP-7 was constructed successfully. Results indicated that hBMP-7 was expressed in BMSCs sufficiently and was involved in inducing differentiation of BMSCs into osteoblasts. The method would provide substantial basement for hBMP-7 gene therapy.