论文部分内容阅读
背景:骨形成蛋白2是骨基质中最重要骨生长因子,主要生物学作用是促进未分化的间充质细胞和骨系细胞的募集和分化,是骨生成的启动因子,可以作为骨修复基因治疗的理想目的基因。目的:使用前期已经构建成功的pIRES2-EGFP-BMP-2真核表达质粒转染兔骨髓间充质干细胞。设计、时间及地点:细胞学观察实验,于2007-12/2008-06在新疆医科大学自治区地方病分子生物学实验室完成。材料:良种新西兰大白兔由新疆医科大学动物试验中心提供,pIRES2-EGFP-BMP-2由本校分子生物学实验室构建、保存。方法:采用贴壁法及密度梯度离心法提取骨髓间充质干细胞。采用脂质体介导转染法将pIRES2-EGFP-BMP-2真核表达质粒转染兔骨髓间充质干细胞。主要观察指标:通过荧光显微镜、反转录聚合酶链式反应、免疫组织化学及Western免疫印迹检测其转录、表达活性。结果:转染的兔骨髓间充质干细胞细胞可见绿色荧光蛋白的持续表达,绿色荧光蛋白分布于整个细胞,呈胞浆分布,说明重组载体构建成功并能在体细胞中表达。转染pIRES2-EGFP-BMP-2的兔骨髓间充质干细胞经G418抗性筛选并传代、培养4周后,RT-PCR反应可扩增出112kb条带,大小与预期相符合。Western免疫印迹检测显示,经pIRES2-EGFP-BMP-2转染的兔骨髓间充质干细胞经在相对分子质量为30×103处显现单一特异性条带,表明为骨形成蛋白2基因表达产物。免疫组织化学显示,转染pIRES2-EGFP-BMP-2后经G418抗性筛选并传代、培养4周后的兔骨髓间充质干细胞细胞质中有棕黄色颗粒阳性信号。结论:用pIRES2-EGFP-BMP-2真核表达质粒成功转染兔骨髓间充质干细胞并获得表达。
BACKGROUND: Bone morphogenic protein 2 is the most important bone growth factor in bone matrix. Its main biological function is to promote the recruitment and differentiation of undifferentiated mesenchymal cells and bone cells. It is a promoter of bone formation and can be used as a bone repair gene The ideal target of treatment. OBJECTIVE: To transfect rabbit bone marrow mesenchymal stem cells using pIRES2-EGFP-BMP-2 eukaryotic expression plasmid which has been successfully constructed in the early stage. DESIGN, TIME AND SETTING: The cytology observation experiment was performed at the Laboratory of Endemic Diseases and Molecular Biology, Xinjiang Medical University from December 2007 to June 2008. MATERIALS: The breeding New Zealand white rabbits were provided by the Animal Experiment Center of Xinjiang Medical University. The pIRES2-EGFP-BMP-2 was constructed and preserved by our molecular biology laboratory. Methods: Bone marrow mesenchymal stem cells were extracted by adherent method and density gradient centrifugation. The pIRES2-EGFP-BMP-2 eukaryotic expression plasmid was transfected into rabbit bone marrow mesenchymal stem cells by liposome-mediated transfection. MAIN OUTCOME MEASURES: The transcription and the expression activity were detected by fluorescence microscopy, reverse transcription polymerase chain reaction, immunohistochemistry and Western blotting. Results: The transfection of rabbit bone marrow mesenchymal stem cells showed the continuous expression of green fluorescent protein, green fluorescent protein distributed throughout the cell, showing cytoplasmic distribution, indicating that the recombinant vector was successfully constructed and expressed in somatic cells. Rabbit bone marrow mesenchymal stem cells transfected with pIRES2-EGFP-BMP-2 were screened and passaged by G418 resistance. After 4 weeks of culture, the 112kb band was amplified by RT-PCR and the size was consistent with the expectation. Western blotting showed that the bone marrow mesenchymal stem cells transfected with pIRES2-EGFP-BMP-2 showed a single specific band at a molecular weight of 30 × 103, indicating that it is the product of bone morphogenetic protein 2 gene expression. Immunohistochemistry showed that pIRES2-EGFP-BMP-2 was screened and passaged after G418 resistance. After 4 weeks of culture, there was a positive signal of brown granules in the cytoplasm of rabbit bone marrow mesenchymal stem cells. Conclusion: The rabbit bone marrow mesenchymal stem cells were successfully transfected with pIRES2-EGFP-BMP-2 eukaryotic expression plasmid.