人类风湿性关节炎滑膜—软骨—NOD/SCID鼠嵌合体模型的制作研究
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摘要
研究背景与目的：类风湿性关节炎(RA)是一种以关节滑膜炎症为病理中心的系统性自身免疫性疾病，全世界发病率约0.4％。目前发病原因仍不明确，亦无特效的治疗措施。由于缺乏敏感、特异性的早期实验室检查指标，现行的诊断主要依靠临床症状及辅助检查，当患者被确诊时，往往已出现不可逆性关节损伤。建立与RA发病机制及病理相同或相似的动物模型在RA研究中的作用也就越来越重要了。
目前国内RA动物模型主要是诱导性炎症模型，如胶原诱导性关节炎(CIA)和佐剂性关节炎(AA)和自发性炎症模型(CJ鼠)，主要从风、寒、湿、热、肾虚等“痹症”角度模拟造模，而未能与病症紧密结合；它们的滑膜增生都是受免疫炎症反应驱动，并不能体现RA滑膜自身增生和侵蚀，与RA实际发病过程仍存在较大的距离。因此，上世纪90年代中期开始，将RA滑膜移植到免疫缺陷动物身上构建免疫缺陷动物／人RA移植物嵌合体模型的研究工作迅速得到重视。该模型在RA滑膜增生的病理机制，特别是筛选以抑制滑膜增生和软骨侵蚀为目标的药物或治疗方法中得到广泛应用。而国内对该模型的研制工作才刚开始起步，免疫缺陷动物主要有裸鼠，T、B细胞功能缺陷的联合重度免疫缺陷鼠(SCID)和T、B、NK细胞三缺陷的NOD／SCID和BNX鼠。目前广泛应用的是SCID鼠，NOD／SCID鼠能否作为RA滑膜移植物的载体尚未见报道。
本实验建立的NOD／SCID鼠-人RA移植物嵌合体模型，目的使RA滑膜组织在NOD／SCID鼠体内形成新的自身免疫性反应，于人体外模拟复制出RA的人源化动物模型，同时对RA发病进程有重要意义的指标进行研究探讨。为我国深入的研究RA提供新的动物模型，尤其为阻止滑膜增生及软骨侵蚀从中医药角度研究RA搭建平台。
方法：
(1)模型的建立：将20只NOD／SCID鼠随机分为2组，各组10只，雌雄不限。RA模型组：用3％戊巴比妥钠腹腔注射(0.2ml／100g)麻醉后，在鼠背部纵行切开(0.8～1.2cm)小口，将修剪好的正常软骨(0.5～0.8cm)植于皮下，再将RA患者滑膜置于软骨之上缝合，术后切口常规消毒，200ml饮水瓶中加入诺氟沙星(8mg／ml)抗生素溶液2ml，一周后拆线。OA模型组：同样方法建立NOD／SCID鼠-人骨关节炎移植物嵌合体模型进行比较。模型鼠术后与术前均在SPF级环境饲养，标准动物饲料，并保证每日摄食量相当，饮水不限。室温22±2℃，湿度保持60％，每周换垫料一次。
(2)血清TNF-a含量检测：10周后，摘眼球法收集模型鼠血液，以3000r／min离心20min，血清于低温环境保存。在液相试剂盒中将~(125) I-TNF、抗体、标准品经缓冲液溶解后，精密吸取浓度(0.3，0.9，2.7，8.1，24.3ng／ml)的标准品和样本各100μl置5ml离心管中。并设T管(总计数管)和NSB管(非特异性结合管)，向T管加入100μl的标记抗原，NSB管加入100μl标记抗原和100μl注射用水后，样品管分别加入标记抗原100μl和抗体100μl充分混匀，置4℃冰箱。24h后取出，除T管外，各管分别加入分离剂500μl，充分混匀。室温下静置15min后，以3500r／min离心20min后弃上清液，在放射免疫γ计数器上检测出样品的TNF-a含量。
(3)组织病理学检测：颈椎脱臼法处死模型鼠，用组织剪和手术刀将移植物剥离。经4％多聚甲醛固定8h，乙二胺四乙酸二钠(EDTA)脱钙，酒精梯度脱水、二甲苯梯度透明，浸蜡包埋，切成厚度4μm切片贴于防脱硅化玻片上，烘干后备用。病理检测指标与步骤：①HE染色：经脱蜡、酒精梯度脱水、水洗、HE染色、脱水和封片固定后，在显微镜下进行组织学观察；并对滑膜增生、软骨侵蚀和软骨降解进行评分；②VEGF原位杂交法检测：脱蜡的切片经后固定、预杂交、标记探针杂交、洗涤、封闭、加抗体、DAB显色以及苏木素复染封片等步骤完成，在显微镜下观察特异性阳性表达；③TUNEL法检测细胞凋亡：切片在脱蜡的基础上经过37℃胰蛋白酶消化30min、加酶标抗体后37℃孵育60min、DAB显色剂以及苏木素复染封片等步骤完成实验的操作。细胞凋亡片经自动图象分析，提取反映阳性信号强弱的平均光密度值(MIOD)和阳性信号多少平均着色面积(MSA)作半定量分析。
(4)统计处理：实验数据采用SPSS13.0软件包进行统计学分析，统计结果以均数±标准差((?)±s)形式表达，模型鼠血清TNF-a放射含量和切片细胞凋亡半定量分析数据进行t检验，滑膜细胞增生程度、软骨侵蚀程度和软骨降解程度分析进行两个独立样本非参数检验，P≤0.05为有显著性差异。
结果：
(1)模型鼠一般情况：实验期间NOD／SCID鼠无手摸皮骨感、活动度差、毛稀疏等典型移植物抗宿主病的表现，模型鼠在SPF环境下10周存活率100％。
(2) TNF-a含量统计分析结果：RA滑膜组与OA滑膜组血清中TNF-a含量分别为0.80±0.06、0.70±0.03，经统计分析有显著性差异(P＜0.001)。
(3)组织病理学观察：①HE染色：在显微镜下OA滑膜组只见少量的滑膜细胞增生和炎症细胞，移植的滑膜组织主要被纤维组织形成的条索状物代替，无明显地软骨被侵蚀破坏发生；在RA滑膜组可明显见到大量的滑膜细胞增生和生化中心形成，病变部位的组织结构间质变为疏松，为境界不清晰的颗粒状或块状无结构强嗜酸性红染物质；软骨边缘被滑膜组织侵蚀破坏明显；OA滑膜组与RA滑膜组炎症积分评价分别为：滑膜增生(1.50±0.51 vs 2.56±1.15)、软骨侵蚀(1.15±0.51 vs 2.42±1.29)和软骨降解(1.50±0.51 vs 2.44±1.04)。两组间差异均达到显著水平(P≤0.040)。②VEGF：镜下OA滑膜组在切片中很少见到细胞胞浆VEGF mRNA的阳性表达；而在RA滑膜组的切片中能清楚见到VEGF mRNA的细胞胞浆着色呈棕黄色的阳性表达。③细胞凋亡：镜下OA滑膜组可明显见到细胞核被染成棕黄色颗粒即细胞凋亡，RA滑膜组细胞凋亡不明显。OA滑膜组与RA滑膜组细胞凋亡半定量分析结果分别为：(OD：959.23±80.89vs 168.37±12.87)、(Area：1890.51±159.14 vs 180.21±8.25)，两组间差异均有统计学意义(P＜0.001)。
结论：
用重度联合免疫缺陷的NOD／SCID鼠为载体，可成功建立人RA滑膜-软骨-NOD／SCID鼠嵌合体动物模型。对模型的相关指标检测结果显示，NOD／SCID鼠一次性接受组织移植后，通过10周的饲养，移植的人RA滑膜继续保持增生和侵蚀能力，且继续高表达TNF-a和VEGF，而细胞凋亡则明显受抑。该模型在体外模拟复制出了自身免疫状态下RA滑膜细胞增生到软骨破坏的病理过程。人源化动物模型的建立为更深入研究RA奠定了一定基础，并具有广泛的临床应用价值。
Background and Objectives: Rheumatoid arthritis (RA) is an autoimmunedisease characterized by joint synovitis and the disease incidence is about 0.4％allover whole world. Nowadays, its etiology is still not clear, and there are not specifictherapeutic measures. The current diagnosis mainly depends on clinical symptoms andauxiliary examinations due to lack of sensitive and specific laboratory indexes atearly stage. Most of patients with a final diagnosis of RA have irreversible jointdamage. It is recognized widely that synovial hyperplasia and angiogenesis are the keyfactors resulting in cartilage and bone destruction. So it is pretty important to establishan animal model which has the identical or similar pathomechanism in the role ofresearches of RA.
At present, in China, the major animal models are inducibility inflammationmodels [for instance: collagen induced arthritis (CIA) and adjuvant arthritis(AA)]andspontaneous inflammation model (Campylobacter Jejuni mice), which simulationmodel mainly in terms of hi-syndromes such as wind, cold, humidity, hot, kidneydeficiency, etc, but fail to combine closely with disorder, the synovial hyperplasia andcartilage invasion can not be isolated, because these pathological changes are totallydrived by immune and inflammatory responses. They still have a long-distance to the actual pathomechanism of RA. Therefore, since the middle 90s of last century, muchattention has been quickly paid to new fields studies. In these researches, RA synovialmembrane was transplanted into immunodeficient animals to bulid animal-HuRAgchimera model. Such kind of model is widely used in exploring the pathomechanismof RA, especially in screening new drugs or medications for purpose of inhibitingsynovial hyperplasia and cartilage invasion. However, in China, the generation of thiskind of animal model is just on its first step. Immunodeficiency animals include nudemice, mice with severe combined immune deficiency (SCID) of T cells and Bcells, NOD/SCID mice and BNX mice with immune deficiency of T cells, B cells ornatural killer cells. Currently, SCID is widely used. As for whether NOD/SCID micecan be used as a carrier of RA synovial grafts or not, no report is found.
The chimera model of NOD/SCID mice-human rheumatiod arthritis grafts whichwas established in this experiment aims at formation of the new autoimmunity reactionwhich was provoked by RA synovial membrane in NOD/SCID mice, and smulation ofhuman RA animal model outside the human body, meanwhile, researching of indexeswhich are significant in the process of the pathogenesis of RA. It provides a new animalmodel for an in-depth study of RA, especially for preventing synovial hyperplasia andcartilage erosion in terms of Chinese medicine.
Methods:
(1) Model production The NOD/SCID mice were radomized into twogroups, with 10 mice in each group, of either sex(gender). The RA group synovial:under general anesthesia (0.2ml/100g),the mice were operated in the back with alongitudinal incision(0.8～1.2cm).The prepared normal cartilage slice(0.5～0.8cm)was planted under the skin of the back, then, the synovial tissue from RA patient wastransplanted on the slice. The skin was sutured. Carry out routine disinfection onincision after operation and add 2ml of norfloxacin antibiosis solution (8mg/ml) into200ml water drinking bottle. Dermal sutures out one week later.The second group isused as control group for experiment. The RA group synovial: after implanting intocartilage,place synovium of Osteoarthritis patients on cartilage and suturation.After operation, mice in experiment after operation should be fed with standard animal feed in the same SPF grade environment as before operation, with correspondentintake amount for each day and without limit on water drinking.Keep room tempera-ture of 22±2℃and humidity of 60％. Padding should be exchanged once a week.
(2) Hematology detection(TNF-a):Collect blood from mice by eyeball remov-ing method 10 weeks later and centrifugate at 3000 r/min for 20 min.Serum should bepreserve in low-temperature environment.After dissolving ~(125)I-TNF, antibody andstandard with buffer in liquid reagent box,accurately and respectively absorb 100μlstandard and sample with concentrations (0.3, 0.9, 2.7, 8.1 and 24.3 ng/ml) into 5 mlcentrifuge tube.Additional set T tube (total counter tube) and NSB tube (nonspecificbinding tube).Add 100μl marked antigen to T tube and add 100μl marked antigen and100μl water for injection. And add 100μl antigen and 100μl antibody to the othertube. Mix well and mark corresponding No.of experimental mice.Culture in 4℃cons-tant temperature cabinet. Take it out 24 hours later, expect that separation agent is notadded to T tube,respectively add 500μl separation agent to the other tubes.Mix welland stand still at room temperature for 15 min before carrying out centrifugation at3500 r/min for 20 min.Discard supematant and detect radioactive counting of sampleswith pre programmed procedure on radio-immunityγcounter.
(3) Histopathology analysis: Model mice were killed by neck vertebrae disloat-ion method and strip plan with tissue scissors and scalpel. Fix with 4％paraformal-dehyde for 8 hours and carry out decalcification with ethylenediamine tetraaceticacid(EDTA). Then carry out dehydration with alcohol gradient and clarify withxylene.Embed with paraffine.Cut into slices of 4μm thickness and paste on silicifiedglass piece. Dry it, ready for use. Pathologic detection index and procedure:①HEstaining: after dewaxing, dehydration of alcohol gradient, water washing, HE dye,dehydration and mounting, carry out histological observation under micros cope,and estimate for synovial hyperplasia,cartilage invasion and cartilaged degradation②Detection of VEGF hybridization in situ method: slice sheet after dewaxing isfinished after procedures such as after fixing,prehybridization, hybridis ation of mark-ed probe,washing, containning,adding antibody, DAB coloration and redye mounting by hematoxylin.Observe specific positive expression under microscope.③Experimentoperation is finished after procedures of detection of apoptosis with Terminal deoxynu-cleotidy transferase-mediated dUTP nick end labeling(TUNEL) method: After digestedfor 30 minutes with trypsase at 37℃, based on dewaxing and adding enzyme markedantibody, enzyme labelled antibody should be incubated for 60 minutes at 37℃, DABcoloration and redye mounting by hematoxylin and so on step. The professional imageanalysis software,Image Pro plus 6.0 was used to analyse the difference of the meanintegrated optical density(MIOD) and mean value of stained (AREA) in the twogroups.
(4) Statistics analysis: Statistics analysis should be carried out on experimentaldata with SPSS13.0 software package, with statistics results denoted in form of(?)±s.model mice blood serum of TNF-a contents and semiquantitative analysis datain T test,synovium hyperplastic degree,cartilage corrosion degree and cartilage degra-dation degree analyzing in 2 independent sample test,P=0.05 for significant difference.
Rusults:
(1) General state of mouse model: There should be no typical graft-versus-hostdisease(GVHD) behavior such as hand touching skin bone sense, bad mobility andsparse capill for NOD/SCID mice during experiment period.Survival rate for 10weeks of model mice under SPF environment is 100％.
(2) Statistical analysis results for TNF-a: There are obviously difference inserum amount of TNF-a between for the RA synovial group and the OA group (0.80±0.06 vs 0.70±0.03),which has significant difference(P＜0.001).
(3) Histopathology indicated that:①HE dye: there is only a little amount ofsynovium cell hyperplastic and inflammatory cell for OA synovium group undermicroscope, implanted synovium tissue is mainly replaced by strip material formedfrom fibroplasia,without manifest corrosion and injury of cartilage.A great amount ofsynovial cell hyperplastic and biochemical center can be obviously seen to be formedin RA synovial group.Histological interstitial substance in disease part becomesloosen.It becomes granulation with unsharp boundary or block strong acidophic red dye substance without structure.Edge of cartilage is obviously damaged by corrosionof vs tissue.The OA synovial group and the RA group synovial hyperlasia (1.50±0.51vs 2.56±1.15),cartilage invasion (1.15±0.51 vs 2.42±1.29) and cartilage degradation(1.50±0.51 vs 2.44±1.04),two sets have significant difference(P=0.040).②VEGF:sp-ecific positive expression of VEGF mRNA of cytolymph is very rare on slice for OAvs group under microscope.And specific behavior to exhibit brown yellow by cytoly-mph expressing VEGF mRNA can be clearly seen on slice of RA synovial group.③Apoptosis: It can be obviously seen for OA synovium group that cell nucleus isdyed to brown yellow granulation under microscope,which is cell apoptosis under.positive expression with specificity is not easy found for RA synovial group. Semiqu-antitative analysis of the RA synovial group and the OA synovial: (MIOD:959.23±80.89 vs 168.37±12.87),(Area:1890.51±159.14 vs 180.21±8.25), they have significantdifference (P＜0.001).
Conclusions:
It can establish the chimera model of NOD/SCID mice-human rheumatoidarthritis grafts successfully through NOD/SCID mice with severe combined immuno-Deficiency. It can be seen from the relative detection result of the model,afterNOD/SCID mice had been done a tissue transplantation and had been raised tenweeks,tansplanted human RA synovial membranes continue to maintain the ability ofHyperplasia and erosion.Furthermore,they continue to highly express TNF-a andVEGF, however, apoptosises are obviously inhibited.The model have SimulatedPathological process from RA synovial cells hyperplasia to cartilage damage in theautoimmune state outside human body.The establishment of human animal model laysa good foundation for further research on RA research and has wide clinical applicationvalue.
目前国内RA动物模型主要是诱导性炎症模型，如胶原诱导性关节炎(CIA)和佐剂性关节炎(AA)和自发性炎症模型(CJ鼠)，主要从风、寒、湿、热、肾虚等“痹症”角度模拟造模，而未能与病症紧密结合；它们的滑膜增生都是受免疫炎症反应驱动，并不能体现RA滑膜自身增生和侵蚀，与RA实际发病过程仍存在较大的距离。因此，上世纪90年代中期开始，将RA滑膜移植到免疫缺陷动物身上构建免疫缺陷动物／人RA移植物嵌合体模型的研究工作迅速得到重视。该模型在RA滑膜增生的病理机制，特别是筛选以抑制滑膜增生和软骨侵蚀为目标的药物或治疗方法中得到广泛应用。而国内对该模型的研制工作才刚开始起步，免疫缺陷动物主要有裸鼠，T、B细胞功能缺陷的联合重度免疫缺陷鼠(SCID)和T、B、NK细胞三缺陷的NOD／SCID和BNX鼠。目前广泛应用的是SCID鼠，NOD／SCID鼠能否作为RA滑膜移植物的载体尚未见报道。
本实验建立的NOD／SCID鼠-人RA移植物嵌合体模型，目的使RA滑膜组织在NOD／SCID鼠体内形成新的自身免疫性反应，于人体外模拟复制出RA的人源化动物模型，同时对RA发病进程有重要意义的指标进行研究探讨。为我国深入的研究RA提供新的动物模型，尤其为阻止滑膜增生及软骨侵蚀从中医药角度研究RA搭建平台。
方法：
(1)模型的建立：将20只NOD／SCID鼠随机分为2组，各组10只，雌雄不限。RA模型组：用3％戊巴比妥钠腹腔注射(0.2ml／100g)麻醉后，在鼠背部纵行切开(0.8～1.2cm)小口，将修剪好的正常软骨(0.5～0.8cm)植于皮下，再将RA患者滑膜置于软骨之上缝合，术后切口常规消毒，200ml饮水瓶中加入诺氟沙星(8mg／ml)抗生素溶液2ml，一周后拆线。OA模型组：同样方法建立NOD／SCID鼠-人骨关节炎移植物嵌合体模型进行比较。模型鼠术后与术前均在SPF级环境饲养，标准动物饲料，并保证每日摄食量相当，饮水不限。室温22±2℃，湿度保持60％，每周换垫料一次。
(2)血清TNF-a含量检测：10周后，摘眼球法收集模型鼠血液，以3000r／min离心20min，血清于低温环境保存。在液相试剂盒中将~(125) I-TNF、抗体、标准品经缓冲液溶解后，精密吸取浓度(0.3，0.9，2.7，8.1，24.3ng／ml)的标准品和样本各100μl置5ml离心管中。并设T管(总计数管)和NSB管(非特异性结合管)，向T管加入100μl的标记抗原，NSB管加入100μl标记抗原和100μl注射用水后，样品管分别加入标记抗原100μl和抗体100μl充分混匀，置4℃冰箱。24h后取出，除T管外，各管分别加入分离剂500μl，充分混匀。室温下静置15min后，以3500r／min离心20min后弃上清液，在放射免疫γ计数器上检测出样品的TNF-a含量。
(3)组织病理学检测：颈椎脱臼法处死模型鼠，用组织剪和手术刀将移植物剥离。经4％多聚甲醛固定8h，乙二胺四乙酸二钠(EDTA)脱钙，酒精梯度脱水、二甲苯梯度透明，浸蜡包埋，切成厚度4μm切片贴于防脱硅化玻片上，烘干后备用。病理检测指标与步骤：①HE染色：经脱蜡、酒精梯度脱水、水洗、HE染色、脱水和封片固定后，在显微镜下进行组织学观察；并对滑膜增生、软骨侵蚀和软骨降解进行评分；②VEGF原位杂交法检测：脱蜡的切片经后固定、预杂交、标记探针杂交、洗涤、封闭、加抗体、DAB显色以及苏木素复染封片等步骤完成，在显微镜下观察特异性阳性表达；③TUNEL法检测细胞凋亡：切片在脱蜡的基础上经过37℃胰蛋白酶消化30min、加酶标抗体后37℃孵育60min、DAB显色剂以及苏木素复染封片等步骤完成实验的操作。细胞凋亡片经自动图象分析，提取反映阳性信号强弱的平均光密度值(MIOD)和阳性信号多少平均着色面积(MSA)作半定量分析。
(4)统计处理：实验数据采用SPSS13.0软件包进行统计学分析，统计结果以均数±标准差((?)±s)形式表达，模型鼠血清TNF-a放射含量和切片细胞凋亡半定量分析数据进行t检验，滑膜细胞增生程度、软骨侵蚀程度和软骨降解程度分析进行两个独立样本非参数检验，P≤0.05为有显著性差异。
结果：
(1)模型鼠一般情况：实验期间NOD／SCID鼠无手摸皮骨感、活动度差、毛稀疏等典型移植物抗宿主病的表现，模型鼠在SPF环境下10周存活率100％。
(2) TNF-a含量统计分析结果：RA滑膜组与OA滑膜组血清中TNF-a含量分别为0.80±0.06、0.70±0.03，经统计分析有显著性差异(P＜0.001)。
(3)组织病理学观察：①HE染色：在显微镜下OA滑膜组只见少量的滑膜细胞增生和炎症细胞，移植的滑膜组织主要被纤维组织形成的条索状物代替，无明显地软骨被侵蚀破坏发生；在RA滑膜组可明显见到大量的滑膜细胞增生和生化中心形成，病变部位的组织结构间质变为疏松，为境界不清晰的颗粒状或块状无结构强嗜酸性红染物质；软骨边缘被滑膜组织侵蚀破坏明显；OA滑膜组与RA滑膜组炎症积分评价分别为：滑膜增生(1.50±0.51 vs 2.56±1.15)、软骨侵蚀(1.15±0.51 vs 2.42±1.29)和软骨降解(1.50±0.51 vs 2.44±1.04)。两组间差异均达到显著水平(P≤0.040)。②VEGF：镜下OA滑膜组在切片中很少见到细胞胞浆VEGF mRNA的阳性表达；而在RA滑膜组的切片中能清楚见到VEGF mRNA的细胞胞浆着色呈棕黄色的阳性表达。③细胞凋亡：镜下OA滑膜组可明显见到细胞核被染成棕黄色颗粒即细胞凋亡，RA滑膜组细胞凋亡不明显。OA滑膜组与RA滑膜组细胞凋亡半定量分析结果分别为：(OD：959.23±80.89vs 168.37±12.87)、(Area：1890.51±159.14 vs 180.21±8.25)，两组间差异均有统计学意义(P＜0.001)。
结论：
用重度联合免疫缺陷的NOD／SCID鼠为载体，可成功建立人RA滑膜-软骨-NOD／SCID鼠嵌合体动物模型。对模型的相关指标检测结果显示，NOD／SCID鼠一次性接受组织移植后，通过10周的饲养，移植的人RA滑膜继续保持增生和侵蚀能力，且继续高表达TNF-a和VEGF，而细胞凋亡则明显受抑。该模型在体外模拟复制出了自身免疫状态下RA滑膜细胞增生到软骨破坏的病理过程。人源化动物模型的建立为更深入研究RA奠定了一定基础，并具有广泛的临床应用价值。
Background and Objectives: Rheumatoid arthritis (RA) is an autoimmunedisease characterized by joint synovitis and the disease incidence is about 0.4％allover whole world. Nowadays, its etiology is still not clear, and there are not specifictherapeutic measures. The current diagnosis mainly depends on clinical symptoms andauxiliary examinations due to lack of sensitive and specific laboratory indexes atearly stage. Most of patients with a final diagnosis of RA have irreversible jointdamage. It is recognized widely that synovial hyperplasia and angiogenesis are the keyfactors resulting in cartilage and bone destruction. So it is pretty important to establishan animal model which has the identical or similar pathomechanism in the role ofresearches of RA.
At present, in China, the major animal models are inducibility inflammationmodels [for instance: collagen induced arthritis (CIA) and adjuvant arthritis(AA)]andspontaneous inflammation model (Campylobacter Jejuni mice), which simulationmodel mainly in terms of hi-syndromes such as wind, cold, humidity, hot, kidneydeficiency, etc, but fail to combine closely with disorder, the synovial hyperplasia andcartilage invasion can not be isolated, because these pathological changes are totallydrived by immune and inflammatory responses. They still have a long-distance to the actual pathomechanism of RA. Therefore, since the middle 90s of last century, muchattention has been quickly paid to new fields studies. In these researches, RA synovialmembrane was transplanted into immunodeficient animals to bulid animal-HuRAgchimera model. Such kind of model is widely used in exploring the pathomechanismof RA, especially in screening new drugs or medications for purpose of inhibitingsynovial hyperplasia and cartilage invasion. However, in China, the generation of thiskind of animal model is just on its first step. Immunodeficiency animals include nudemice, mice with severe combined immune deficiency (SCID) of T cells and Bcells, NOD/SCID mice and BNX mice with immune deficiency of T cells, B cells ornatural killer cells. Currently, SCID is widely used. As for whether NOD/SCID micecan be used as a carrier of RA synovial grafts or not, no report is found.
The chimera model of NOD/SCID mice-human rheumatiod arthritis grafts whichwas established in this experiment aims at formation of the new autoimmunity reactionwhich was provoked by RA synovial membrane in NOD/SCID mice, and smulation ofhuman RA animal model outside the human body, meanwhile, researching of indexeswhich are significant in the process of the pathogenesis of RA. It provides a new animalmodel for an in-depth study of RA, especially for preventing synovial hyperplasia andcartilage erosion in terms of Chinese medicine.
Methods:
(1) Model production The NOD/SCID mice were radomized into twogroups, with 10 mice in each group, of either sex(gender). The RA group synovial:under general anesthesia (0.2ml/100g),the mice were operated in the back with alongitudinal incision(0.8～1.2cm).The prepared normal cartilage slice(0.5～0.8cm)was planted under the skin of the back, then, the synovial tissue from RA patient wastransplanted on the slice. The skin was sutured. Carry out routine disinfection onincision after operation and add 2ml of norfloxacin antibiosis solution (8mg/ml) into200ml water drinking bottle. Dermal sutures out one week later.The second group isused as control group for experiment. The RA group synovial: after implanting intocartilage,place synovium of Osteoarthritis patients on cartilage and suturation.After operation, mice in experiment after operation should be fed with standard animal feed in the same SPF grade environment as before operation, with correspondentintake amount for each day and without limit on water drinking.Keep room tempera-ture of 22±2℃and humidity of 60％. Padding should be exchanged once a week.
(2) Hematology detection(TNF-a):Collect blood from mice by eyeball remov-ing method 10 weeks later and centrifugate at 3000 r/min for 20 min.Serum should bepreserve in low-temperature environment.After dissolving ~(125)I-TNF, antibody andstandard with buffer in liquid reagent box,accurately and respectively absorb 100μlstandard and sample with concentrations (0.3, 0.9, 2.7, 8.1 and 24.3 ng/ml) into 5 mlcentrifuge tube.Additional set T tube (total counter tube) and NSB tube (nonspecificbinding tube).Add 100μl marked antigen to T tube and add 100μl marked antigen and100μl water for injection. And add 100μl antigen and 100μl antibody to the othertube. Mix well and mark corresponding No.of experimental mice.Culture in 4℃cons-tant temperature cabinet. Take it out 24 hours later, expect that separation agent is notadded to T tube,respectively add 500μl separation agent to the other tubes.Mix welland stand still at room temperature for 15 min before carrying out centrifugation at3500 r/min for 20 min.Discard supematant and detect radioactive counting of sampleswith pre programmed procedure on radio-immunityγcounter.
(3) Histopathology analysis: Model mice were killed by neck vertebrae disloat-ion method and strip plan with tissue scissors and scalpel. Fix with 4％paraformal-dehyde for 8 hours and carry out decalcification with ethylenediamine tetraaceticacid(EDTA). Then carry out dehydration with alcohol gradient and clarify withxylene.Embed with paraffine.Cut into slices of 4μm thickness and paste on silicifiedglass piece. Dry it, ready for use. Pathologic detection index and procedure:①HEstaining: after dewaxing, dehydration of alcohol gradient, water washing, HE dye,dehydration and mounting, carry out histological observation under micros cope,and estimate for synovial hyperplasia,cartilage invasion and cartilaged degradation②Detection of VEGF hybridization in situ method: slice sheet after dewaxing isfinished after procedures such as after fixing,prehybridization, hybridis ation of mark-ed probe,washing, containning,adding antibody, DAB coloration and redye mounting by hematoxylin.Observe specific positive expression under microscope.③Experimentoperation is finished after procedures of detection of apoptosis with Terminal deoxynu-cleotidy transferase-mediated dUTP nick end labeling(TUNEL) method: After digestedfor 30 minutes with trypsase at 37℃, based on dewaxing and adding enzyme markedantibody, enzyme labelled antibody should be incubated for 60 minutes at 37℃, DABcoloration and redye mounting by hematoxylin and so on step. The professional imageanalysis software,Image Pro plus 6.0 was used to analyse the difference of the meanintegrated optical density(MIOD) and mean value of stained (AREA) in the twogroups.
(4) Statistics analysis: Statistics analysis should be carried out on experimentaldata with SPSS13.0 software package, with statistics results denoted in form of(?)±s.model mice blood serum of TNF-a contents and semiquantitative analysis datain T test,synovium hyperplastic degree,cartilage corrosion degree and cartilage degra-dation degree analyzing in 2 independent sample test,P=0.05 for significant difference.
Rusults:
(1) General state of mouse model: There should be no typical graft-versus-hostdisease(GVHD) behavior such as hand touching skin bone sense, bad mobility andsparse capill for NOD/SCID mice during experiment period.Survival rate for 10weeks of model mice under SPF environment is 100％.
(2) Statistical analysis results for TNF-a: There are obviously difference inserum amount of TNF-a between for the RA synovial group and the OA group (0.80±0.06 vs 0.70±0.03),which has significant difference(P＜0.001).
(3) Histopathology indicated that:①HE dye: there is only a little amount ofsynovium cell hyperplastic and inflammatory cell for OA synovium group undermicroscope, implanted synovium tissue is mainly replaced by strip material formedfrom fibroplasia,without manifest corrosion and injury of cartilage.A great amount ofsynovial cell hyperplastic and biochemical center can be obviously seen to be formedin RA synovial group.Histological interstitial substance in disease part becomesloosen.It becomes granulation with unsharp boundary or block strong acidophic red dye substance without structure.Edge of cartilage is obviously damaged by corrosionof vs tissue.The OA synovial group and the RA group synovial hyperlasia (1.50±0.51vs 2.56±1.15),cartilage invasion (1.15±0.51 vs 2.42±1.29) and cartilage degradation(1.50±0.51 vs 2.44±1.04),two sets have significant difference(P=0.040).②VEGF:sp-ecific positive expression of VEGF mRNA of cytolymph is very rare on slice for OAvs group under microscope.And specific behavior to exhibit brown yellow by cytoly-mph expressing VEGF mRNA can be clearly seen on slice of RA synovial group.③Apoptosis: It can be obviously seen for OA synovium group that cell nucleus isdyed to brown yellow granulation under microscope,which is cell apoptosis under.positive expression with specificity is not easy found for RA synovial group. Semiqu-antitative analysis of the RA synovial group and the OA synovial: (MIOD:959.23±80.89 vs 168.37±12.87),(Area:1890.51±159.14 vs 180.21±8.25), they have significantdifference (P＜0.001).
Conclusions:
It can establish the chimera model of NOD/SCID mice-human rheumatoidarthritis grafts successfully through NOD/SCID mice with severe combined immuno-Deficiency. It can be seen from the relative detection result of the model,afterNOD/SCID mice had been done a tissue transplantation and had been raised tenweeks,tansplanted human RA synovial membranes continue to maintain the ability ofHyperplasia and erosion.Furthermore,they continue to highly express TNF-a andVEGF, however, apoptosises are obviously inhibited.The model have SimulatedPathological process from RA synovial cells hyperplasia to cartilage damage in theautoimmune state outside human body.The establishment of human animal model laysa good foundation for further research on RA research and has wide clinical applicationvalue.
引文
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[1] Aupperle KR,Boyle DC,Hendrixx M,et al.Regulation of synovivocyte proliferation, apoptosis,and invasion by the p53 tumor suppressor gene[J].Am J Pathol,1998,152(4):1091-1098.
[2] Okamoto K,Asahara H,Kobayashi T,et al. Induction of apoptosis in the rheumatoid synovium by Fas ligand gene transfer[J].Gene Ther,1998,5(3):331-338.
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[4] Akbar AN,Savill J,Gombert W,et al.The specific recognition by macrophages of CD8 + CD45RO + T cells undergoing apoptosis:a potential mechanismfor T cell clearance during resolution of viral infections[J].J Exp Med,1994,180(5):1943-1947.
[5] Mastuno H,Yudoh K,Nakazawa F,et al.Antirheumat ic effects of humanized anti-Fas monoclonal antibody in human rheumatoid arthritis/SCID mouse chimera[J].Rheumato logy,2002, 29(8):1609-1614.
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