腺病毒介导BMP2/7基因共转染构建MSCs成骨分化及其信号传导机制的研究

BMP2/7 heterodimer, a bone structure formation protein, has stronger osteogenesis inductive potential than its homodimer. So the study of BMP2/7 heterodimer has been one of the most important research directions in tissue engineering osteogenesis now. After several years of preliminary research in the Molecular Orthopaedics Research Center of Harvard University School of Medicine applicants have proved that the target cell of BMP2/7 heterodimer and homodimer is different. And also BMP2/7 heterodimer stimulates different signal transduction pathways in osteogenesis induction. As target cells, rats bone marrow mesenchymal stem cells are proved to be useful, reasonable and promising scientifically in the study of the signal transduction pathway of BMP2/7 induced osteogenesis. In this research adenovirus infected BMP2/7 heterodimers is used to induce the formation of BMSC and then stimulates the osteogenesis potential. The biological expression pathway is studied and analyzed by using RNAi inhibited BMP receptor expression system. This study will firstly modify mouse BMSC combined calcium alginate gel with BMP2/7 heterodimer gene to regenerate tissue-engineered bone for the repairing of serial skull bone defects of the mouse. All the achievements and experimental technique will provide an important experimental theoretical basis for the clinical application of tissue-engineered bone.

骨形态发生蛋白2/7（BMP2/7）异源二聚体较其同源二聚体有更强的诱导成骨潜能，是目前组织工骨研究的重要分支方向之一。申请者在哈佛大学医学院分子矫形外科研究中心几年的前期研究证实：由于异源二聚体BMP2/7和同源二聚体作用的靶细胞不同，BMP2/7异源二聚体激活了不同的信号传导途径而发挥了其成骨潜能，利用非建系大鼠骨髓间充质干细胞做为效应靶细胞研究异源二聚体BMP2/7诱骨信号传导被证实是科学合理而可行的实验途径，并将最终证实其生物学机制。本课题应用腺病毒转染异源二聚体BMP2/7诱导BMSC及成骨分化潜能，应用RNAi靶向抑制BMP受体表达系统研究和分析其生物学表达途径，课题将首次应用异源二聚体BMP2/7基因修饰大鼠BMSC复合藻酸钙凝胶，以构建基因技术完成组织工程骨并完成大鼠系列的颅骨缺损实验研究，课题研究及相关技术对组织工程骨最终的临床应用将提供重要的实验基础和理论依据。

骨形态发生蛋白2/7（BMP2/7）异源二聚体较其同源二聚体有更强的诱导成骨潜能，是目前组织工骨研究的重要分支方向，但其信号传导机制仍不明了。课题组利用AdMax腺病毒介导BMP2、BMP7基因共转染大鼠骨髓间充质干细胞，首次在大鼠骨髓间充质干细胞中分泌表达BMP2/7异源二聚体重组蛋白，且具有成骨诱导分化的生物学活性。BMP2/7异源二聚体蛋白基因修饰大鼠BMSCs细胞上调表达早期成骨标志物碱性磷酸酶及晚期骨性标志物I型胶原及骨钙素，与BMP2及BMP7同源二聚体相比，腺病毒介导的BMP2/7异源二聚体重组蛋白的分泌表达对大鼠BMSCs表现出增强的成骨诱导分化潜能。BMP信号通路实验结果表明，该成骨潜能与BMP2/7异源二聚体能快速激活BMP信号传导因子 Smad、JNK和ERK等磷酸激酶的活化尤其是ERK信号通路的磷酸化激活有关。通过BMP信号通路阻断实验，进一步确认了BMP2/7异源二聚体可能通过更有效地激活ERK信号传导通路而发挥其增强的诱导BMSCs细胞的成骨分化潜能；腺病毒介导BMP2/7异源二聚体基因修饰大鼠BMSCs细胞并复合脱钙骨基质可以在大鼠颅骨缺损中形成新生骨组织，且与其同源二聚体BMP2、BMP7相比，BMP2/7异源二聚体蛋白具有更强的体内成骨能力，而其修复骨缺损所需的有效剂量明显较低以及其修复骨缺损时间明显缩短。本研究成果及开发的基因共转染技术使基因治疗增加骨诱导细胞因子的局部分泌以促进骨修复及组织工程骨的未来临床应用提供了更加有效的新途径。