FGF2调控软骨下骨活性促进关节软骨损伤修复的研究

发布时间：2018-06-08 18:51

本文选题：关节软骨修复 + 碱性成纤维细胞生长因子(FGF2/bFGF)　；参考：《北京协和医学院》2017年硕士论文

【摘要】：由于缺少血供,关节软骨损伤难以修复,最终会发展为骨性关节炎甚至运动障碍。目前,关节软骨损伤后的再生修复是临床医学的一个巨大挑战,也是再生医学研究的一个热点。多种生长因子参与了关节软骨的形成及发育过程,它们也被广泛应用于关节软骨损伤修复的研究中。本课题组在前期构建了一种时空控释碱性成纤维细胞生长因子(bFGF/FGF2)的胶原基复合材料发现,FGF2是通过上调内源性生长因子表达水平进而促进了关节软骨的损伤修复。进一步分析研究结果,我们推测上调的骨形态发生蛋白2和4(BMP2和BMP4)是FGF2促进关节软骨修复的主要原因。近期,通过实时定量PCR(qRT-PCR),我们发现升高的骨形态发生蛋白2(BMP2)和BMP4主要来源于软骨下骨,提示FGF2是通过调控软骨下骨活性,上调其BMP表达水平、进而促进了关节软骨的损伤修复。我们还发现BMP2和BMP4的受体竞争性抑制分子BMP3加重模型动物损伤区软骨下骨的破坏程度,抑制关节软骨修复。基于以上发现,我们提出了软骨下骨在关节软骨损伤修复中具有重要作用,FGF2可以调控其活性进而促进关节软骨的损伤修复,靶向递送FGF2至软骨下骨可以改善关节软骨缺损的修复。本研究针对这一假说开展了研究工作,首先通过软骨下骨区域直接注射FGF2考察了其对关节软骨修复的作用;进一步在此基础上,结合前期研究工作基础,构建了一种具有定向释放FGF2的胶原基复合材料,使FGF2可以只作用于软骨下骨;最后通过动物实验考察该复合材料对关节软骨损伤修复的影响及其机制。通过本研究工作,以期为FGF2在关节软骨损伤修复中的合理应用提供新思路,也为关节软骨损伤修复机制的深入理解提供新的依据。现阶段主要研究结果和结论如下:1.本研究首先通过关节软骨缺损区域直接注射低剂量FGF2至软骨下骨局部并联合应用BMP信号通路抑制剂Dorsomorphin,研究和分析了 FGF2在关节软骨损伤修复中的作用及其机制。研究发现,FGF2是通过调控软骨下骨的BMP信号通路,促进软骨的再生修复;而Dorsomorphin由于抑制了 BMP信号通路,减弱了 FGF2促进关节软骨修复的作用。2.为了进一步考察软骨下骨在FGF2促进关节软骨修复中的作用,我们又构建了一种定向释放FGF2的胶原基复合材料。通过纤维蛋白胶与胶原特异性相互作用的特点,结合物理吸附法构建了定向释放FGF2的胶原基双层膜材料,该胶原基双层膜具有表层致密的上层胶原膜和具有疏松结构的下层胶原膜,FGF2负载于下层疏松胶原膜。体外释放研究发现,双层膜所负载的分子具有定向释放的特点。3.本研究将上述定向释放FGF2的胶原基复合材料植入家兔关节软骨损伤区域。通过酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)方法检测收集到的复合材料植入后的动物关节液,发现定向释放FGF2的胶原基复合材料植入组的早期BMP2和BMP4的含量较对照组显著升高。进一步证实了早期的研究,FGF2可以上调BMP2和BMP4的表达水平。4.最后,本研究将上述定向释放FGF2的胶原基复合材料植入家兔关节软骨损伤区域,考察了该材料对关节软骨损伤修复的影响。研究发现,与对照组相比,该材料在植入8周后明显改善了关节软骨损伤修复的效果,有效促进了透明软骨和软骨下骨组织的再生以及新生组织与周围组织的整合。综上所述,本文证明了软骨下骨在关节软骨修复过程中具有重要作用,FGF2通过调控软骨下骨活性、上调BMP2和BMP4的表达水平,从而促进关节软骨的损伤修复;本研究成功构建了具有定向释放FGF2作用的胶原基双层膜材料,显著改善关节软骨的损伤修复效果。因此,由于其作用机理相对明确、使用剂量可以相对减少,并且作用周期较短,本研究所构建的定向释放FGF2的胶原基复合材料可能成为一种有应用前景的促进关节软骨修复的新型生物材料。
[Abstract]:Due to lack of blood supply, articular cartilage injury is difficult to repair and eventually develops into osteoarthritis and even dyskinesia. At present, regenerative repair after articular cartilage injury is a great challenge in clinical medicine and a hot spot in the research of regenerative medicine. In the study of the repair of articular cartilage injury, a collagen based composite of time and space controlled basic fibroblast growth factor (bFGF/FGF2) was constructed in our group, and FGF2 was found to promote the repair of articular cartilage by up regulating the expression level of endogenous growth factors and further analysis of the results. We speculate that the up-regulated bone morphogenetic protein 2 and 4 (BMP2 and BMP4) are the main reasons for FGF2 to promote articular cartilage repair. Recently, by real-time quantitative PCR (qRT-PCR), we found that increased bone morphogenetic protein 2 (BMP2) and BMP4 are mainly derived from subchondral bone, suggesting that FGF2 regulates the activity of subchondral bone and up-regulated its BMP expression. It further promotes the repair of articular cartilage damage. We also found that BMP2 and BMP4 receptor competitive inhibitor BMP3 aggravates the damage degree of subchondral bone in the damaged area of model animals and inhibits articular cartilage repair. Based on the above findings, we suggest that subchondral bone plays an important role in the repair of articular cartilage injury, and FGF2 can regulate it. The activity further promotes the repair of articular cartilage damage. The target delivery of FGF2 to subchondral bone can improve the repair of articular cartilage defects. This study aims at this hypothesis. First, a direct injection of FGF2 into the subchondral bone area was used to investigate the repair of articular cartilage. On the basis of work, a collagen based composite material with directional release of FGF2 was constructed to make FGF2 only act on the subchondral bone. Finally, the effect of the composite on the repair of articular cartilage injury and its mechanism were investigated by animal experiments. Through this work, a new thought was provided for the rational application of FGF2 in the repair of articular cartilage injury. The main research results and conclusions at this stage are as follows: 1. first of all, we studied and analyzed FGF2 in joint soft tissue by direct injection of low dose FGF2 to subchondral bone and combined with BMP signaling pathway inhibitor Dorsomorphin through articular cartilage defect area. The role and mechanism in repair of bone injury. It is found that FGF2 promotes the regeneration of cartilage by regulating the BMP signaling pathway of the subchondral bone, and Dorsomorphin reduces the role of FGF2 to promote articular cartilage repair due to the inhibition of BMP signaling pathway,.2. in order to further examine the subchondral bone in FGF2 to promote articular cartilage repair. We have also constructed a collagen based composite material for the directional release of FGF2. Through the characteristics of the specific interaction between fibrin glue and collagen, a collagen based double layer membrane material directed to release FGF2 is constructed by physical adsorption. The collagen based double layer membrane has a dense layer of collagen membrane in the surface layer and a loose structure of the lower layer glue. The original membrane, FGF2 loaded with the loose collagen membrane in the lower layer. In vitro release studies have found that the molecules loaded in the double layer membrane have the characteristics of directional release..3. this study implants the collagenous matrix composite of the directional release of FGF2 in rabbit articular cartilage damage area. By enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) Fang Fajian The content of early BMP2 and BMP4 in the group of collagen based composite implanted FGF2 was significantly higher than that of the control group. Further confirmed the early study, FGF2 could up-regulate the expression level of BMP2 and BMP4 at the end of.4.. This study released the above directed release of FGF2 in the collagen basis. The effect of the material on articular cartilage damage repair in rabbits was investigated. It was found that, compared with the control group, the material obviously improved the repair of articular cartilage injury after 8 weeks of implantation, effectively promoting the regeneration of the hyaline cartilage and subchondral bone tissue, as well as the new tissue and surrounding tissue. In summary, this paper shows that the subchondral bone plays an important role in the repair of articular cartilage. FGF2 promotes the repair of articular cartilage by regulating the activity of subchondral bone and up regulating the expression level of BMP2 and BMP4. This study has successfully constructed a collagen based double layer membrane material with the effect of directional release of FGF2. Therefore, because of the relatively clear action mechanism and the relative reduction in the use of the articular cartilage, the collagen based composite of the directional release of FGF2 in this study may be a promising new biomaterial to promote the repair of articular cartilage.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68;R318.08

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