【中文摘要】 骨关节炎(osteoarthritis,OA)是一种严重危害中老年人健康的慢性退行性骨关节病,临床上以膝关节发病最常见。随着社会人口的老龄化,该病的发病率日益上升,我国约有6 000万～7 000万人患不同程度的OA。硫酸软骨素(chondroitin sulfate,CS)是关节软骨的重要组成成分,它具有保护软骨、促进软骨基质的形成、缓解关节疼痛等多种药理作用与生理功能,体外及体内实验中显示它具有保护关节软骨的特性,临床应用中能够减轻骨性关节炎的症状。玻璃酸钠(sodium hyaluronate,SH)是关节滑液及软骨的重要组成成分,广泛存在于软骨多形细胞、滑膜细胞中,对关节、软骨起润滑、缓冲和屏障作用,组成和修复关节软骨。关节腔注射SH已成为OA的主要治疗方法,其能够增加关节滑液的黏弹性和润滑性,较好的改善OA,恢复滑膜和软骨基质的黏弹性,缓解炎症等。本课题研究了CS对软骨细胞增生及Ⅱ型胶原合成的影响,制备了硫酸软骨素玻璃酸钠注射剂(chondroitin sulfate and sodium hyaluronate injection,CS-SHinjection),考察了该制...更多剂对OA动物模型的治疗作用及其机制。本课题取得了以下几方面的结果。1研究了CS对于软骨细胞增生及Ⅱ型胶原合成的影响本研究考察了CS对于兔膝关节软骨细胞增生及Ⅱ型胶原合成的影响。结果显示,CS对软骨细胞的增生无明显影响,且能促进Ⅱ型胶原的合成。2研究制备了硫酸软骨素玻璃酸钠注射剂(CS-SH注射剂)制备了CS-SH注射液剂,建立了CS-SH注射剂的生产工艺。CS-SH注射液的性质稳定,质量可控。3研究了CS-SH注射剂对OA动物模型的治疗作用采用木瓜蛋白酶诱导的兔OA模型,用CS-SH注射剂兔膝关节腔内注射,以生理盐水和SH注射液为对照。给药5周后取材,做光镜观察Mankin评分、观察关节软骨变化情况。结果发现,CS-SH治疗可显著减轻兔OA症状,其疗效好于SH治疗组。4研究了CS-SH注射剂对OA的部分作用机制通过碱水解、蛋白酶裂解、超速离心的方法提取经CS-SH等治疗的OA模型兔关节软骨的糖胺聚糖(glycosaminoglycan,GAG),用天青A比色法检测GAG总量,考察治疗后关节软骨基质蛋白聚糖的变化。结果显示,CS-SH注射液可使病变关节软骨中蛋白聚糖含量增加。研究还检测了治疗后OA模型兔关节滑液中的IL-1β、TNF-α、TLMP-1水平,结果显示CS-SH注射液可显著降低IL-1β和TNF-α的表达水平,同时提高TIMP-1的表达水平。研究还检测了治疗后OA模型兔关节滑液中的NO、iNOS的水平,发现CS-SH注射液可显著降低NO、iNOS的表达水平。根据研究结果推测,CS-SH可能是通过促进软骨基质的合成,保护软骨细胞,降低炎性细胞因子IL-1β和TNF-α的表达水平,提高TIMP-1的表达水平,降低iNOS、NO的表达水平,进而减轻关节炎症,从而对OA产生治疗作用。本研究取得的主要成果和结论有:(1)体外培养中CS能促进软骨细胞Ⅱ型胶原的合成,对于软骨细胞的增殖没有影响。(2) CS-SH注射液制备工艺简单,质量可控,具有良好的稳定性。(3) CS-SH注射液可保护关节软骨,显著改善兔OA症状,减轻炎症。(4) CS-SH注射液可显著降低OA模型兔关节滑液中IL-1β和TNF-α的表达水平,提高TIMP-1的表达水平,显著降低NO、iNOS的表达水平,从而对软骨蛋白聚糖的降解起延缓作用,其作用效果强于SH注射液。 

【英文摘要】 Osteoarthritis (OA) is one of chronic degenerative joint diseases, which is especially harmful to the health of aged people. OA often occurs in knee joints. With the aging of social population, the incidence rate of OA is increasing. Nowadays, there are about 60 million to 70 million people suffer from this kind of diseases to some extent in our country.Chondroitin sulfate is an important component of articular cartilage. CS possesses various pharmacological actions and physiological functions, such as protecting cartilage, promoting the formation of cartilage matrix and relieving joint pain, etc. The protective effect of CS on articular cartilage was manifested in vitro and in vivo. CS can improve the symptoms of osteoarthritis in clinical application.Sodium hyaluronate (SH) is an important component of synovial fluid and articular cartilage with the extensive presence in polymorphic cartilage cells and synovial cells. SH plays an important role in lubrication, buffering and screening... of joints and cartilage, as well as construction and repair of articular cartilage. SH injection into articular cavity has become the main method in treating OA, which can increase the viscoelasticity and lubricity of joint synovial fluid, improve the symptoms of OA, restore the viscoelasticity of synovial membrane and cartilage matrix, and reduce inflammation.The influence of CS on proliferation and collagen II synthesis of chondrocytes in vitro was examined. Effects and mechanisms of CS and SH (CS-SH) injection on OA in rabbits were also explored. The main results of this study are listed as follows.1. Influence of CS on proliferation and collagen II synthesis of chondrocytesIt was shown that CS could promote the synthesis of collagen type II and had no influence on proliferation of chondrocytes.2. Preparation of CS-SH injection CS-SH injection was prepared and stable and quality controllable preparation was obtained.3. Effect of CS-SH injection on OA in rabbitsEffects of CS-SH injection on knee OA in rabbits induced with papain were evaluated. The animals were randomized into for groups and treated with CS-SH injection, CS injection, SH injection or NS injection, respectively. After 5 weeks of treatment, the knee joints of animals were collected and histopathological evaluation was performed. It was shown that treatment with CS-SH injection could significantly improve the symptoms of OA which was assessed by Mankin grade. The effect of CS-SH injection was better than that of SH injection.4. Mechanism of CS-SH injection on OAThe content of glycosaminoglycans (GAG), which was extracted by alkaline hydrolysis and protease digestion from knee joint cartilage of OA in rabbits treated with injections was determined by azur A colorimetry method. The results indicated that the content of prostaglandin in cartilage of animals treated with CS-SH injection was greatly increased.The effects of CS-SH injection on the expression levels of IL-1β, TNF-αand TMP-1 in synovial fluid after treatment were assessed. It was shown that CS-SH injection could significantly inhibit the expression of IL-1βand TNF-αand enhance the expression of TIMP-1. The results also indicated that CS-SH injection could significantly inhibit the expression of NO and iNOS.The results indicate that CS-SH injection may exert therapeutic action on OA by the mechanisms including the protective effect of CS on chondrocytes and decreased expression of IL-1βand TNF-α, increased expression of TIMP-1, as well as decreased expression of NO and iNOS in synovial fluid after treatment with CS-SH injection, and the lessening of inflammation of knee joint thereafter.The main achievements and conclusions of this paper are as follows.(1) CS can promote the synthesis of collagen type II and has no influence on proliferation of chondrocytes.(2) Stable and quality controllable CS-SH injection was prepared. (3) CS-SH injection could protect articular cartilage, lessen inflammation and significantly improve OA symptoms.(4) The mechanisms of CS-SH on osteoarthritis may owing to the expression decrease of IL-1βand TNF-αand the expression enhancement of TIMP-1, as well as the expression decrease of NO and iNOS in synovial fluid of OA animals. Thus the the degradation of OA cartilage was inhibited. The effect of CS-SH injection was better than that of SH injection. 

【中文关键词】 骨关节炎; 硫酸软骨素; 玻璃酸钠; 氧化氮
【英文关键词】 osteoarthritis; Chondroitin sulfate; sodium hyaluronate; NO