基于脾肾相关理论防治航天失重性骨丢失的miRNAs网络调控机制研究

As astronauts in the airspace enviroment, decreased bone mineral density can be caused by weightlessness, resulting in osteoporosis. This continued bone loss will seriously affect the health and efficiency of the astronauts，and even fracture. One of the critical pathogenesis of the bone loss is the decrease in bone formation. It is believed that the kidney dominating bone and manufacturing marrow and the spleen in charge of generation and transformation qi and blood to enrich the kidney essence in Chinese medicine. It is highly valuable for academic study and application that we research the relationship between the restoration of bone mass and the essence of interdependence of spleen and kidney using simulated weightlessness animal model on the ground and bone marrow mesenchymal stem cells in vitro techniques. In the previous study we found that invigorating kidney and spleen prescription can increase bone density and promote bone marrow mesenchymal stem cells changing into bone in tail suspended rats simulated weightlessness. The prevention mechanism of the invigorating kidney and spleen prescription on bone loss by weightlessness and the effects of the invigorating spleen prescription on the tonifing kidney prescription will be studied from regulating bone marrow mesenchymal stem cells osteogenesis differentiation of microRNAs by Wnt/β-catenin signal pathways adoptting real-time quantitative PCR, Western blot and dual luciferase reporter gene detection technique in this project. And the essence of interdependence of spleen and kidney to maintain bone mass will be revealed. At the cellular and molecular level it will be explained that the theory of correlation between spleen and kidney.

航天员在进入太空失重环境后，会出现骨密度下降，导致骨质疏松，这种持续性的骨丢失会严重影响航天员的健康和工作效率，甚至出现骨折。失重性骨丢失的关键发病机制之一是骨形成减少。中医学认为肾主骨生髓，脾主化生气血，充养肾精。利用地面模拟失重动物模型和骨髓间充质干细胞体外培养技术，研究骨量恢复的机制与脾肾先后天互根的本质，具有学术和应用价值。本项目前期研究发现补肾健脾方能增加尾部悬吊模拟失重大鼠骨密度和促进骨髓间充质干细胞成骨分化，在此基础上，拟采用实时定量PCR、Western blot、双荧光素酶报告基因检测等技术，从骨髓间充质干细胞成骨分化的miRNAs经Wnt/β-catenin信号通路网络调控途径，探查补肾健脾方防治失重性骨丢失的分子机制，健脾方对补肾方的影响，揭示脾肾互根维持骨量恒定的机制，为认识“脾肾相关”理论的科学内涵提供细胞和分子水平上的依据。

航天员在进入太空失重环境中会出现骨丢失，严重影响航天员的健康和工作效率。失重性骨丢失的中医病机与脾土失运、肾精不固有关。本项目依据“脾肾相关”理论提出以补肾健脾法防治失重性骨丢失，探查其作用机制，健脾法对补肾法的影响。完成的主要研究内容包括：健脾法、补肾法和补肾健脾法对模拟失重骨丢失大鼠的防治效用比较研究，对模拟失重骨丢失大鼠miRNAs经Wnt/β-catenin信号通路调控BMSCs成骨分化的研究，对体外培养BMSCs的miRNAs经Wnt/β-catenin信号通路调控成骨分化的研究。体内研究发现，健脾法、补肾法和补肾健脾法能增加模拟失重大鼠骨密度，促进BMSCs成骨分化，促进miRNA29、miRNA142-3p、miRNA335-5p表达，增加Runx2、Osterix、ALP、β-catenin、sFRP2、kremen2 mRNA和蛋白表达，补肾法和补肾健脾法促进miRNA27表达，降低DKK1 mRNA和蛋白表达。体外研究发现，健脾方、补肾方和补肾健脾方含药血清可促进大鼠BMSCs成骨分化，促进miRNA29、miRNA335-5p表达，增加Runx2、Osterix、ALP、APC、β-catenin mRNA和蛋白、细胞核β-catenin mRNA和蛋白表达；补肾方和补肾健脾方含药血清可促进miRNA27、miRNA142-3p表达，抑制DKK1 mRNA和蛋白表达；健脾方和补肾健脾方含药血清可促进sFRP2 mRNA和蛋白表达，补肾健脾方含药血清可抑制kremen2 mRNA和蛋白表达。研究结果提示健脾法、补肾法和补肾健脾法具有防治失重性骨丢失的作用，健脾法对补肾法存在协同作用，作用机制与调控miRNAs，激活经典Wnt信号通路，促进BMSCs成骨分化有关。但是在作用机制方面，健脾法对补肾法存在协同和选择作用。