基于Wnt/β-catenin信号通路探讨补肾活血汤调控激素性股骨头缺血坏死BMSCs成骨-成脂分化的机制

It was found in the previous experiments and clinical research that the pathogenesis of hormonal avascular necrosis of femoral head and bone marrow BMSCs differentiation imbalance was closely related. Bushen Huoxue decoction could effectively prevent and treat the disease, but the mechanism had not been illuminated. Based on Wnt/β-catenin signaling pathway regulation of steroid-induced rabbit BMSCs osteogenic and adipogenic differentiation, the rabbit model of hormonal avascular necrosis of femoral head was established, then treated with Bushen Huoxue decoction. The content of main component, biomechanical properties and fat content in marrow cavity of femoral head were observed, the associated factors gene and protein expression in bone tissue were detected, and the relationship between them was studied. In vitro experiment, rabbits BMSCs were seperated and identified, received osteogenic and adipogenic differentiation induction, then treated with Bushen Huoxue decoction containing serum. The Wnt/β-catenin signaling pathway associated factors gene and protein expression in cells were detected, and the mechanism was studied in the view of osteogenic and adipogenic differentiation.

前期实验和临床研究中发现，激素性股骨头缺血坏死发病机制与股骨头髓腔BMSCs成骨-成脂分化密切相关；补肾活血汤可有效防治激素性股骨头缺血坏死，但其作用机制尚未阐明。本研究基于Wnt/β-catenin信号通路在BMSCs成骨-成脂分化中的重要调控作用，①建立激素性股骨头缺血坏死兔模型，用补肾活血汤干预，观察补肾活血汤对股骨头内主要成分含量、生物力学性质及骨髓腔脂肪含量的影响，同时检测骨组织中Wnt/β-catenin信号通路上相关因子的基因和蛋白表达，并探讨二者的相关性。②离体实验中新西兰大白兔分离出BMSCs，成骨和成脂诱导后，用补肾活血汤药物血清干预。观察细胞中Wnt/β-catenin信号通路上相关因子的基因和蛋白表达变化，探讨补肾活血汤对不同分化方向的BMSCs Wnt/β-catenin信号通路上相关因子表达的影响，从成骨成脂分化角度探讨补肾活血汤防治激素性股骨头缺血坏死的机理。

本课题通过补肾活血汤对激素性股骨头缺血坏死兔模型及成骨－成脂诱导后BMSCs的干预，探讨补肾活血汤对激素性股骨头缺血坏死及体外不同分化方向的BMSCs Wnt/β-catenin信号通路上成骨－成脂相关因子的影响机制。在体实验中，新西兰大白兔90只，随机分为空白组、模型组、治疗组。采用贺氏造模法臀肌注射醋酸泼尼松龙建立激素性股骨头缺血坏死兔模型，造模成功后，对治疗组使用补肾活血汤进行干预，其他2组使用生理盐水干预，8周后通过HE染色组织形态学观察及血清碱性磷酸酶、甘油三脂生化检测，观察补肾活血汤对激素性股骨头缺血坏死的影响。离体实验中，采用5月龄健康新西兰大白兔，分离鉴定其BMSCs，分别进行成骨成脂诱导，油红０染色、碱性磷酸酶染色观察成骨成脂诱导状态。诱导成功后用补肾活血汤含药血清进行干预，通过光镜和透射电镜观察BMSCs的形态和超微结构，通过检测细胞中甘油三脂、碱性磷酸酶含量观察BMSCs成骨成脂分化情况，并应用RT—PCR、Western Blotting法检测细胞中Wnt/β-catenin信号通路上相关因子的基因和蛋白表达变化，探讨补肾活血汤对不同分化方向的BMSCs Wnt/β-catenin信号通路上相关因子表达的影响。从成骨成脂分化角度探讨补肾活血汤防治激素性股骨头缺血坏死的作用机理。..统计数据，分析得出：（1）补肾活血汤对兔激素性股骨头缺血坏死有治疗作用，并且它可以促进BMSCs向成骨细胞分化，同时抑制其向脂肪细胞分化。（2）补肾活血汤调控BMSCs成骨-成脂分化的机制主要是通过增强Wnt/β-catenin信号通路中成骨因子Wnt10b、Wntl、β-catenin、FZD6和Ror2的基因和蛋白表达，同时抑制成脂因子DDKl、sFRP4的表达来实现。