论文部分内容阅读
背景:能否通过病毒或者非病毒载体将骨形态发生蛋白2转导到骨髓基质细胞发挥成骨作用?目的:观察真核表达载体pcDNA3-hBMP2转染兔骨髓基质细胞后体外表达,同时将MSC转染后但未筛选兔骨髓基质细胞自体回植入肌组织,利用X线观察其成骨情况。设计、时间及地点:观察对比实验。实验于2004-11/2005-04在辽宁医学院骨科研究室完成。材料:6只成年新西兰大白兔,雌雄不拘,体质量2.0～3.0kg。BMP2抗体为美国Sanaka公司产品,pcDNA3-hBMP2由解放军第四军医大学生化教研室蒲勤教授提供,限制性内切酶购于大连宝生物工程有限公司。方法:从大肠杆菌提取超纯质粒pcDNA3-hBMP2,从成年兔股骨抽取骨髓,密度梯度分离法分离培养骨髓基质干细胞,将细胞分成4组:A组:pcDNA3-hBMP2转染进行G418筛选;B组:pcDNA3-hBMP2转染未用G418筛选;C组:给予pcDNA3空载体转染;D组:仅加入脂质体转染试剂Fugene6。主要观察指标:①应用免疫组织化学法检测转染后瞬时表达。②应用免疫组织化学法检测细胞骨钙素表达,分别应用原位杂交法检测细胞Ⅰ型胶原表达。③转染2周后将B组细胞自体回植入兔后腿肌组织中,移植4周后应用X射线观察成骨情况。结果:①pcDNA3-hBMP2成功转染入骨髓基质干细胞内并100%瞬间表达BMP2。②基因转染4周后,A组细胞骨钙素及Ⅰ型胶原表达高于C组及D组。③B组细胞回植肌组织4周后,X射线可显示新骨形成。结论:pcDNA3-hBMP2能安全有效转染兔骨髓基质干细胞,通过其分泌物BMP2来作用诱导细胞加速分化为成骨细胞。
BACKGROUND: Is it possible to transduce bone morphogenetic protein-2 into bone marrow stromal cells through virus or non-viral vector to exert osteogenesis? OBJECTIVE: To observe the in vitro expression of eukaryotic expression vector pcDNA3-hBMP2 transfected rabbit bone marrow stromal cells, Transfected but not screened rabbit bone marrow stromal cells autologous transplantation into the muscle tissue, the use of X-ray observation of its osteogenic situation. Design, time and place: observe the contrast experiment. The experiment was performed at the Department of Orthopedics, Liaoning Medical University from November 2004 to April 2005. Material: 6 adult New Zealand white rabbits, both male and female, weight 2.0 ~ 3.0kg. BMP2 antibody Sanaka company products, pcDNA3-hBMP2 by the Fourth Military Medical University, Department of Biochemical Professor Pu Qin provide, restriction endonucleases purchased from Dalian Bao Biological Engineering Co., Methods: The supernatant plasmid pcDNA3-hBMP2 was extracted from Escherichia coli and bone marrow was extracted from the femur of adult rabbits. BMSCs were isolated and cultured by density gradient centrifugation. The cells were divided into 4 groups: group A: pcDNA3-hBMP2 transfected for G418 selection; group B : pcDNA3-hBMP2 transfected without G418 selection; group C: pcDNA3 empty vector transfected; D: liposome transfection reagent Fugene6 only. MAIN OUTCOME MEASURES: ①Immunohistochemistry was used to detect transient expression after transfection. ② The expression of osteocalcin was detected by immunohistochemistry, and the expression of type Ⅰ collagen was detected by in situ hybridization. ③ After 2 weeks of transfection, the cells in group B were transplanted into the hindlimb hindlimb muscle autograft, and the bone formation was observed by X-ray after 4 weeks. Results: ①pcDNA3-hBMP2 was successfully transfected into bone marrow stromal cells and transiently expressed BMP2 at 100%. ② After 4 weeks of gene transfection, the expression of osteocalcin and type Ⅰ collagen in group A was higher than that in group C and group D. ③B group of cells back to muscle tissue after 4 weeks, X-ray can show new bone formation. CONCLUSION: pcDNA3-hBMP2 can transfect rabbit bone marrow stromal stem cells safely and effectively and induce the cells to differentiate into osteoblasts through its secreted BMP2.