C57BL/6小鼠CIA模型的建立及其监测体系的初步筛选

目的 建立稳定的C57BL／6小鼠胶原诱导关节炎(CIA)模型,构建进一步进行类风湿关节炎(RA)发病机制及治疗研究的体系。方法 取40只雄性C57BL／6小鼠,随机分为模型组和对照组。模型组采用鸡Ⅱ型胶原与完全弗氏佐剂在尾部皮内注射,21天后同样方法加强免疫。定期观察两组小鼠的关节和关节外表现以及病理学改变;用三重免疫荧光标记、胞内细胞因子测定流式细胞术检测外周血T细胞亚群。结果 对照组小鼠均未发病。模型组小鼠在50天的发病率达70％,关节炎评分在3～8分,持续时间大约100天;病理切片显示典型的炎性细胞浸润,滑膜增生,软骨及软骨下骨破坏．外周血Th1／Th2比值升高。结论 应用Ⅱ型胶原能在C57BL／6小鼠上成功建立CIA模型,该模型是进行RA研究的良好动物模型。     

Ⅱ型胶原诱导的大鼠关节炎模型关节炎症及血清抗体的动态变化

目的:制备Ⅱ型胶原诱导的大鼠关节炎模型(CIA模型)并观察其关节炎症和血清中抗体的变化规律.方法:于第0天和第10天分2次多点皮内注射牛Ⅱ型胶原和完全弗氏佐剂以诱导大鼠CIA模型,应用足爪肿胀容积测定仪动态观察关节肿胀度,采用0～7级关节炎评分法进行关节炎评分,应用组织病理学、X线影像学动态观察踝关节组织的病理变化,应用ELISA方法动态检测血清中多种抗体.结果:CIA模型大鼠免疫后第12天即出现踝关节肿胀,第23天达高峰,持续至第72天(动物处死);X线检查显示:免疫后第30天,软组织明显肿胀,踝关节骨骼结构排列紊乱,间隙不清,可见囊性骨吸收和骨侵蚀;第60天后踝关节骨骼结构排列更加紊乱,骨质疏松和新骨形成并存,提示侵蚀性慢性关节炎.组织病理学观察显示,第72天踝关节滑膜炎性细胞浸润、滑膜增生,关节软骨及骨破坏,显示较明显的慢性关节炎症.血清中总IgG,IgM型类风湿因子(RF),IgG型RF和Ⅱ型胶原抗体于免疫后第15天均明显升高,以后维持在较高水平.结论:应用牛Ⅱ型胶原成功制备大鼠CIA模型,免疫后第12天即加强免疫之后即出现关节炎症的病理变化,至少可持续至第72天.     

胶原性关节炎大鼠关节石蜡切片快速制备方法的建立

胶原性关节炎(collagen-induced arthritis,CIA)大鼠模型是人类类风湿性关节炎疾病的重要动物模型之一,对其关节进行病理组织学评估是研究药物抗类风湿关节炎作用的重要内容。CIA大鼠关节石蜡切片的质量是实现准确评估的关键,传统的EDTA脱钙方法周期较长,本研究尝试采用低浓度甲酸和混合脱钙液对CIA大鼠关节脱钙,以期提高CIA大鼠关节石蜡切片的制备效率和质量。1材料与方法将牛Ⅱ型胶原与弗氏不完全佐剂乳化后多点皮内免疫Wistar大鼠,复制CIA大鼠模型。待大鼠出现关节炎症状后脱颈处死,取双侧后足4%甲醛固定48h。将大鼠分为甲酸组、混合脱钙液组和EDTA组(n=6),进行脱     

类风湿性关节炎大鼠模型的制备

目的 建立简便实用的类风湿性关节炎大鼠模型.方法 利用小牛软骨Ⅱ型胶原加福氏完全佐剂充分乳化后作为造模剂,对Wistar大鼠足底、尾根部、背部多点皮内注射,7天后加强注射一次.结果 二次注射后2周,大鼠四肢严重肿胀,踝关节直径、脚爪体积增大,血清TNF-α水平明显升高,胫骨远端骨小梁吸收破坏,可累及骨皮质,关节软骨可见坏死脱落、纤维化,关节软骨可见坏死脱落、纤维化,关节腔狭窄,动物造模全部成功.结论 Ⅱ型胶原加完全费氏佐剂是一种良好的类风湿性关节炎大鼠模型造模剂.     

类风湿关节炎患者蛋白聚糖aggrecan G1区特异性T细胞诱导SCID小鼠产生膝关节炎的实验研究

目的探讨针对蛋白聚糖的自身免疫反应在类风湿关节炎（RA）发病中的作用。方法从人软骨细胞中克隆获得aggrecan G1区的cDNA片段,应用杆状病毒表达系统获取重组人aggrecan G1区蛋白（rAG1）。用rAG1刺激来自RA患者关节液的单个核细胞,采用β-液闪计数仪对培养细胞进行放射性检测并计算刺激增长指数（SI）,取SI超过3者为rAG1特异性T细胞。将取自3个人的rAG1特异性T细胞克隆腹腔注射至8只SCID Beige小鼠,同时在小鼠左膝注射rAG1。于注射后第8天处死小鼠,取双膝关节行组织病理学检查,观察炎细胞浸润及软骨和骨糜烂情况。结果从RA患者关节液单个核细胞分离得到rAG1特异性T细胞,主要为CD4^＋CD8^-T细胞,分泌Th1型细胞因子（γ-干扰素）。部分腹腔注射人rAG1特异性T细胞的小鼠左膝出现了关节滑膜单个核细胞浸润和早期软骨侵蚀改变。结论人特异性rAG1反应性T细胞在rAG1刺激下可向关节局部归巢而导致关节炎症反应,提示针对rAG1的自身免疫反应可能参与了RA的发病。     

海蛇乙醇浸出物灌胃对大鼠胶原性关节炎的防治作用(论著)

目的：探讨海蛇乙醇浸出物（ＡＥＢＦＳＳ）灌胃对大鼠胶原性关节炎（ＣＩＡ）的预防和治疗作用。方法：用自制的可溶性猪尾Ⅱ型胶原注入实验大鼠左后足造成胶原诱导性大鼠关节炎模型，观察用（预防性或治疗性）与不用ＡＥＢＦＳＳ对ＣＩＡ的影响。结果：在致炎前ＡＥＢＦＳＳ灌胃，当剂量达浸出蛋白４．０ｍｇ／ｋｇ，可明显推迟ＣＩＡ发病时间，降低ＣＩＡ患病率和减轻关节炎症状；在致炎后ＡＥＢＦＳＳ灌胃，可明显减轻关节炎症的程度，其作用在用药７天后开始表现明显。关节组织病理检查显示，ＡＥＢＦＳＳ显著减轻了患鼠关节的炎症程度。初步分析表明，ＡＥＢＦＳＳ灌胃既降低了ＣＩＡ患鼠血清抗ＣⅡ抗体水平，又对患鼠耳迟发型超敏反应（ＤＴＨ）产生了明显的抑制作用。结论：ＡＥＢＦＳＳ灌胃对大鼠胶原性关节炎有显著的防治作用；而其机理同时涉及Ｔ和Ｂ淋巴细胞免疫。     

类风湿关节炎

类风湿关节炎（Rheumatoidarthritis，简称RA）是一种慢性、炎症性、进行性和破坏性关节疾病。关节病变主要表现在四肢大、小关节，也可累及颈椎和颞颌关节。早期症状为受累关节的疼痛、肿胀、发僵及活动困难，长期不愈的晚期症状则为关节畸形和强直，最终导致终身残疾。小部分患者发病后有自发缓解趋势。多数患者病情反复，时轻时重。另有小部分患者病情呈持续性加剧，罕有静止。RA为一常见病，根据我国流行病学调查的初步结果，如以RA患病率0．4％计算，全国患者至少是440万。各国的统计资料证明。RA以女性多发，女性是男性的2～3倍。…     

99mTc-MDP显像对胶原诱导型关节炎大鼠的早期诊断作用

目的研究^99mTc-MDP显像在关节炎大鼠病程中的影像特点及其早期诊断价值。方法70只雄性Wistar大鼠随机分为对照组（n=28）与模型组（n=42）,两组再按不同时间点各分为7组,模型组大鼠通过皮内注射胶原与佐剂制作胶原诱导型关节炎（CIA）大鼠模型。尾静脉注射^99mTc-MDP进行核素显像,感兴趣区法（ROI）计算双膝关节与全身放射性比（R=T／tT）。观察不同时间点核素浓聚情况,并与关节指数（AI）、足爪厚度、病理变化及X线结果比较。结果CIA大鼠膝关节核素浓聚明显增加（P〈0．05）,达高峰后逐渐下降,42天后又出现一个小峰值。结论^99mTc-MDP显像可反映CIA早期滑膜病变,与早期病理变化一致,早于足爪厚度的改变,且比X线检查敏感,对早期关节病变的诊断价值较高。     

胶原诱导性关节炎伴贫血大鼠模型的建立及沙利度胺对其血红蛋白的影响

目的建立胶原诱导性关节炎(CIA)伴贫血大鼠模型,并研究沙利度胺对其血红蛋白的影响。方法128只雄性Wist-ar大鼠随机分为5组:Ⅰ组(正常对照组,n=24)、Ⅱ组(模型组,n=26)、Ⅲ组(模型 沙利度胺高剂量组,n=26,200mg.kg-1.d-1)、Ⅳ组(模型 沙利度胺低剂量组,n=26,100mg.kg-1.d-1)、Ⅴ组(模型 甲氨蝶呤组,n=26,每周2.7mg/kg)。每组再按不同免疫后不同时间点各分为14、21、28、35、42、60d6个亚组。模型组大鼠皮内多点、分次注射Ⅱ型胶原与完全弗氏佐剂,建立CIA伴贫血大鼠模型。对Ⅰ、Ⅱ组大鼠红细胞生成素(EPO)、血清铁(SI)、总铁结合力(TIBC),骨髓形态学及骨髓内、外铁的情况进行观察与测定。造模后12d给药,观察大鼠血红蛋白水平的变化。结果Ⅱ组大鼠在造模后28d出现贫血,与Ⅰ组比较,血红蛋白水平明显降低(P<0.05),贫血表现为SI、TIBC、骨髓内铁降低,血清铁蛋白水平升高(P<0.05),而骨髓外铁无显著变化。造模后60dⅢ组大鼠血红蛋白水平明显高于Ⅱ组(P<0.05)。结论通过改良CIA大鼠模型的方法,可成功建立关节炎伴贫血的大鼠模型,关节炎越明显,贫血越显著。沙利度胺能够改善关节炎大鼠的贫血情况,为RA患者的临床治疗提供了实验依据。     

口服Ⅱ型胶原诱导免疫耐受对大鼠类风湿性关节炎和创伤性骨性关节炎的影响

目的 :探讨应用口服Ⅱ型胶原诱导免疫耐受对创伤性骨性关节炎的影响。材料和方法 :应用切断大鼠膝关节内侧副韧带和内侧半月板的方法制备大鼠膝关节骨性关节炎的动物模型 ,以关节软骨组织学和组织化学染色以及T淋巴细胞转化实验为观察指标 ,观察口服Ⅱ型胶原对其病程发展的影响 ,并以经典的大鼠胶原引发关节炎症的类风湿性关节炎模型为对照。结果 :在创伤性骨性关节炎时 ,大鼠的T淋巴细胞增殖程度受到口服Ⅱ型胶原的抑制 ,从一个侧面反映针对Ⅱ型胶原的自家免疫可能参与了病变的发生和发展过程。但是 ,从病理改变来看 ,口服Ⅱ型胶原却不能抑制OA的发展 ,因此 ,创伤性骨性关节炎虽然在病变发生和发展过程中可能有免疫因素的参与 ,但是 ,相比RA ,这种参与不是主要的 ,OA的发病过程仍然是由于机械因素导致关节软骨破坏 ,在此基础上逐步发展起来的。口服Ⅱ胶原不能减轻OA的发病程度 ,因此不能应用口服Ⅱ型胶原的方法来治疗OA。     

Molecular MR Imaging for Visualizing ICAM-1 Expression in the Inflamed Synovium of Collagen-Induced Arthritic Mice

Objective

To determine the utility of intercellular adhesion molecule (ICAM)-1 antibody-conjugated gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA-anti-ICAM-1) as a targeted contrast agent for the molecular magnetic resonance imaging (MRI) in collagen-induced arthritis (CIA).

Materials and Methods

Three groups of mice were used: non-arthritic normal, CIA mice in both the early inflammatory and chronic destructive phases. The MR images of knee joints were obtained before and after injection of Gd-DTPA-anti-ICAM-1, Gd-DTPA, and Gd-DTPA-Immunoglobulin G (Ig G) and were analyzed quantitatively. The patterns of enhancement on the MR images were compared with the histological and immunohistochemical ICAM-1 staining.

Results

The images obtained after injection of Gd-DTPA-anti-ICAM-1 displayed gradually increasing signal enhancement from the moment following injection (mean ± standard deviation [SD]: 424.3 ± 35.2, n = 3) to 24 hours (532 ± 11.3), rather than on pre-enhanced images (293 ± 37.6) in the early inflammatory phase of CIA mice. However, signal enhancement by Gd-DTPA and Gd-DTPA-IgG disappeared after 80 minutes and 24 hours, respectively. In addition, no significant enhancement was seen in the chronic destructive phase of CIA mice, even though they also showed inflammatory changes on T2-weighted MR images. ICAM-1 expression was demonstrated in the endothelium and proliferating synovium of the early inflammatory phase of CIA mice, but not in the chronic destructive phase.

Conclusion

Molecular MRI with Gd-DTPA-anti-ICAM-1 displays specific images targeted to ICAM-1 that is expressed in the inflamed synovium of CIA. This novel tool may be useful for the early diagnosis and differentiation of the various stages of rheumatoid arthritis.     

Indium-111 leukocyte imaging in patients with rheumatoid arthritis

This study evaluates the usefulness of labeled leukocyte imaging in patients with rheumatoid arthritis. In 33 patients, the incidence of pain and swelling in 66 wrist joints and 66 knee joints was compared with the accumulation of [111In]leukocytes. No accumulation of [111In]leukocytes was seen in any of the patients' wrists (0/12) or knee joints (0/14) when both pain and swelling were absent. In contrast, 93% (25/27) of wrist joints and 80% (24/30) of knee joints with both pain and swelling were positive by [111In]leukocyte scintigraphy. There was little correlation between the stage of the disease, as determined by radiography, and [111In]leukocyte accumulation. This study suggests that [111In]leukocyte imaging may be a reliable procedure for monitoring the activity of rheumatoid arthritis, especially for confirming the lack of an ongoing inflammatory response.     

Imaging cell death with radiolabeled annexin V in an experimental model of rheumatoid arthritis.

Rheumatoid arthritis is associated with chronic synovial inflammation due to the abnormal accumulation of macrophages and autoreactive T lymphocytes in joints. The autoreactive cells cause an inflammatory proapoptotic response to self-antigens resulting in eventual bone, cartilage, and soft-tissue loss and destruction. The goal of our study was to determine the timing and intensity of apoptosis in joints using 99mTc-labeled annexin V, an in vivo marker of apoptosis, in a murine model of immune arthritis. METHODS: We used 99mTc-annexin V and autoradiography to study the extent and severity of apoptosis in the front and rear paws of DBA/1 mice with type II collagen-induced rheumatoid arthritis. RESULTS: Compared with control values (n = 10), there was a significant (P < 0.002) nearly 3-fold increase in uptake of 99mTc-annexin V in the front foot pads, rear toes, rear foot pads, and heels at the time of maximal extremity swelling as determined by serial caliper measurements at 4 wk after inoculation with type II bovine collagen (n = 9). The front toes had a 5- to 6-fold increase in uptake compared with control values (P < 0.001). Histologic analysis revealed only scattered rare lymphocytes in the periarticular soft tissues, without joint destruction. Dual autoradiography with 125I-bovine serum albumin as a control showed that 99mTc-annexin V localization was specific. Treatment with methylprednisolone for 1 wk (n = 8) at 4 wk after immunization with type II collagen decreased 99mTc-annexin V uptake by 3- to 6-fold compared with control values (P < 0.002). CONCLUSION: 99mTc-annexin V can detect collagen-induced immune arthritis and its response to steroid therapy before joint destruction.     

Pertechnetate uptake of joints in rheumatoid arthritis.

A new isotopic method for evaluation of inflammatory activity in rheumatoid arthritis is reported. By use of repeated whole body profile scanning a double tracer examination with 99mTc-pertechnetate and 113mIn-transferrin was performed. Total pertechnetate activity and total indium activity were measured over both hands, both feet, the ankles, and over the knee joints. In addition, the extravascular component of pertechnetate uptake and the ratio extravascular/total pertechnetate activity were calculated for these regions. The activity curves were followed up to 2 h after tracer injection in seven female patients presenting with rheumatoid arthritis and in six female controls. The four parameters calculated were compared and statistical analysis revealed the most significant discrimination of both groups for the ratio extravascular/total pertechnetate activity 2 h after injection (P less than 0.002).     

Pertechnetate uptake of joints in rheumatoid arthritis

A new isotopic method for evaluation of inflammatory activity in rheumatoid arthritis is reported. By use of repeated whole body profile scanning a double tracer examination with 99mTc-pertechnetate and 113mIn-transferrin was performed. Total pertechnetate activity and total indium activity were measured over both hands, both feet, the ankles, and over the knee joints. In addition, the extravascular component of pertechnetate uptake and the ratio extravascular/total pertechnetate activity were calculated for these regions. The activity curves were followed up to 2 h after tracer injection in seven female patients presenting with rheumatoid arthritis and in six female controls. The four parameters calculated were compared and statistical analysis revealed the most significant discrimination of both groups for the ratio extravascular/total pertechnetate activity 2 h after injection (P(0.002).     

Collagen-induced arthritis in rats. Assessment by serial magnification radiography

Serial whole body and lateral hindpaw magnification radiographs were obtained on 15 rats with collagen-induced arthritis. The radiographs were evaluated for findings of inflammatory arthritis. Soft tissue swelling and juxta-articular osteopenia usually developed two weeks after immunization. The swelling remained stable for the remaining seven weeks of the study, but the osteopenia apparently improved. Three weeks after immunization, erosions and periostitis began to develop in ten of the rats. These latter radiographic changes rapidly worsened over the next three weeks and then stabilized. The most frequent and severe changes were seen in the intertarsal, tibiotalar, and metatarsophalangeal joints. The toes, forepaws, and knees were infrequently involved. Other peripheral joints and the axial skeleton were spared. Five rats developed soft tissue swelling but had no articular erosions or periostitis. Serial radiography has potential usefulness for monitoring the effects of treatment in this experimental model for inflammatory arthritis.     

MRI of the wrist and finger joints in inflammatory joint diseases at 1-year interval: MRI features to predict bone erosions

The aim of this study was to assess the ability of MRI determined synovial volumes and bone marrow oedema to predict progressions in bone erosions after 1 year in patients with different types of inflammatory joint diseases. Eighty-four patients underwent MRI, laboratory and clinical examination at baseline and 1 year later. Magnetic resonance imaging of the wrist and finger joints was performed in 22 patients with rheumatoid arthritis less than 3 years (group 1) who fulfilled the American College of Rheumatology (ACR) criteria for rheumatoid arthritis, 18 patients with reactive arthritis or psoriatic arthritis (group 2), 22 patients with more than 3 years duration of rheumatoid arthritis, who fulfilled the ACR criteria for rheumatoid arthritis (group 3), and 20 patients with arthralgia (group 4). The volume of the synovial membrane was outlined manually before and after gadodiamide injection on the T1-weighted sequences in the finger joints. Bones with marrow oedema were summed up in the wrist and fingers on short-tau inversion recovery sequences. These MRI features was compared with the number of bone erosions 1 year later. The MR images were scored independently under masked conditions. The synovial volumes in the finger joints assessed on pre-contrast images was highly predictive of bone erosions 1 year later in patients with rheumatoid arthritis (groups 1 and 3). The strongest individual predictor of bone erosions at 1-year follow-up was bone marrow oedema, if present at the wrist at baseline. Bone erosions on baseline MRI were in few cases reversible at follow-up MRI. The total synovial volume in the finger joints, and the presence of bone oedema in the wrist bones, seems to be predictive for the number of bone erosions 1 year later and may be used in screening. The importance of very early bone changes on MRI and the importance of the reversibility of these findings remain to be clarified.     

光动力疗法对大鼠类风湿性关节炎治疗作用的观察

目的　观察以血啉甲醚 (HMME)为光敏剂的光动力效应对类风湿性关节炎动物模型的治疗作用。方法　LEW大鼠 30只 ,随机分为正常对照组 5只、关节炎组 10只、治疗组 15只。关节炎组和治疗组用Ⅱ型胶原蛋白诱发关节炎 (CIA)。治疗组在大鼠出现踝关节红肿后 1周 ,炎症达到高峰时进行光动力治疗 (PDT)。随机治疗大鼠一侧的踝关节 ,对侧作为单纯HMME对照。治疗方法是大鼠麻醉后尾静脉注入HMME 10mg kg ,2 0min后踝关节照光 ,激光波长 6 2 7 8nm ,功率密度 10 0mW cm2 ,照射时间 10 0 0s,能量密度 10 0J cm2 。治疗后 2周取关节进行病理评分。结果　关节炎组滑膜细胞增生 ,血管翳形成 ,并侵蚀和破坏软骨和骨 ;治疗组滑膜细胞增生不明显 ,软骨和骨完整。病理评分治疗组滑膜增生、血管翳形成及软骨破坏、骨破坏和总分较关节炎组和HMME组低 ,统计学检验差异有显著意义 (P <0 0 1)。结论　在HMME 10mg kg,激光功率密度 10 0mW cm2 、能量密度 10 0J cm2 的条件下 ,光动力疗法能够有效地减轻CIA的滑膜细胞增生 ,减轻血管翳的形成及对软骨和骨的破坏 ,起到保护软骨和骨的作用。     

Synovectomy of the knee with 90Y

In 33 patients with chronic arthritis of the knee, 48 knees were treated with an intra-articular injection of 5 mCi yttrium silicate (90Y). There were 27 patients with rheumatoid arthritis (RA) and 6 with osteoarthrosis (OA); the mean follow-up period was 33 months. At clinical investigation after 1 year, no signs of pain or swelling were found in 15 knees. In most cases, pain and swelling improved subjectively, with a mean duration of 11 months; in 20 knees, the improvement lasted more than 22 months. When radiographs showed severe destruction, 90Y treatment was unsuccessful, but an important new finding was that most patients with mild or moderate radiological abnormalities appeared to have a long-lasting improvement. The result did not correlate with erythrocyte sedimentation rate (ESR), haemoglobin or Rose titre at the time of injection or at follow up, suggesting that the result of the treatment is more dependent on local factors than on the disease activity. The results of 90Y treatment in 6 OA knees with persistent swelling were promising regarding swelling, even in patients with moderate radiological abnormalities. The main side-effect was a sometimes painful swelling of the knee, which was always successfully treated with an intraarticular corticosteroid injection. In 90Y-treated knees, the incidence of unstable joints was not significantly higher than in non-treated knees. In conclusion, 90Y synovectomy may be a successful treatment for patients older than 50 years with chronic arthritis of the knee due to RA and probably also OA, even when moderate radiological abnormalities are present.     

The ‘pre-erosive’ radiologic signs of rheumatoid arthritis in soft tissue radiography of the hands

Soft tissue radiography of the hands using the mammographic immersion technique was performed on 119 patients, having definite or classical rheumatoid arthritis, and on 123 controls of matching age, sex, and professional distribution. A total of 7744 finger joints and carpal borders were investigated for joint swelling, periarticular edema, pre-erosive and erosive bone changes, joint space narrowing, and osteoarthritic joint margin spurs. Slight joint swelling and pre-erosive bone changes were found in connection with osteoarthritic joint changes in elderly control patients. Periarticular edema and moderate to massive joint swelling were quite reliable signs for synovitis. The incidence of pre-erosive bone signs was significantly greater in the rheumatoid arthritis group than in the control group, especially in patients less than 60 years old. These signs can be regarded as suggestive of rheumatoid arthritis; probability diagnosis could be performed using these signs and the Bayesian approach.