补肾活血法促骨质疏松大鼠成骨细胞增殖的经典Wnt/β-catenin信号通路机制研究

Osteoporosis is a common and frequently encountered disease，however ，At present, There is no effective drug treatment. traditional Chinese medicine （TCM ）hold the special theory that " the kidney-essence is sufficient, formation of the marrow will be nourished"Making use of the method of BUSHEN HUOXUE appeared valid efficency. many papers point out that the mechanism of BUSHEN HUOXUE FA to treat osteoporosis was through Promoting proliferation of osteoblast cell and the canonical Wnt/β-catenin signaling pathway key point of osteoblast cell proliferation ,our previous research found that BUSHEN HUOXUE FA markedly up-regulated the protein expression of β-catenin, LRP5, TCF in the canonical Wnt/β-catenin signaling pathway,however the further mechanism need to be proved。On basis of a large number of previous researches,this study through BUSHEN HUOXUE FA Promoting proliferation of osteoblast cell in vivo and in vitro experiments,to proved that BUSHEN HUOXUE FA to treat osteoporosis was through Promoting proliferation of osteoblast cell,and use Dickkopf 1(Dkk1) which was the classics blocking agent of the Wnt/β-catenin signaling pathway to illustrate the mechanism of bushenhuoxuefa Promoting osteoblast cell proliferation ,further to explore the regulation of osteoblasts role targets and provides the evidence for Herb treatment for osteoporosis in TCM. From the angle of the molecular to provide experimental evidence for expounding the scientific connotation of "the kidney dominating the bones" in TCM.

骨质疏松症是常见病，多发病；目前尚缺乏有效的治疗药物。中医以其独特性的理论,以"肾藏精, 主骨生髓"为理论基础, 运用补肾活血法显现出优势，大量研究证实补肾活血法治疗骨质疏松症主要是通过促进成骨细胞增殖来实现的，而经典WNT信号通路是成骨细胞增殖的关键，我们的前期研究发现补肾活血法能调节经典Wnt信号通路中Wnt蛋白、β-catenin、LRP5相关因子的表达，但其具体的作用机制仍需进一步研究。基于前期的大量研究，本项目通过补肾活血法代表方益骨汤体内外促进成骨细胞增殖，防治骨质疏松症的实验研究，来证实补肾活血法通过促进成骨细胞增殖达到治疗骨质疏松症的目的，并在wnt通路抑制剂DKK1干预下，进一步研究补肾活血法通过Wnt/β-catenin信号通路促进成骨细胞增殖的作用机制，阐明其调控成骨细胞的作用靶点，为补肾活血中药治疗骨质疏松提供依据，并从分子角度来阐释 "肾主骨，生髓"理论。

本项目通过体外分离培养成骨细胞，通过Wnt信号通路的抑制剂 DKK1来干预对成骨的影响，并定量检测经典Wnt/β-catenin通路中Wnt10b, β-catenin、LRP-5蛋白基因的表达，分析补肾活血法基于经典Wnt/β-catenin通路对成骨细胞增殖的调控机制。同时进一步建立去势大鼠骨质骨松症动物模型，通过补肾活血法进行干预，检测各组成骨细胞数量来分析补肾活血法体内对成骨细胞增殖的影响并从组织病理学、荧光定量PCR等技术分析补肾活血法通过经典Wnt/β-catenin信号通路体内促进成骨细胞增殖的作用机制。结果说明：益骨汤可以通过经典Wnt/β-catenin信号通路促进成骨细胞增殖，其机制为益骨汤通过增强WNT10b、LRP5、β-catenin基因的表达，从转入水平上影响成骨细胞的增殖，进一步通过下调DKK1的表达，抑制DKK1和LRP5/6的结合，减弱DKK1阻断WNT通路的作用，从而促进成骨细胞增殖。与本项目为补肾活血中药治疗骨质疏松提供依据，并从分子角度来阐释 “肾主骨，生髓”理论.