黄芪三仙汤对绝经后骨质疏松大鼠成骨细胞作用靶点蛋白质组学研究

Kidney deficiency and blood stasis are the main pathological basis of postmenopausal osteoporosis.Osteoblast is key cell in the occurrence of osteoporosis.Form the respect of osteoblast,we think there are possible that differential expression of key functional proteins exist in osteoblast of postmenopausal osteoporosis.The functional proteins are the targets of prescription for invigorating the kidney and activating the blood. Astragalus Sanxian Decoction is an old prescription for invigorating kidney and activating blood.Through long-term clinical and experimental studies it have been confirmed that there is obvious effect on postmenopausal osteoporosis.On this basis,this study is executed by culturing osteoporosis osteoblast in vitro system, combined with proteomics technique and serum pharmacology technology in vivo and vitro experiments,studying the differential expression of functional proteins on osteoblast with Astragalus Sanxian Decoction. The generality and specificity of functional proteins expression on osteoblasts will be studied with different serum，to aim at searching the targets of prescription for invigorating kidney and activating blood and reveal the essence of deficiency and blood stasis on postmenopausal osteoporosis.That can study part of mechanism of osteoporosis formation and Chinese traditional medicine on postmenopausal osteoporosis from level of the functional protein expression,which will lay the foundation on screening the treatment of postmenopausal osteoporosis with traditional Chinese medicine and develop new thinking to research Chinese herbal recipe.

肾虚血瘀是绝经后骨质疏松症的主要病理基础。成骨细胞是骨质疏松发生的关键细胞，从成骨细胞的角度来看，我们认为绝经后骨质疏松症成骨细胞中很可能存在差异性关键功能蛋白表达，这种关键功能蛋白点，正是补肾活血方药的作用靶点。.黄芪三仙汤是补肾活血古方，通过长期的临床和实验研究证实其对绝经后骨质疏松症有明显的疗效。在此基础上，本研究拟建立骨质疏松成骨细胞体外培养体系，利用蛋白质组学方法和血清药理学技术，结合体内、体外实验，进一步观察黄芪三仙汤对成骨细胞功能蛋白质差异表达，了解不同血清对成骨细胞功能蛋白质表达的共性和特异性，旨在寻找补肾活血方的作用靶点，揭示绝经后骨质疏松肾虚血瘀证的实质；从功能蛋白质表达层面研究骨质疏松形成的部分机制和中药对绝经后骨质疏松症的作用机理，为筛选治疗绝经后骨质疏松症的中药奠定基础，为中药复方的研究开拓新的思路。

本项目通过在体实验发现黄芪三仙汤能改善去卵巢的绝经后骨质疏松模型大鼠的体重、子宫重量、股骨和腰椎骨密度、骨矿含量、骨生物力学及大鼠生化指标。体外实验中，通过BCIP/NBT染色和碱性磷酸酶活性检测观察到黄芪三仙汤能促进成骨细胞分化；通过矿化结节染色和钙离子浓度检测观察到黄芪三仙汤有增加成骨细胞骨矿化作用。在作用靶点研究中，应用蛋白组学方法从绝经后骨质疏松大鼠成骨细胞中分析鉴定出4个比较可信的蛋白，分别是：抗增殖蛋白（prohibitin）、转录延伸因子B多肽1（transcription elongation factor B(SIII), polypeptide 1）、核苷二磷酸激酶（nucleoside diphosphate kinase）、肥大细胞羧肽酶A（mast cell carboxypeptidase A）。黄芪三仙汤可能通过调节这些差异蛋白质所涉及的能量代谢、细胞增殖和信号转导等多方面的功能，来实现对去卵巢所致大鼠绝经后骨质疏松症的防治作用。本项目全面肯定了黄芪三仙汤对绝经后骨质疏松症的防治作用。并将蛋白质组学研究和中药血清药理学研究有机结合，探讨中药复方的作用机制和作用靶点，为中药复方的研究开拓了新的思路。本研究发现的差异表达明显的蛋白质-抗增殖蛋白 (Prohibitin，PHB)广泛分布在各种生物细胞和组织中，PHB具有多种生物学功能，主要表现对于细胞分化和凋亡具有双向调节机制，并且与衰老关系密切，又可调控雌激素及其受体，可以应用于老年性疾病的细胞分化和凋亡的研究中，特别是绝经后骨质疏松症凋亡途径的研究。