【作者】 杨庆秋；

【导师】 胡侦明；

【作者基本信息】 昆明医学院 ， 外科学， 2011， 博士

【摘要】 研究背景：云南白药的主要组成成分有三七、重楼、麝香等名贵中草药,对于骨伤治疗有其独特的疗效,但对其促进骨折愈合的作用机理至今尚无明确的生物学机理解释。目前,有关白药治疗骨折的许多研究仍停留在临床疗效观察阶段,对于白药复方的有效成分的分析及作用机理却很少见报道。由于云南白药的组成配方是保密的原因,有关白药复方的药理、药效研究难以深入,更缺乏从分子、细胞水平上进行深入系统的研究,缺少良好的现代病理生理学基础。同时,由于药理研究模式基本上沿用西药药理学的模型,不能客观地反映白药复方的主要药效,无法切实表达复方的临床疗效,这也是白药复方研究能否突破低水平重复的关键。研究目的：作为我国著名中成药-云南白药,有必要通过体外细胞培养方法以及进一步的动物实验研究,采用现代分子生物学技术对其主要成分对骨折愈合过程中的成骨作用和作用机理进行深入研究,进一步揭示云南白药对骨组织愈合修复作用的分子机制。研究方法：本课题设计首先通过拆方研究,观察云南白药及十种组成成分(代号)对体外鼠骨髓基质细胞分化、增殖以及对骨基质蛋白及相关生长因子表达变化的影响,阐明云南白药各原药成分在MSC成骨诱导分化中的作用、促进骨折修复的细胞、分子的作用机理。在此基础上,从前期实验中寻找出高效低毒成分,将分析和综合研究有机结合起来,进行重新配伍组方,并将新配伍局部应用到兔骨折愈合过程中,通过动态观察骨折修复的情况,x线检测,组织学、以及免疫组化等分子生物学等方法,观察重组配伍的云南白药对骨组织修复过程中几种细胞因子及胶原的影响,明确云南白药复方制剂中各原药成分所处的地位和作用,进一步探讨云南白药促进骨折愈合的作用机制。研究结果：1.云南白药的多种成分通过促进鼠骨髓基质细胞产生BMP和TGF-β等因子,从而诱导MSCs向成骨细胞转化,促进成骨细胞活性,同时促进骨基质合成,其中云南白药E、G、H、I、J五种原药成分细胞毒性较小,促进基质细胞转化作用较明显。2.在实验性兔骨折愈合的早期、中期阶段,1到6号组方白药能促进内皮细胞增殖,诱导新生血管的形成,从而促进骨折的愈合过程。3.本研究中还发现云南白药还通过促进Ⅰ型胶原与其受体整合素αβ结合,激活FAK途径,促进成骨细胞的增殖和分化,并具有促进局部血管生成,改善局部供氧条件的作用,这可能有助于抑制骨折愈合过程中的软骨化骨,促进膜内成骨以加速新骨形成的速度和质量,缩短骨折愈合的过程。研究结论：1.骨折愈合过程早、中期,云南白药主要通过直接影响及间接影响促进骨髓基质细胞向成骨细胞分化,促进成骨细胞活性,诱导骨折周围组织新血管生成,引起骨折局部血供丰富及细胞活跃等机制,从而加快骨折的愈合。2.云南白药的多种成分中可能具有某些类似骨生长因子和血管生长因子的作用,其中E、G、H、I、J五种原药成分细胞毒性较小,促进骨髓基质细胞转化作用较明显,具有进一步的研究开发基础。3.白药单个原药成分之间、E、G、H、I、J五种原药成分重新组方及白药复方的药效学比较实验结果较为复杂。新组方白药对骨折愈合的促进作用虽然是共同的,但各新组方之间却没有显示出明显的相关性及差异性,白药中各单个原药成分对复方药效起不起作用、起多大作用,其具体有效成分,尤其是白药复方制剂的有效成分和作用机制尚待进一步深人研究。更多还原

【Abstract】 BackgroundThe essential component of the famous "Yunnan Baiyao" are san qi (Panax notoginseng) and other Chinese Herbal Medicine, and it is already used widely in clinical almost one hundred years, and has unique, reliable curative effect especially in treating of bone fracture. But we know less about the biological Mechanism of action.At present, a lot of studies about Baiyao are stay at the observing stage of clinical effects, rarely study about the analysis of effective constituent and mechanism of action. And the study about pharmacology and pesticide effect of Compound Baiyao is Low levels and repeated. And short of systematic and Intensive study at molecular and cellular level, and lake of Good and modern basis of Pathophysiology.The Pathophysiologic study model is practically the same as Western medicine. So is unable to reflect principal clinical and pesticide effects of compound Baiyao objectively, and this is strategic point of to can make a breakthrough an indicator of at the study of compound Baiyao.ObjectiveAs our country Famous "Yunnan baiyao", it is necessary to intensive study the bone formation and mechanism of Yunnan Baiyao and major components on treating bone fracture by means of making external cells (MSCs) cultivation and further animal experiment with adopting the contemporary Molecular biology technique, to explore the mechanism of actions of Yunnan Baiyao and major components on promoting bone tissue repair.MethodsThis project, first, put a broken prescriptions study into practice by observing the Yunnan Baiyao and ten major components (code name) expect that proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from SD rats and the express change of bone Matrix Protein and related growth factor. To explore the effect of Yunnan Baiyao and each component on that proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and the cellular molecule mechanism of promoting the healing of bone fracture.Second, to look for efficient and harmfulless component from earlier studies, and design the new compound Baiyao application locally in the healing of rabbit bone fracture by fall together analyse and comprehensive. To clear and definite the located position and action of Yunnan Baiyao and each component, and explore the mechanism of promoting the healing of bone fracture through dynamically observe the situation of bone tissue repairing by detection of x-Ray, histology, immunohistochemistry, molecular biology and the effect of Yunnan Baiyao and each component on some kinds of cell factor and collagen during the repairing of bone fracture.Results1. Variety of Yunnan Baiyao components stimulate the differentiation of osteoblasts derived from bone marrow stromal dells by that facilitate the production of some factors such as TGF-β,BMP, and encourage the activity of osteoblast, advance the composition of bone matrix. E, G, H, I, J components have obvious effect on the differentiation of bone marrow stromal dells and have lower cytotoxicity in all of Yunnan components.2.One to six number new formulating Baiyao has obvious effect on promoting the healing of bone fracture by proliferation of endothelial cell and formation of new blood vessels in early and medium term during the repairing of rabbit bone fracture.3.This study find that Baiyao also have obvious effect on proliferation and differentiation of osteoblast by promoting combination of collagen I and integrainαβand activating FAK-way and formatiing local new blood vessels and improving condition of local oxygen supply. It is possible contribute to accelarte speed and quality and reduce the process of the healing of bone fracture by that new formulating Baiyao suppresed the process of cartilagenous and promoted that of intramembranous ossification.Conclusions 1. In early and medium term during the repairing of rabbit bone fracture, the possible mechanism of Yunnan Baiyao and major components in promoting bone healing was to directly or indirectly effect on the proliferation of BMSCs, enhance the activity of osteoblast, and the formation of new blood vessels, therefore, lead to blood supply plentiful and cell vigorous of fracture local.2. It is possible the Yunnan Baiyao and major components had same stimulative fouction similar as bone growth factor and vascular growth factor. Among all of components, E、G、H、I、J had obvious stimulative effect on the differentiation of bone marrow stromal dells and have lower cytotoxicity, which had foundation of farther research and development.3. The experimental result that the Compare pharmacological action of in each Baiyao component, new formulating Baiyao and compound Baiyao is complicated. Although the 1 to 6 number new formulating Baiyao has a common effect on promoting the healing of bone fracture, but this research did not show obvious relativity and Differentiation both between and within new formulating Baiyao components as we thought before. We did not understand that each Baiyao component play what kind of role in the compound Baiyao, and what is the specific active constituent in all of Baiyao components. Especially the definite constituent in which and the possible mechanism of promoting the healing of bone fracture need more intensive work to do.更多还原