基于Wnt/β-catenin信号通路的补肾活血胶囊干预激素性股骨头坏死骨修复机制研究

The hip-preserve of young adult necrosis of the femoral head is the research focus of modern medicine. The bone marrow stromal cells ( BMSCS ) osteogenic differentiation prevent the collapse of femoral head, is a difficult problem. After years of clinical research, Chinese medicine of Bushen and Huoxue for treatment of the necrosis of the femoral head and its preparation in the hip-perserve curative effect is distinct. But the molecular mechanism of bone repair is not know.In this paper, with Bushen Huoxue Capsule, combined with somatic cells, using RT-PCR, Western blot, flow cytometry, fluorescence staining and confocal microscopy method, detection of intracellular contributed to Wnt,β -catenin osteogenic differentiation of classic Wnt/ -catenin signaling pathway, cyclinD1 protein gene mRNA, protein expression level and protein activation state, deeply studying the mechanism of signal transduction pathway in the pathogenesis of osteonecrosis of femoral and Chinese medicine intervention in. Trying to find out the disease incidence of key links and traditional Chinese medicine interventions targeting; Chinese medicine Bushen Huoxue rule is added to the theory of cell signal transduction of new evidence.

青壮年股骨头坏死的保髋治疗是现代医学的研究焦点。其中促骨修复，预防股骨头塌陷，是难点问题。我们多年前期临床研究发现，补肾活血类中药复方及其制剂在股骨头坏死保髋治疗中疗效显著，但是其促进骨修复的分子生物学机制尚不了解。本课题拟用补肾活血胶囊，结合离体细胞，利用RT-PCR、Western blot、流式细胞仪、荧光染色＋共聚焦显微镜等方法，检测细胞内促成骨分化经典Wnt/β-catenin信号通路中Wnt、β-catenin,cyclinD1等蛋白基因mRNA、蛋白表达水平及蛋白激活状态，深入研究该信号通路在补肾活血胶囊促进骨修复的作用机制。试图寻找出补肾活血胶囊的干预作用靶点；为中医补肾活血治则治疗股骨头坏死理论增添细胞信号转导新证据。

本课题阐明了补肾活血胶囊经Wnt/β-catenin信号通路促进骨髓基质干细胞（BMSCs）向成骨细胞分化，抑制成骨细胞凋亡，从而保护激素性股骨头坏死中的成骨细胞，初步证明了 Wnt/β-catenin信号通路在本病中的修复机制。. 制备不同浓度的补肾活血胶囊含药血清：根据Meeh-Rubner公式A（体表面积）=K（系数）.W(体重g)2/3.10-4,计算出大鼠服用中药补肾活血胶囊的剂量，将补肾活血胶囊中药按照比列1:2：4 进行给药后获取不同浓度的含药血清。分离、培养、传代Wistar雄性大鼠BMSCs，观察BMSCs生长情况和形态特征。加入1×10-6mmol/L 地塞米松磷酸钠模拟激素单一因素损伤BMSCs模型。MTT法检测不同浓度补肾活血胶囊含药血清对BMSCs以及经地塞米松磷酸钠干预后BMSCs的增值及活性影响。流式细胞术检测不同浓度补肾活血胶囊含药血清对BMSCs以及经地塞米松磷酸钠干预后BMSCs细胞周期的影响。不同浓度补肾活血胶囊含药血清对BMSCs及经地塞米松磷酸钠干预后BMSCs成骨分化效果的检测：用钙钴法染色检测碱性磷酸酶、用茜素红法染色检测钙结节、用免疫组化检测Ι型胶原蛋白。采用Real-time PCR及Western blot分别检测含药血清培养的BMSCs及激素干预后BMSCs 的Wnt/β-catenin信号通路（Wnt、β-catenin、细胞周期蛋白CyclinD1等)基因表达水平及蛋白激活状态。. 研究结论如下：1.补肾活血胶囊能促进BMSCS的增值活性，无细胞毒性。2.补肾活血胶囊可显著促进BMSCs向成骨方向分化；3.补肾活血胶囊可以抑制骨髓基质干细胞凋亡；4.补肾活血胶囊可提高激素损伤后BMSCs 中Wnt、β-catenin和细胞周期蛋白CyclinD1 mRNA表达和蛋白的激活，诱导向成骨细胞方向分化。