【作者】 张磊；

【导师】 陈百成；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:骨关节炎(osteoarthritis, OA)是一种以关节软骨损伤为特点的、原因不明的慢性退行性疾病。OA是全球分布最广、最为流行的疾病之一,严重影响了患者的日常和社会生活。OA的主要病理改变为关节软骨的损伤、退变及软骨下骨的硬化、增生、囊变,继而导致关节间隙狭窄。关节软骨是成熟的透明软骨没有血管和修复细胞,软骨细胞的分裂潜能及其修复能力非常有限,当关节软骨发生严重退行性变时进行治疗很困难。目前OA的保守治疗方法虽然很多,但是治疗效果都有限。对于严重OA的病例只能采用人工关节置换来治疗,然而关节置换术存在着诸如年龄、手术损伤大、费用高、长期预后、再次翻修等一系列问题。因此寻找有效的保守治疗骨关节炎的方法至关重要。碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)是最有效的体外软骨细胞分裂素之一,具有加速软骨代谢,促进关节软骨再生的作用,但bFGF体内半衰期很短仅为3~5min,全身或局部应用的效果均不能满足临床上治疗需要。因此应用bFGF缓释制剂对长期维持损伤局部的有效药物浓度有重要意义。目前研究发现关节软骨组织中的基质金属蛋白酶-13(matrix metalloproteinases-13, MMP-13)、基质金属蛋白酶抑制剂-1(tissue inhibitors of metallomatrix-1, TIMP-1)可调控软骨细胞外基质的合成与降解,参与关节软骨的损伤过程。因此,本实验应用壳聚糖包被bFGF制作缓释微球以长时间维持关节内的有效药物浓度,并观察其抑制OA进展的作用及其对MMP-13、TIMP-1 mRNA表达的影响。探讨bFGF持续释放对实验性兔膝关节OA的治疗作用的部分作用机制。方法:健康成年新西兰大白兔54只随机选取9只作为对照组,其余45只作为实验组给予实验干预。于兔双后腿膝关节腔内注射木瓜蛋白酶建立兔膝骨关节炎模型,将其随机分为5组,每组9只,分别为模型组、PBS微球组(PBS-M)、bFGF溶液组(bFGF-S)、10μg bFGF微球组(10-bFGF-M)及100μg bFGF微球组(100-bFGF-M)。用壳聚糖制成含PBS及含bFGF的缓释微球,干冻保存备用。分别于第3周及第6周两次于各治疗组动物关节腔内注射溶液或微球,于第9周处死动物后取材。肉眼观察兔软骨组织大体形态,采用印度Ink评分法比较各组动物大体软骨损伤程度;通过HE染色及PAS染色方法观察软骨组织病理改变,采用Mankin评分系统评价;应用实时定量RT-PCR检测关节软骨组织MMP-13、TIMP-1 mRNA表达变化。结果:1微球的外观形态及粒径分布光学及电子显微镜观察到壳聚糖微球外观圆整,粒径分布均匀,大部分微球粒径分布在3~7μm范围内,平均粒径为4.69±0.06μm。2各组关节软骨标本Ink评分结果:与对照组相比,模型组关节软骨损伤程度明显加重(3.38±0.65 vs. 0.15±0.37, P<0.05);PBS-M组、bFGF-S组与模型组相比,关节软骨损伤程度无明显减轻(3.08±0.64, 3.31±0.63 vs. 3.38±0.65, P值均>0.05),而10-bFGF-M组和100-bFGF-M组关节软骨损伤程度较模型组明显减轻(1.92±0.49, 1.31±0.48 vs. 3.38±0.65, P值均<0.05)。并且100-bFGF-M组与10-bFGF-M组相比关节软骨损伤程度明显减轻(1.31±0.48 vs. 1.92±0.49, P<0.05)。3各组关节软骨标本镜下Mankin评分结果:与对照组相比,模型组关节软骨损伤程度明显加重(13.08±0.64 vs. 0.53±0.51, P<0.05);bFGF-S组、PBS-M组与模型组相比,关节软骨损伤程度无明显减轻(12.15±1.07, 12.85±0.80 vs. 13.08±0.64, P值均>0.05),而关节软骨损伤程度较模型组明显减轻(8.08±0.49, 5.85±0.69 vs. 13.08±0.64, P值均<0.05),100-bFGF-M组与10-bFGF-M组相比关节软骨损伤程度明显减轻(5.85±0.69 vs. 8.08±0.49, P<0.05)。4关节软骨组织MMP-13、TIMP-1mRNA表达改变:与对照组相比,模型组MMP-13 mRNA表达明显上调, TIMP-1 mRNA表达明显下降(P值均<0.05)。PBS-M组、bFGF-S组MMP-13、TIMP-1mRNA表达与模型组比较无明显差异(P值均>0.05),10-bFGF-M组和100-bFGF-M组较模型组MMP-13 mRNA表达明显下降,TIMP-1 mRNA表达明显上调(P值均<0.05)。并且100-bFGF-M组较10-bFGF-M组MMP-13 mRNA表达明显下降,TIMP-1 mRNA表达明显上调(P值均<0.05)。结论:1关节腔内注射木瓜蛋白酶,可建立膝骨关节炎模型。用沉淀/凝聚法成功制备bFGF壳聚糖微球。2壳聚糖包被bFGF与单纯bFGF溶液注射组相比,具有明显减轻兔膝骨关节炎软骨损伤的作用,提示本实验制备的bFGF缓释微球能维持bFGF相对稳定的关节腔内浓度,保证了软骨损伤的修复。3兔膝骨关节炎模型组膝关节软骨组织MMP-13 mRNA表达明显增加,TIMP-1 mRNA表达明显下降。而通过bFGF缓释微球的治疗,可明显降低MMP-13 mRNA表达,增加TIMP-1 mRNA表达,推测bFGF缓释微球治疗骨关节炎的作用可能通过调节MMP-13/TIMP-1 mRNA表达实现。更多还原

【Abstract】 Object: Osteoarthritis (OA) is a chronic, degenerative disorder of unknown cause characterized by gradual loss of articular cartilage. It is the most prevalent disease in our society, with a worldwide distribution. Since joint function is remarkably affected, daily living and social activities of patients with OA are restricted. OA process results in cystic degeneration of the bone surrounding the joint, with loss of cartilage and irregular, abnormal bone formation at the edges of the joint and narrowing of the joint space. The articular cartilage, which is the matured hyaline cartilage, has no vessels, and the cells for repair cannot be provided. There are limits to the division potential of the cartilage cells and their ability to be repaired. Therefore, it becomes difficult to treat OA when it progresses and the articular cartilage degenerates. At present, there are many conservative treatments for OA, but their clinical outcomes are limited. Advanced OA only be managed by artificial joint replacements, but there are problems concerning the degree of invasion, cost, and long-term prognosis. For these reasons, an effective conservative treatment to inhibit OA progression is needed. Basic fibroblast growth factor (bFGF) is regarded as one of the most potent mitogens for chondrocytes in vitro, which can accelerate cartilage metabolism or promote cartilage regeneration. The effect of bFGF cannot be maintained, even when it is locally injected, because its biologic half-life is short (3~5 minutes). Therefore, it is important to maintain the bFGF concentrations by using a sustained-release system of gelatin hydrogel microspheres. At present, many studies showed that the tissue inhibitors of metallomatrix-1 (TIMP-1) and tissue inhibitors of metallomatrix-1 (TIMP-1) could regulate the synthesis and degradation metabolism of extra-cellular matrix (ECM), and play important roles in the development the degradation of articular cartilage. So, in this experiment, bFGF was made into chitosan microspheres to maintain the bFGF local effective concentrations. To observe the inhibitory effects of bFGF in microspheres in treating OA in the rabbit knee and influence to express of TIMP-1 and MMP-13 mRNA. Our objective was to investigate the partial mechanism by intraarticular injections of bFGF in microspheres.Methods: The experiment was performed on 54 adult New Zealand white rabbits. We used intra-articular injection caroid in rabbit knee to induce osteoarthritis models. These animals were divided into a model group and four therapeutic groups (PBS-M, bFGF-S, bFGF-10-M, bFGF-100-M) at random, at the same time we made a control group. Meanwhile, we used chitosan to make microsphere with PBS or bFGF impregnated. We injected the PBS-impregnated or bFGF impregnated microsphere or bFGF-liquid into rabbit knee at week 3 and 6, and killed the animals at week 9. Cartilage pathology was scored by Indian Ink system in naked eyes. And scored by Mankin system in HE or PAS staining. The expression of cartilage TIMP-1 and MMP-13 mRNA was monitored by real-time PCR.Results:1 Shape and size of Chitosan microspheresIt can be got that chitosan microspheres made from the optimal formula have good sphericity and narrow size distribution by optical microscopy and SEM photography. The size distribution was range from 3μm to 7μm. The average diameter was 4.69±0.06μm.2 The result of Ink score in each group: Compared with control animals, the injury of model animals was dramatic (3.38±0.65 vs. 0.15±0.37, P<0.05), There were no differences between the model group and the therapeutic groups that used PBS-impregnated microsphere or bFGF-liquid at the injury of knee (3.08±0.64, 3.31±0.63 vs. 3.38±0.65, P>0.05). However, in bFGF-impregnated microsphere group the injury of cartilage decreased (1.92±0.49, 1.31±0.48 vs. 3.38±0.65, P<0.05). And compared with 10-bFGF-impregnated microsphere group, in 100-bFGF-impregnated microsphere group the injury of cartilage was notably decreased (1.31±0.48 vs. 1.92±0.49, P <0.05).3 The result of Mankin score in each group: Compared with control animals, the injury of model animals was dramatic (13.08±0.64 vs. 0.53±0.51, P<0.05), There were no differences between the model group and the therapeutic groups that used PBS-impregnated microsphere or bFGF-liquid at the injury of knee (12.15±1.07, 12.85±0.80 vs. 13.08±0.64, P>0.05). However, in bFGF-impregnated microsphere group the injury of cartilage decreased (8.08±0.49, 5.85±0.69 vs. 13.08±0.64, P<0.05). And compared with 10-bFGF-impregnated micro- sphere group, in 100-bFGF-impregnated microsphere group the injury of cartilage was notably decreased (5.85±0.69 vs. 8.08±0.49, P<0.05).4 Charges of cartilage TIMP-1、MMP-13 mRNA expression in each group: Compared with control group, the injury of model animals was dramatic, and the expression of TIMP-1 mRNA was decreased, meanwhile MMP-13 mRNA was increased (P<0.05). There were no differences between the model group and the therapeutic groups that used PBS-impregnated microsphere or bFGF-liquid at the expression of TIMP-1 and MMP-13 mRNA (P>0.05). However, in bFGF-impregnated microsphere group the expression of TIMP-1 mRNA was increased, and MMP-13 mRNA was notably decreased (P <0.05). And compared with 10-bFGF- impregnated microsphere group, in 100-bFGF-impregnated microsphere group the expression of TIMP-1 mRNA was increased, and MMP-13 mRNA was notably decreased (P <0.05).Conclusion: 1 The intra-articular injection caroid can successfully induce osteoarthritis model in the rabbit knee. bFGF-impregnated microsphere is successfully made from Chitosan.2 The bFGF can Sustained release to recovery the damage of cartilage of rabbit osteoarthritis only when it impregnated in microsphere.3 Compared with control animals, the injury of model animals was dramatic, and the expression of TIMP-1 mRNA was decreased, meanwhile MMP-13 mRNA was increased. However, the injury of knee was released in the bFGF-impregnated microsphere group, the expression of TIMP-1 mRNA was increased, and MMP-13 mRNA was notably decreased. So the pathogenesis of this role may through up-regulated TIMP-1 mRNA and down-regulated MMP-13 mRNA.更多还原