【作者】 王林龙；

【导师】 陈廖斌；

【作者基本信息】 武汉大学 ， 外科学， 2014， 博士

【摘要】 骨关节炎(osteoarthritis, OA)是一种以软骨退行性病变为主要病理特征的慢性关节疾病,是老年人群疼痛和致残的主要原因。OA病因复杂,传统观点认为主要由机械因素及年龄相关的软骨退变所致。但近年来越来越多的证据显示,代谢综合征(]metabolic syndrome)与OA有密切联系。流行病学调查研究发现,OA患者的代谢综合征发病率明显增加。作为代谢综合征的重要组成部分,糖尿病(Diabetes Mellitus, DM)与OA关系密切,并可能是OA发病的一个独立危险因素。流行病学调查研究发现,非胰岛素依赖型糖尿病患者更易患OA,且OA人群患糖尿病的风险较对照组人群患糖尿病的风险高。此外,有研究发现,以链脲佐菌素(Streptozotocin, STZ)诱导糖尿病,大鼠软骨出现胰岛素样生长因子1(insulin-like growth factor1, IGF-1)抵抗,70天后滑膜结构重塑,胶原沉积。这些研究均提示糖尿病与OA之间存在密切关联,而高血糖作为糖尿病最直接的表现,其在糖尿病性OA发生发展中的作用目前鲜有报道。OA不仅仅是关节软骨的损伤,也可观察到其它关节组织(如滑膜)的病理性改变。OA的初始表现大多是滑膜病变,这些病变一般要早于关节软骨的X线或MRI的影像学表现。滑膜炎与OA的许多临床症状相关,可反映疾病的结构性进展。滑膜炎的严重程度可提示髌股关节软骨病将如何进展。由于一些可溶性介质的活性,滑膜炎成为OA发病机制的关键因素。参与OA发病机制的两个主要细胞因子,IL-1β和TNF,主要由活化的滑膜细胞、单核细胞和关节软骨细胞产生。此外,在OA进程中,滑膜可产生基质金属蛋白酶(matrix metalloproteinases, MMPs)和聚集蛋白聚糖酶(aggrecanase),促进软骨基质降解。滑膜在OA发病中的重要作用及其作为治疗靶标的可能性已经受到广泛关注。内质网(endoplasmic reticulum, ER)应激是真核细胞在受到外界刺激时的一种保护性应激反应,通过诱导未折叠蛋白反应(unfolded protein response, UPR)来进行适应性调节,UPR则通过抑制蛋白的合成,诱导内质网伴侣蛋白表达以此促进蛋白折叠以及加速未折叠蛋白或错误折叠的蛋白的降解来缓解内质网应激。研究表明,内质网应激偶联炎症反应与多种疾病的发生发展密切相关,包括糖尿病和OA。OA软骨的内质网应激标志物葡萄糖调节蛋白-78(Glucose regulation protein78,Grp78)和Bcl-2交互蛋白(Bc12-Interacting Protein-1,Bag-1)较正常软骨表达增高。更有研究表明,细胞内晚期糖基化终产物的聚集通过内质网应激诱导软骨细胞凋亡。此外,有研究证明了关节炎滑膜和滑膜细胞高表达内质网应激相关基因。为证实糖尿病慢性高糖暴露可致OA,且滑膜内质网应激在其中发挥关键作用,本研究通过动物实验建立1型糖尿病和OA模型,并在细胞和分子水平证实高糖的损伤作用及其可能的内质网应激机制,在整体和细胞水平证实我们的假说。第一部分糖尿病大鼠OA易感现象目的：流行病学调查和临床试验研究提示糖尿病与OA关系密切,本部分拟通过整体动物实验,证实糖尿病大鼠关节软骨质量下降,长距离跑步刺激下OA易感现象。方法：成年wistar雄性大鼠STZ(60mg/kg)一次性腹腔注射,2周后大鼠断尾取血检测糖尿病造模成功率。糖尿病造模成功大鼠正常饮食一月后,给予长距离跑步(每日以20m/mmin的速度跑55mmin,共6周,总跑步里程30Km)诱导OA。跑步结束后,异氟烷麻醉处死大鼠。取部分膝关软骨节行墨汁染色,观察大体形态改变并进行软骨大体形态评分。剩余膝关节组织固定、包埋、脱钙切片后,行HE和番红O染色,Ⅱ型胶原免疫组化,光镜下观察软骨病理改变,并行OA的Mankin’s评分。此外,膝关节滑膜行HE和MMP-13、NF-KB免疫组化。结果：①糖尿病大鼠OA造模前后血糖变化：大鼠STZ腹腔注射后一周尾静脉血糖为23.83±1.34mmol/L,对照组血糖为5.90+1.34mmo1/L,两组间统计学差异显著(P<0.01),糖尿病造模成功率100%；跑步6周后,糖尿病组大鼠血糖为32.16+1.14mmol/L,对照组血糖为7.19±0.47mmo1/L,糖尿病大鼠在整个实验过程中均处于高血糖状态；②关节软骨大体改变及评分：与对照组相比,正常大鼠接受跑步刺激后,其关节软骨大体形态正常,关节表面较光滑；糖尿病大鼠接受跑步刺激后,其关节软骨表面出现较严重磨损,行墨汁染色后发现关节软骨表面明显毛糙,糖尿病组软骨大体评分均较对照组明显升高(P<0.01)；③关节软骨病理学改变及评分：跑步后大鼠膝关节石蜡切片行番红0染色后发现,对照组基质染色明显,表面出现轻度毛糙；与对照组相比,糖尿病组出现不同程度的软骨表面缺损,软骨基质结构紊乱,软骨厚度变薄,基质淡染,软骨细胞数明显减少；免疫组化示糖尿病鼠关节软骨Ⅱ型胶原蛋白表达降低。跑步诱导OA后,糖尿病组软骨OA病理Mankin’s评分升高至11.9分,较对照组(1.6分)明显增高(P<0.01)；④滑膜病理改变：糖尿病大鼠膝关节滑膜衬里细胞层增厚,间质细胞数增多,出现炎性细胞浸润,滑膜炎组织学评分明显增高(P<0.01),免疫组织化学结果显示,MMP-13和NF-κB的蛋白表达显著升高(P<0.01)。结论：糖尿病损伤大鼠关节软骨和滑膜,在接受过量跑步负荷诱导OA后,膝关节出现典型的OA病理改变,提示,糖尿病高糖状态可致大鼠OA易感,滑膜炎可能参与糖尿病OA发生。第二部分高糖通过损伤滑膜引起软骨细胞病理变化目的：本部分研究拟在细胞水平证实高糖对关节软骨和滑膜的损伤作用及其相互作用机制。方法：以不同浓度葡萄糖(10、20、30、40mM)分别处理大鼠膝关节软骨细胞和成纤维样滑膜细胞(Fibroblast-Like Synoviocytes, FLS)3天和10天,实时定量PCR检测细胞基质合成(col2al、aggrecan)、基质降解(MMP-3、MMP-13、ADAMTS-4、 ADAMTS、 TIMP-1和TIMP-3),以及炎症因子(IL-6/TNF-a)和凋亡相关基因(caspase-3、 caspase-8、 caspase-9和Bcl-2)的表达。检测FLS培养液中MDA和SOD的改变。然后,以30mM葡萄糖处理FLS3天的细胞培养液刺激软骨细胞3天,检测软骨细胞基质合成、基质降解、炎症和凋亡改变。结果：①对软骨细胞：高糖刺激3天和10天对软骨细胞的活力无明显影响；3天时软骨基质合成相关基因col2al和aggrecan表达增加,10天时增加更为显著；基质降解重要基因在3天时显著降低,但高糖刺激10则转为升高；凋亡相关基因在3天和10天均未见明显变化；IL-6和TNF-α在10天明显升高。②对FLS：高糖刺激3天时对滑膜细胞活力有促进作用,10天时转为抑制：高糖处理3天时FLS促炎、降解和凋亡相关基因即明显升高,并且升高的程度在高糖刺激10天时更为显著；此外,高糖培养基培养3天的FLS培养液上清中SOD含量呈葡萄糖浓度依赖性降低,MDA浓度依赖性升高。③滑膜培养液对软骨细胞：30mM高糖的滑膜培养液培养软骨细胞后,软骨细胞基质合成显著下降,促基质降解酶表达增强,促炎因子IL-6和TNF-α显著升高；凋亡相关基因均未见明显变化。结论：FLS较软骨细胞更易受高糖损伤。高糖可能通过刺激FLS分泌炎症和降解因子,从而引起软骨细胞病理损害。氧化损伤可能参与了这一过程。第三部分内质网应激参与高糖所致滑膜损伤目的：拟在细胞分子水平,进一步探讨高糖损伤FLS的机制,证实内质网应激参与糖尿病性OA。方法：①高糖诱导内质网应激：高糖分别处理软骨细胞和FLS24/48/72小时,检测细胞内质网应激标志基因表达；②内质网应激效应的证实：实验分四组,正常对照组(10mM葡萄糖)、内质网应激诱导组(10mM葡萄糖+内质网应激诱导剂衣霉素5μg/m)、高糖对照组(30mM葡萄糖)和内质网应激抑制组(30mM葡萄糖+内质网应激抑制剂4-丁酸苯酯1mM),分别处理FLS2/4/8/16小时,检测滑膜细胞内质网应激、凋亡和基质降解相关基因表达情况。结果：①高糖诱导关节软骨细胞和FLS内质网应激：软骨细胞高糖处理24、48和72小时结果显示,内质网应激标志基因C/EBP同源蛋白(CCAAT-enhancer-binding protein homologous protein, CHOP)呈时间依赖性升高,升高在72小时具有统计学意义(P<0.05)。与对照组相比,NF-κB和生长静止DNA损伤诱导基因34(growth arrest and DNA damage-inducible gene34,GADD34)在葡萄糖浓度20和30mM时出现显著性升高;GRP78在20mM葡萄糖浓度处理72小时时升高。对于FLS,在30mM高糖处理24小时时,所有检测的内质网应激标志基因的表达明显上调,而当处理时间延长到48和72小时后,这些基因表达恢复至对照组水平。比较发现,高糖诱导FLS应激的强度明显大于对软骨细胞的强度。②衣霉素诱导FLS内质网应激并出现类似高糖暴露的细胞损伤：与正常对照组相比,衣霉素处理组内质网应激标志基因中脑星形胶质细胞源性神经营养因子(mesencephalic astrocyte-deriVed neurotrophic factor,MANF)和GADD34出现时间依赖性升高,在处理8和16小时时升高具有显著统计学意义(P<0.01)；MMP-3、MMP-13和ADAMTS5升高,同时TIMP-1也明显升高；caspase-3禾□caspase-9在衣霉素处理16小时明显升高,caspase-8则在4小时明显升高,Bcl-2则呈时间依赖性降低,降低在8和16小时具有显著统计学意义(P<0.01)；此外,葡萄糖转运体(Glucose transporter,GLUT)-1在8和16小时明显升高,同时伴有iNOS的升高(P<0.05)。③4-丁酸苯酯抑制高糖所致的FLS内质网应激及细胞炎症降解改变：与高糖对照组相比,4-丁酸苯酯处理组内质网应激标志基因显著降低(P<0.01)；MMP-3明显降低,但同时TIMP-1也明显降低(P<0.01),MMP-13和ADAMTS-5无明显改变；caspase-3呈时间依赖性降低,且降低在处理16小时具有统计学意义(P<0.01),抗凋亡基因Bcl-2升高,在4小时和8小时差异具有显著统计学意义；此外,葡萄糖转运体GLUT-1在16小时明显降低(P<0.01)。结论：高糖可诱导软骨细胞和FLS内质网应激,且对FLS的影响更为显著。内质网应激介导了高糖所致的FLS降解和凋亡等病理损伤。综上,糖尿病高血糖可诱导FLS内质网应激从而损伤滑膜,病变滑膜分泌炎症和降解因子加剧高糖对关节软骨的损害,使关节软骨质量下降,长距离跑步刺激下OA易感。更多还原

【Abstract】 Osteoarthritis (OA), a joint disorder characterized by cartilage degradation and osteophyte formation, is considered a major public health issue today. Millions of people suffer from this musculoskeletal degenerative disorder, which may lead to disability, deterioration of quality of life and a heavy economical impact on every nation’s health systems. The lack of an effective treatment reflects the need for better understanding of the pathophysiology and aetiopathology of OA.The articular joint includes three different but closely linked tissues:cartilage, synovial membrane, and subchondral bone, all of which are known to play significant roles in the pathogenesis of osteoarthritis. Chondrocytes are the only cells in cartilage and interdispersed within extracellular matrix. Cartilage contains a number of matrix metalloproteinases (MMPs) involved in matrix turnover. These include MMP-3, MMP-8, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motif4(ADAMTS-4) and ADAMTS-5. OA is not only due to articular cartilage damage, but also related to pathological changes in other joint tissues (such as synovium). Most of the initial performance of OA are synovial lesions and these lesions are generally appeared ealier than X-ray or MRI imaging findings of articular cartilage. In the process of OA, synovium can produce MMPs and aggrecanase, promoting cartilage matrix degradation.OA is a multi-factorial disease and conventionally considerated to be induced mainly by mechanical factors and age-related degeneration of cartilage. However, recent study indicates that diabetes has compacted relationship with OA and may be an independent risk factor of OA. Epidemiological studies have found that patients with non-insulin-dependent diabetes are more susceptible to OA, and the OA population are more susceptible to suffer diabetes than the control population. Epidemiological studies revealed that the incidence of metabolic syndrome with OA significantly increased in patients (2.2to5.3-fold).As an important part of the metabolic syndrome, diabetes is closely related to OA. It may represent an independent risk factor for OA. The main features of DM characterized with chronic hyperglycemia accompanied with insulin secretion or metabolic disorders induced by lack of function. The number of world DM patients was approximately285million in2010, and it will be up to439million in2030. In addition, studies have found that STZ induced DM with IGF-1resistance in rat cartilage, which occurs synovial remodeling and collagen deposition in70days.These studies strongly suggest that there is a close association between DM and OA, and hypoglycemic as the direct representation of DM, its role in the development of diabetic O A rarely reported in the present.However, the role of hyperglycemia, the most direct manifestation of diabetes, in diabetes-induced OA has been rarely reported. The present study was designed to test the adverse effects of high glucose on articular cartilage and explore the underline mechanism, so as to demonstrater the diabetes induced OA.PART ONEDiabetes increased the susceptibility of OA in adult male ratsObjective Correlations between diabetes and osteoarthritis were found by epidemiological investigations and clinical case studies. This part was designed to confirm the decline of articular cartilage quality in diabetic rats, and the increased the susceptibility of OA in adult male rats under long distance running stimulation.Methods Adult male wistar rats were administrated with STZ (60mg/kg) through intraperitoneal injection. Two week later, the blood of tails was detected to confirm if the diabetes model is achived successfully. The rats were fed with normal diet. One month after confirmation of diabetes model, rats were given the long distance running to induce OA (with speed of20m/min and time of55min daily, a total of six weeks, with a total running mileage30Km). After running, the rats were sacrificed by isoflurane anesthesia. A portion of the knee cartilage carried ink staining, observed the gross morphological changes and scored the cartilage gross morphology. For HE and safranin O staining experiments, the rest tissue of knee were fixed, embedded, decalcified sections. col2al, MMP-13was on immunohistochemistry, cartilage pathological changes were observed by light microscopy, and Mankin’s score was carried out to assess OA. Results①changes of blood glucose and body weight in diabetic rats before and after OA modeling:23.83±1.34mM VS5.90±1.34mM (P<O.01) before running and32.16±1.14mM VS7.19±0.47mM after running (P<0.0), thus the success rate of diabetes modeling was100%and diabetic rats were under hyperglycemia throughout the experiment. Diabetic rats appeared with polydipsia, polyuria and decreased weight after diabetes modeling. The weight of control group were409-438g, and diabetic rats’ weight were265-283g (P<0.01);②score by surgical microscope:after receiving running stimulation, compared with the control group, normal rats’general morphology of cartilage are normal, articular’s surface is smooth; however, ink staining showed that, the cartilage surface, especially the surface of the tibial plateau of diabetic rats wron seriously, and the articular cartilage surface was dull and rough. The general score of cartilage of diabetic group was significantly higher than the control group (P<O.01);③athological changes of articular cartilage and score: safranin O staining revealed that matrix staning in the knees of control rats were obvious after running, with surface mild rough; compared with the control group, the cartilage surface of diabetic group showed varying degrees of damage, structural disorder of cartilage matrix, thinner cartilage, lightly stained matrix, and significantly decreased numbers of cartilage cells. After running-induced OA, Mankin’s score of OA cartilage pathology of diabetic group increased to11.9points, which significantly higher than the control group (1.6points)(P<0.01).Conclusion Diabetes possess advers effects on articular cartilage of rats. After receiving excessive running, rats with diabetes suffered typical OA, suggesting that diabetes increased the susceptibility of OA in adult male rats.PART TWOCatabolic effect of high glucose on rat chondrocytes potentiated by fibroblast-like synoviocytesObjective The present study was designed to test the adverse effects of high glucose on articular cartilage and explore the underline mechanism.Methods Rat articular chondrocytes and fibroblast-like synoviocytes (FLS) were cultured in media with different concentrations of glucose (10,20,30and40mM) for three and ten days during the2nd passage, respectively. Expression levels of matrix metalloproteinase3(MMP-3), MMP-13, a disintegrin and metalloproteinase with thrombospondin motif4(ADAMTS-4), ADAMTS-5, interleukin6(IL-6) and tumor necrosis factor a (TNF-a) were detected. Malondialdehyde (MDA) and Superoxide Dismutase (SOD) contents in synoviocytic culture media were detected. Culture media of synoviocytes stimulated by30mM glucose for three days were collected and administrated on another part of chondrocytes for three days. Catabolic profiles of chondrocytes were detected.Results High glucose inhibited matrix degradation of chondrocytes in three days, but favored in ten days. In FLS, high glucose promoted inflammatary cytokines and MMPs secretion, more obviously in long term. MDA was increased and SOD was reduced in synoviocytic media. Chondrocytes administrated with synoviocytic media showed increased matrix degradation.Conclusion FLS was more vulnerable to high glucose than chondrocyte. High glucose might impair articular cartilage by stimulating secretion of catabolic and proinflammatory factors of FLS. Oxidative damage might be involved in the process.PART THREEEndoplasmic reticulum stress involved in synovial injury induced by high glucoseObjective High glucose can induce endoplasmic reticulum stress, and it exists in OA. This part intended to explore the mechanism of synovial injury induced by high glucose on cellular and molecular level, and confirme the involved endoplasmic reticulum stress in diabetic OA.Methods Fibroblast-like synovial cells were treated by Tunicamycin5ug/ml+10mM glucose and4-Phenylbutyric acid lmM+30mM glucose for2/4/8/16hours, and the gene expression of endoplasmic reticulum stress, apoptosis and matrix degradation were detected by real-time quantitative PCR.Results①TM induced ERS in fibroblast-like synovial cells accompanied by similar cells injury exposed to high glucose:Compared with the control group, ERS marker genes MANF and GADD34in TM treatment group appeared time-dependent increase, and there was a statistically significant increase after treatment with8and16hours (P<0.01). CHOP increased slightly but there was no statistically significance, and GRP78had no significant change. Matrix degradation-related genes MMP-3, MMP-13and ADAMTS5increased. However, anti-matrix-degrading enzyme TIMP-1increased significantly. Apoptosis-related genes caspase3and caspase9in TM treatment group were significantly increased after processing16hours, and caspase8 increased significantly after processing4hours. Anti-apoptotic gene Bcl2had a time-dependent decrease, with a statistically significant lower after treatment with8and16hours (P<O.01). Besides, glucose transporter GLUT1increased significantly after treatment with8and16hours, accompanied by the increase of iNOS (P<O.05).②4-PBA inhibited ERS induced by high glucose and change of cell inflammation and degradation:Compared with the control group, ERS marker genes MANF, GADD34and GRP78in4-PBA treatment group had no significant change. CHOP had a statistically significant decrease after treatment with4hours (P<0.0l). Matrix degradation-related gene MMP-3decreased significantly and anti-matrix-degrading enzyme TIMP-1also decreased significantly (P<O.01). However, MMP-13and ADAMTS-5had no significant change. Apoptosis-related gene caspase3had a time-dependent decrease, with a statistically significant lower after treatment with16hours (P<0.01). Anti-apoptotic gene Bcl2increased, and there was a statistically significant increase after treatment with4and8hours. Besides, glucose transporter GLUT1decreased significantly after treatment with16hours (P<0.01).Conclusion TM can induce ERS in fibroblast-like synovial cells, accompanied by cell apoptosis and degradation pathological changes.4-PBA can inhibit ERS and subsequent matrix degradation and apoptosis changes induced by high glucose. Thus, high glucose can induce ERS, leading to cell apoptosis and matrix degradation, and finally resulting in disease.更多还原