人类风湿关节炎滑膜成纤维细胞-软骨-重度联合免疫缺陷鼠模型的研制

目的 建立人类风湿关节炎(RA)滑膜成纤维细胞(SFs)-软骨-重度联合免疫缺陷(SCID)小鼠模型,以骨关节炎滑膜成纤维细胞(OASFs)作对照,探讨RASFs在RA发病中的作用及机制.方法 将培养传代至第4代的RASFs/OASFs经5-溴脱氧尿嘧啶核苷体(5-Brdu)标记后注入可吸收明胶海绵,与人的正常软骨一起移植入SCID小鼠背部皮下,第30天处死模型,剪取移植物和鼠双膝关节作组织学观察,免疫组织化学检测5-Brdu和Vimentin阳性细胞,酶联免疫吸附试验(ELISA)检测血清中人基质金属蛋白酶(MMP)-3和人白细胞介素(IL)-6含量.统计学方法采用两独立样本秩和检验.结果 ①2组血清中均可检测出人MMP-3,仅在RASFs组检测到1例IL-6.②RASFs组移植的软骨侵蚀程度(0.6±0.7与0.3±0.5)和软骨降解程度(2.3±0.8与1.7±1.0)较OASFs组有增高趋势,但差异均无统计学意义(P＞0.05).③RASFs组鼠膝关节的滑膜增生程度(3.1±0.8与1.7±1.0,P＜0.01)和软骨侵蚀程度(1.6±1.7与0.6±1.4,P＜0.05)均显著高于OASFs组.④在SCID鼠皮下、移植软骨处、鼠膝关节滑膜及骨髓中检测出5-Brdu及Vimentin阳性的SFs.结论 传代培养的RASFs在SCID鼠皮下存活并保持其侵袭性生长的特点,且能迁移至远处关节中诱发关节炎症.     

类风湿关节炎滑膜侵蚀软骨SCID鼠模型的建立及初步实验研究

目的建立类风湿关节炎(RA)-SCID小鼠模型,观察滑膜对软骨的侵蚀情况,为探讨RA滑膜侵蚀软骨机制及治疗奠定基础。方法70只SCID小鼠,3～6周龄。无菌获得12例非关节炎患者正常关节软骨,修剪成约2 mm×2 mm×1 mm后备用。同时取10例RA患者(膝关节或髋关节)滑膜组织修剪成约3 mm×6 mm的小块(片),这些小块包裹软骨后共植入SCID鼠肾包囊处,4例骨关节炎(OA)患者滑膜及2例正常滑膜同样处理作对照。术后观察小鼠和移植物的一般情况,从第4周开始分批处死SCID小鼠并切除移植物,进行苏木素-伊红(HE)染色病理分析。结果大部分滑膜及软骨在SCID小鼠体内存活,4周开始滑膜黏附并侵蚀软骨,12周后RA滑膜明显破坏软骨,移植物细胞浸润、软骨破坏与RA病理类似,OA滑膜轻微破坏软骨,而正常滑膜几乎不破坏软骨。结论RA滑膜植入SCID可成活并能侵蚀软骨,RA-SCID小鼠可作为人源化滑膜侵蚀软骨模型,在探讨机制特别是软骨破坏及治疗方面可能有明显优势。     

Etanercept抑制BNX鼠-人RA移植物嵌合体模型中的滑膜增生和软骨侵蚀及降解

目的观察肿瘤坏死因子（TNF）-α拮抗剂Etanercept能否抑制类风湿关节炎（RA）的滑膜增生和软骨破坏，并探讨作用机制。方法将人RA滑膜和正常关节软骨移植到BNX小鼠皮下构建BNX鼠-人RA移植物嵌合体模型，造模4周后给予Etanercept（100μg）皮下注射，连续4周，对照组给予注射用水。评价移植物中的滑膜增生、滑膜细胞对软骨的侵蚀和软骨细胞周围软骨降解的组织学积分，并检测血清TNF-α含量，滑膜的TNF-α和人血管内皮生长因子（VEGF）的表达以及滑膜细胞凋亡情况。结果与对照组比较．Etanercept组中滑膜增生、软骨侵蚀、软骨降解积分以及血清TNF-α含量均显著降低：滑膜细胞的TNF-α和VEGF表达水平也显著下降。但滑膜细胞凋亡程度差异无统计学意义。结论Etanercept能抑制嵌合体模型中的滑膜增生和软骨侵蚀及降解。其作用机制除了中和滑膜组织和血液中的TNF-α外．还可能与下调滑膜细胞的VEGF表达有关。     

骨桥蛋白在类风湿关节炎发病机制中的研究进展

类风湿关节炎(rheumatoid arthritis,RA)为慢性炎症性自身免疫病,主要病理特点为滑膜炎、血管翳的形成,以及软骨和骨的破坏.RA病变核心部位在关节滑膜,滑膜主要的组织细胞学改变有:滑膜内膜层细胞[成纤维样滑膜细胞(FLS)和巨噬样细胞]的增生、滑膜内膜下层炎性细胞(巨噬细胞、淋巴细胞等)的浸润,侵蚀关节面的血管翳形成,破骨细胞活化并介导矿化软骨和软骨下骨的侵蚀.病变中涉及到的分子有细胞因子、黏附分子、趋化因子、蛋白酶等.     

间充质干细胞免疫调节类风湿关节炎的研究进展

<正>类风湿关节炎(RA)是一种慢性全身性疾病,主要影响滑膜关节,导致关节破坏和功能障碍。据报道全世界RA的患病率是0.5%~1%,每年有400万新发病例~(〔1〕)。RA的发展是由免疫系统功能障碍所介导的,但确切的致病机制仍不清楚。RA的病理特征是关节滑膜的慢性炎症(滑膜炎),通过浸润型炎症、免疫细胞以及成纤维样滑膜细胞(FLSs)之间的相互作用导致软骨和骨组织受侵蚀。RA一旦形成,侵蚀不会停止,会造     

血管内皮生长因子在类风湿关节炎的表达

类风湿关节炎 (rheumatoidarthritis,RA)是一种常见的以关节滑膜慢性炎症病变为主要表现的自身免疫性疾病 ,其滑膜增生形成血管翳 ,有似肿瘤样生长的特点 ,造成对骨关节的侵蚀破坏。Paleolog[1 ] 证实血管内皮生长因子 (VEGF)在RA的滑膜血管翳的形成过程中可能起关键作用 ,它是联系各种炎性因子的枢纽 ,直接促进滑膜组织新血管形成 ,增强血管通透性 ,引起一系列病理改变。文献 [2 5 ]中已有血清VEGF水平在RA进展不同时期及与疾病活动性、类风湿因子 (RF)滴度关系的报道。但用VEGF和RA检索Medline和PubMed均未见反映不同病期血清…     

高频超声在类风湿关节炎早期诊断中的应用研究

目的：探讨高频超声在类风湿关节炎（RA）患者早期诊断中的应用价值。方法：RA患者36例,根据病程分为病程〈2年和病程≥2年两组。健康对照30名,分别应用高频超声检测双侧腕关节、第2掌指关节、第2和第3近端指间关节。测量滑膜厚度、示指屈肌腱及尺侧腕伸肌腱腱鞘厚度;观察有无滑膜增殖、滑膜血流增多、关节软骨及软骨下骨侵蚀、肌腱病变。结果：RA组各关节滑膜及腱鞘厚度同健康对照组比较差异有统计学意义（P均〈0.01）。超声检查未发现健康对照组关节内有血流信号、关节腔积液、软骨和软骨下骨侵蚀及肌腱病变。RA组超声发现77%的关节有滑膜增生,67%的关节增厚的滑膜内部及周边血流增多,12个腕关节可见关节腔积液,40%的关节骨皮质有缺损改变。在病程〈2年组,超声发现41个关节有骨侵蚀,而X线只发现21个关节有骨侵蚀。超声发现的肌腱病变主要有腱鞘增宽、肌腱失去正常的纤维状回声结构、肌腱边缘不规则或模糊、不同程度的肌腱撕裂,检出率分别为58%、56%、44%、30%。在病程〈2年组,超声表现主要为滑膜炎和肌腱病变,骨侵蚀性病变多出现在病程≥2年组。结论：高频超声在RA病程早期即可检测出滑膜炎症、肌腱病变、骨侵蚀,较体格检查和X线检测更为敏感,有利于RA的早期诊断。     

类风湿关节炎患者血清和滑膜骨桥蛋白的测定

目的 研究骨桥蛋白(OPN)在类风湿关节炎(RA)患者外周血中的浓度变化及在滑膜的表达,探讨OPN在RA患者中的发病机制.方法 收集91例RA患者以及29名正常对照人群临床资料和血清,用酶联免疫吸附试验(ELIsA)方法检测OPN在RA患者外周血中的浓度,分析其变化和RA临床及实验室指标的关系.收集7例RA患者和1名正常对照的滑膜组织,用免疫组织化学的方法观察OPN的表达情况.结果 与正常对照组相比,活动组和非活动组的RA患者外周血中OPN水平均明显升高(p＜0.01),且与RA患者的部分临床指标有相关性:与压痛关节数(r=0.435,P=0.005)、关节的X线分期(r=-0.415,P=0.007)、关节功能(r=0.394,P=0.012)显著相关.OPN在RA滑膜组织大量表达,而在正常对照仅见OPN的少量表达.结论 OPN在RA患者外周血中浓度显著升高,且在滑膜表达明显,OPN可能与RA滑膜增生、骨侵蚀有关.     

类风湿关节炎滑膜组织人糖皮质激素诱导型肿瘤坏死因子受体表达的免疫组织化学研究

目的 观察研究人糖皮质激素诱导型肿瘤坏死因子受体(hGITR)在类风湿关节炎(RA)滑膜组织和软骨组织中的表达及其与滑膜炎性病变程度的相关性.方法 用免疫组织化学的方法对16例RA患者、9例骨关节炎(OA)患者及4例无关节病变的截肢患者滑膜组织和软骨组织中hGITR的表达进行描述分析,并对hGITR表达情况与RA患者滑膜炎性病变程度相关性进行分析.结果 hGITR在RA滑膜组织主要分布于血管翳周围,如血管内皮细胞和炎性细胞等,hGITR阳性细胞数约为69%.而该受体在RA患者软骨组织细胞中的表达率与对照组比较差异无统计学意义.OA滑膜组织和软骨组织细胞中hGITR也呈现不同程度的阳性表达,但与RA比较阳性程度较弱,阳性细胞数量较少;对hGITR表达与RA滑膜炎性程度的相关性分析发现,hGITR与RA滑膜炎性程度呈正相关(r=0.895,P<0.01).结论 hGITR在RA滑膜组织炎性细胞及血管内皮细胞上的异常表达可能是其参与RA血管翳的形成及滑膜组织的侵袭等病理过程导致RA患者滑膜损伤的一个重要机制.     

葡萄糖-6-磷酸异构酶软骨寡聚基质蛋白抗突变型瓜氨酸波形蛋白抗体和抗环瓜氨酸肽抗体联合检测在类风湿关节炎诊断中的应用价值

类风湿关节炎(rheumatoid arthritis,RA)是常见的以关节滑膜慢性炎症病变为主要表现的全身性自身免疫性疾病目前诊断RA主要依靠临床症状和血清学诊断指标.近来发现葡萄糖-6-磷酸异构酶(GPI)及抗突变型瓜氨酸化波形蛋白(MCV)抗体与RA有一定相关性.     

The role of TNF-alpha in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera

OBJECTIVE: In order to elucidate which cytokine preferentially stimulates the synovium in patients with rheumatoid arthritis (RA), we investigated the roles of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: The SCID-HuRAg mice were prepared according to our previously described method. First, SCID-HuRAg mice were treated with chimeric anti-TNF-alpha monoclonal antibody (mAb, 100 microg/mouse) and histological changes were examined 4 weeks after the initial treatment. Secondly, a total of 100 microg of recombinant TNF-alpha or IL-6 (0.6 microg/h) was administered daily to mice using an osmium pump. The histological changes and serum cytokine levels were examined 4 weeks after the initial administration. Human immunoglobulin G (IgG) was administered to mice as a control. RESULTS: Synovial inflammatory cells were significantly decreased after the anti-TNF-alpha mAb treatment; conversely, the degree of synovial inflammation was significantly exacerbated by TNF-alpha administration. The levels of both IL-6 and TNF-alpha in sera were significantly increased by recombinant TNF-alpha administration, while TNF-alpha levels were unchanged by IL-6 administration. This suggests that TNF-alpha controls IL-6 production. Despite the profound changes in inflammation, we found no effects on bone and no articular cartilage damage was produced by TNF-alpha. CONCLUSION: This study provides strong evidence that TNF-alpha is a key molecule in the control of the inflammatory changes that occur in the RA synovium. In addition, TNF-alpha regulates IL-6 production. However, other inflammatory pathways independent of TNF-alpha may contribute to the bone and cartilage damage seen in RA.     

Methotrexate inhibits rheumatoid synovitis by inducing apoptosis

OBJECTIVE: To clarify the pharmacological action of methotrexate (MTX) on the synovium of patients with rheumatoid arthritis (RA) using severe combined immunodeficient (SCID) mice in which human RA synovial tissue had been grafted (SCID-HuRAg). METHODS: One month after engraftment of human RA tissue into SCID mice, MTX (0.3 mg/kg) was administered orally, then the appearance of apoptosis in the grafted tissue was examined by TdT mediated dUTP nick end labeling (TUNEL) staining and electron microscopy at various time points after MTX administration. In cultured synovial cells, synovial apoptotic changes after MTX treatment were studied by agarose gel electrophoresis and flow cytometric analysis. To compare the histological changes induced by MTX with those induced by other disease modifying antirheumatic drugs (DMARD) and a nonsteroidal antiinflammatory drug, histological examination of the grafted synovial tissues from SCID-HuRAg mice was conducted after 4 weeks of oral administration of MTX (0.3 mg/kg/week), salazosulfapyridine (30 mg/kg/day), auranofin (0.2 mg/kg/day), bucillamine (10 mg/kg/day), or indomethacin (2 mg/kg/day). RESULTS: A significant decrease in the number of inflammatory cells was observed in the grafted synovial tissue of MTX treated SCID-HuRAg. A similar antiinflammatory effect was not observed with the other DMARD. Induction of apoptosis was noted with MTX treatment but not with the others. The pro-apoptotic effect of MTX was also observed in synovial cell cultures. CONCLUSION: MTX induces apoptosis in RA synovium that, in turn, may contribute to its antiinflammatory effect on RA synovitis.     

"Inverse wrap": an improved implantation technique for virus-transduced synovial fibroblasts in the SCID mouse model for rheumatoid arthritis

The SCID mouse model for rheumatoid arthritis (RA) is an established and reliable approach to examining the distinct mechanisms operative in RA synovium, and evaluating novel gene therapy strategies. However, serum concentrations of circulating gene therapy products following gene transfer are frequently too low to allow detection. This problem stimulated us to develop a novel implantation technique to improve the yield of these soluble gene products. Synovial fibroblasts from patients with RA were cultured, passaged, and transduced with Ad5 sTNFRp55:Ig. sTNFRp55:Ig production was confirmed by ELISA, and then cells were implanted into SCID mice using a novel implantation strategy in which pieces of human cartilage were engrafted into a fibroblast-saturated inert sponge. Thereafter, the sponges were implanted under the skin of the mice instead of under the kidney capsule, as in the original approach, allowing co-implantation of larger pieces of cartilage together with higher numbers of adenovirus-transduced RA synovial fibroblasts. The improved implantation technique not only resulted in a reduction in the number of mice needed in each experiment by approximately 60%, and a reduction of the time taken for surgery by about 50%, but also considerably enhanced the serum concentrations of the gene product sTNFRp55-Ig, allowing detection of the soluble TNF receptor p55 by standard ELISA. In summary, the improved implantation technique for the SCID mouse model for RA results in more economic animal treatment, and facilitates the detection and quantification of circulating gene products following adenovirus-based gene transfer into synovial fibroblasts. Received: August 7, 2000 / Accepted: December 21, 2000     

“Inverse wrap”: an improved implantation technique for virus-transduced synovial fibroblasts in the SCID mouse model for rheumatoid arthritis

Abstract

The SCID mouse model for rheumatoid arthritis (RA) is an established and reliable approach to examining the distinct mechanisms operative in RA synovium, and evaluating novel gene therapy strategies. However, serum concentrations of circulating gene therapy products following gene transfer are frequently too low to allow detection. This problem stimulated us to develop a novel implantation technique to improve the yield of these soluble gene products. Synovial fibroblasts from patients with RA were cultured, passaged, and transduced with Ad5 sTNFRp55:Ig. sTNFRp55:Ig production was confirmed by ELISA, and then cells were implanted into SCID mice using a novel implantation strategy in which pieces of human cartilage were engrafted into a fibroblast-saturated inert sponge. Thereafter, the sponges were implanted under the skin of the mice instead of under the kidney capsule, as in the original approach, allowing co-implantation of larger pieces of cartilage together with higher numbers of adenovirus-transduced RA synovial fibroblasts. The improved implantation technique not only resulted in a reduction in the number of mice needed in each experiment by approximately 60%, and a reduction of the time taken for surgery by about 50%, but also considerably enhanced the serum concentrations of the gene product sTNFRp55-Ig, allowing detection of the soluble TNF receptor p55 by standard ELISA. In summary, the improved implantation technique for the SCID mouse model for RA results in more economic animal treatment, and facilitates the detection and quantification of circulating gene products following adenovirus-based gene transfer into synovial fibroblasts.     

Treatment with the angiogenesis inhibitor endostatin: a novel therapy in rheumatoid arthritis

OBJECTIVE: An endostatin that inhibits angiogenesis dependent tumor growth is being tested as an antitumor agent. The neoangiogenesis condition of cancer is essentially identical to that of rheumatoid arthritis (RA). Thus antiangiogenic treatment has potential for treatment of RA. We investigated the effects of human recombinant endostatin on human RA synovial tissue by use of a novel model of RA, in which human RA tissue is grafted into SCID mice (SCID-HuRAg). METHODS: Ten or 50 mg/kg of human recombinant endostatin was administered by percutaneous direct intrasynovial injection in each of 7 SCID-HuRAg mice. We examined the volume of the grafted tissue mass and the histological changes 7 days after endostatin administration. Six control mice received phosphate buffered saline in the same manner. RESULTS: The grafted synovial volume of SCID-HuRAg mice was significantly decreased by endostatin administration. The number of inflammatory cells (macrophages and lymphocytes) was also significantly reduced in a dose dependent manner. The number of vessels that were counted by von Willebrand factor VIII and type IV collagen positive cells was decreased, although apoptotic cells were increased in RA synovia. CONCLUSION: The results suggest that antiangiogenesis treatment using endostatin represents a potential new therapeutic strategy for RA.     

A new model for rheumatoid arthritis generated by engraftment of rheumatoid synovial tissue and normal human cartilage into scid mice

Objective. A new animal model was used to study the interaction between rheumatoid synovial cells and cartilage and to explore the cellular basis of rheumatoid joint destruction. Methods. Fresh synovial tissue derived from patients with rheumatoid arthritis was implanted with normal human cartilage into SCID mice, either subcutaneously or under the renal capsule, for up to 304 days. The implants were analyzed by light and electron microscopy, as well as by immunohistochemistry and in situ hybridization. Results. Human synovial tissue and cartilage implanted in SCID mice are maintained by the animals for up to 304 days. After 35 days, focal erosions occur at the site of attachment of synovial lining cells to the cartilage. After 105 days, a pannus-like formation, consisting of proliferating synovial fibroblast-like cells invading the cartilage, is observed. The fibroblast nature of these cells was supported by observation of only focal expression of the macrophage markers CD14 and CD68. Cells at the immediate site of cartilage destruction express messenger RNA for cathepsin L, whereas cathepsin D messenger RNA was detected in subsynovial regions away from the site of destruction. The human origin of the tissue involved in cartilage destruction was demonstrated using monoclonal antibodies to HLA-ABC and human type IV collagen. Conclusion. The present approach introduces a novel in vivo model of rheumatoid arthritis for the study of the molecular and cellular mechanisms of rheumatoid joint destruction at sites of synovial attachment to cartilage. In this model, the SCID mouse acts as a useful host for studying the properties of rheumatoid synovium in the absence of circulating human blood components.     

Inhibitory effects of an anti-rheumatic agent T-614 on immunoglobulin production by cultured B cells and rheumatoid synovial tissues engrafted into SCID mice

OBJECTIVE: To clarify the pharmacological action of an anti-rheumatic agent T-614, we investigated its effects on immunoglobulin (Ig) production by cultured B cells and Ig secretion from synovial tissues of patients with rheumatoid arthritis (RA) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: Murine B cells were prepared from mouse spleen by a T-cell depletion method. The cells were cultured with lipopolysaccharide (LPS) and/or interleukin 4 (IL-4) in the absence or presence of T-614. Human B cells were isolated from peripheral blood of healthy donors and the Ig production was induced by co-culture with autologous T cells and anti-CD3 antibody. SCID-HuRAg was prepared according to our previous method. T-614 was orally administered to the mice once daily for 4 weeks starting on the fourth week after the implantation. Then, peripheral blood was obtained and the implanted tissues were removed. Igs in the culture media or the sera were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In murine B-cell cultures, T-614 significantly decreased not only the IgM production stimulated with LPS but IgG1 production induced by LPS and IL-4. Regarding human B cells stimulated with T cells, it also inhibited IgM and IgG production. In SCID-HuRAg mice, high concentrations of polyclonal human IgG were detectable in the sera of all mice. A significant decrease in the IgG level was observed in the T-614-treated group compared with the control group. CONCLUSIONS: We showed that T-614 inhibited Ig production by the cultured B cells and also decreased the high level of human IgG observed in SCID-HuRAg mice. These results may support its effect on plasma Ig in RA patients and provide insights into the mechanisms of its anti-rheumatic effect.     

Methotrexate (MTX) and albumin coupled with MTX (MTX-HSA) suppress synovial fibroblast invasion and cartilage degradation in vivo

OBJECTIVE: To investigate the effect of methotrexate (MTX) and albumin coupled with methotrexate (MTX-HSA) on cartilage invasion and induction of perichondrocytic degradation by rheumatoid arthritis synovial fibroblasts (RA SF) in vivo. METHODS: Human cartilage and RA SF were co-transplanted in three groups of severe combined immunodeficient mice (SCID), which received 1 mg/kg free MTX (n = 9), 1 mg/kg MTX-HSA (n = 6), or 0.9% NaCl (n = 5), respectively, intraperitoneally twice a week. After 4 weeks' treatment, the mice were killed and the implants analysed histologically. RESULTS: The control group had a mean (SEM) score for cartilage invasion of RA SF of 2.0 (0.26) and for perichondrocytic cartilage degradation of 1.5 (0.34). In contrast, mice which received MTX showed a significantly reduced invasion (0.78 (0.14), p<0.01) and a reduction in perichondrocytic cartilage degradation scores (0.69 (0.2), p<0.05) in comparison with the control group. Mice treated with MTX-HSA also had significantly reduced scores for RA SF invasion into the cartilage (0.92 (0.41), p<0.05) and for cartilage degradation (0.83 (0.44), p<0.05) compared with controls. The effects of MTX and MTX-HSA were not significantly different between these two groups. CONCLUSION: Treatment with MTX or MTX-HSA significantly ameliorates cartilage destruction in the SCID mouse model for human RA.     

The SCID–HuRAg mouse as a model for rheumatoid arthritis

Many animal models have been developed for a study of rheumatoid arthritis (RA). However, RA animal models are not always similar to RA patients in their response to antirheumatic drugs. Recently, humanized monoclonal antibody (mAb) has been developed for the treatment of RA, but at present there is no animal model on which to screen this mAb therapy because of problems with cross-reactivity. We therefore considered the development of a novel animal model for the screening of antirheumatic drugs using the severe combined immunodeficiency (SCID) mouse in order to prevent the rejection of human transplant cells. Following subcutaneous implantation of synovial tissue in the SCID mouse, all target cells within the SCID–HuRAg mouse were of human origin, having migrated from the implanted tissue. Moreover, human interlukin-6 and rheumatoid factor were detected in this mouse serum. We therefore propose that this SCID–HuRAg mouse is a novel, useful animal model for the study and development of new drugs for RA patients. This novel RA animal model is reviewed in this chapter.