【作者】 王贵霞；

【导师】 张秀英；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2010， 博士

【摘要】 类风湿性关节炎(rheumatoid arthritis,RA)是一种以关节滑膜炎、破坏性关节病变为主要特征的慢性、自身免疫性疾病,致畸、致残率极高。对于RA至今尚无特效疗法,东北农业大学生命科学学院生物制药教研室正在研发的治疗RA的重组基因工程抗体药物,是以抗引起RA主要致炎因子IL-1β,IL-17为靶标的双价抗体药物,它的研制成功将填补国内治疗RA疾病的双价抗体药物的空白,缩短我国生物制药与世界总体制药水平的差距。为了保证生物技术药物的安全,有效和质量监控,需要进行临床前药理学的研究,动物模型的构建则是其中最为重要的组成环节之一,本研究的首要目的是建立能较全面反映RA特点的动物疾病模型。IL-1β在RA炎的发病进程中的主要作用机理已经得到了明确的阐述,主要是使滑膜细胞和软骨细胞合成和释放胶原酶和其他蛋白溶解酶,并抑制软骨细胞合成蛋白多糖基质,从而在RA炎的发病进程中起重要作用;而近年来在RA病人滑膜上清液中发现大量存在的具有生物活性的IL-17,能显著促进炎症细胞分泌TNF-α、IL-1β,与其他炎症细胞因子有协同作用,间接促进RA炎症的发展。而多聚蛋白聚糖酶,即带有血小板凝血酶原敏感基序的解整连素和金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS) ADAMTS-4对RA疾病中细胞外基质蛋白聚糖的降解起着重要的作用。在构建动物模型的基础上,本研究将通过IL-17诱导体外培养的软骨细胞,检测ADAMTS-4的表达情况,深入的研究IL-17在RA疾病中的是否在关节局部发挥直接作用,以期进一步探讨RA在分子水平的发病机理,更好的为创新药物的临床前药理学研究提供基础。首先建立类风湿性关节炎动物模型,方法为:45只SD大鼠随机分成3组:免疫诱导组,免疫诱导加寒湿刺激组和对照组。免疫诱导组用鸡Ⅱ型胶原和弗氏完全佐剂乳化混合后0.25mL注入大鼠足跖部和背部皮下,7d后以0.1mL乳化剂加强免疫一次。寒湿刺激组在免疫诱导的基础上,每天将大鼠放入冰水中1h,连续14d。以相同体积的溶剂冰醋酸注射做为对照组。诱导后每周测量大鼠体重和足爪肿胀程度,21d和42d分别采集大鼠血清,测定抗Ⅱ型胶原抗体、TNF-α、IL-1β和IL-17表达水平,并对踝关节进行组织病理学、X-ray放射学检查。成功建立RA的整体动物模型后,本研究进一步采用体外试验研究法,从分子水平单因素的考察IL-17在关节局部的致病作用,首先以胰蛋白酶和胶原酶两步消化法无菌取得大鼠软骨细胞,然后进行体外培养,经IL-17诱导后,在不同的时间点采集细胞样品,从蛋白质水平分析RA中IL-17能否诱导破坏软骨基质重要酶ADAMTS-4的表达,再从基因水平检测ADAMTS-4表达量的变化。具体试验方法为:将体外培养软骨细胞分为二组,即IL-17诱导组和未诱导对照组,分别取24h,48h的细胞培养上清液,离心后,采用蛋白质免疫印迹法检测ADAMTS-4蛋白质的表达情况,采用实时定量PCR法检测ADAMTS-4的mRNA的表达量的变化。首先采用Trizol法提取软骨细胞RNA,紫外分光光度仪测定RNA的OD值,并进行琼脂糖凝胶电泳检测提取RNA的质量。将质量合格的RNA逆转录为cDNA,采用PCR扩增ADAMTS-4片段,经琼脂糖凝胶电泳,紫外检测仪下观察PCR扩增结果。分别对两个时间点IL-17处理组软骨细胞与未处理组软骨细胞的cDNA在ABI 7500 real time PCR仪上进行实时定量PCR扩增,同时对每个样品做内参GAPDH的扩增。每个样品重复检测3次,用2-△ΔCt相对定量法分析其mRNA转录改变。制备的类风湿性关节炎动物模型显示,试验大鼠在免疫后,足爪在次日严重红肿,随后肿胀逐渐消退,第7天加强免疫之后,双侧后足爪肿胀明显,前爪也发生继发性肿胀。胶原诱导组发病率为80%,胶原诱导加寒湿刺激组发病率为85%,溶剂对照组没有发病。与对照组相比,两个模型组间血清中抗CⅡ抗体、TNF-α、IL-1β和IL-17的水平均升高,差异极显著(P<0.01),而两个模型组的大鼠血清中抗CⅡ抗体、TNF-α、IL-1β和IL-17水平相比较,胶原诱导加寒湿刺激组比单纯诱导组稍有升高,但是差异不显著(P>0.05)。对照组的大鼠踝关节组织切片可见清晰的关节腔,而两个模型组大鼠踝关节组织切片可见滑膜细胞增生,排列疏散,紊乱,软骨细胞增生,关节腔可见炎性细胞浸润,滑膜组织中淋巴细胞严重浸润。发病大鼠踝关节处X光片的骨关节边缘模糊、软骨破坏、骨变形。采用胰蛋白酶和胶原酶两步消化法成功获得原代的软骨细胞,对软骨细胞进行传代培养后,细胞生长状况良好,软骨细胞经冻存复苏后,活细胞率可以达到87%。经蛋白质免疫印迹分析表明,无论是经IL-17诱导还是未诱导组的软骨细胞均分泌表达ADAMTS-4蛋白,以Trizol法提取软骨细胞中的总RNA经鉴定纯度较好,经逆转录获得的cDNA为模版,以ADAMTS-4的上下游引物进行PCR扩增,可获得约99bp的预期长度的产物片段,条带清晰,无杂带。实时定量PCR检测致炎细胞因子IL-17诱导体外培养软骨细胞的ADAMTS-4的转录水平,发现破坏软骨细胞基质的ADAMTS-4的mRNA转录水平在诱导后24h和48h均显著升高,分别升高了6.37,8.62倍,与对照组相比差异极显著(P<0.01)。通过对临床症状、关节炎指数、抗CⅡ抗体滴度、致炎细胞因子水平、组织病理变化和影像学变化的综合指标评价,本研究成功建立了大鼠类风湿性关节炎疾病模型,为创新药物的临床前研究奠定了基础。在RA整体动物模型中,以ELISA方法检测发现发病大鼠血清中IL-1β、IL-17的水平与对照组相比显著升高,说明创新药物构建的双价抗体以这两种致炎因子为靶标,将会有效的阻断RA的炎症进程。由于本研究旨在为抗IL-1β、IL-17双价的基因工程抗体药物的临床前药理学研究提供基础,考虑到整体动物模型体内环境复杂,为排除各种其他因素的干扰,本研究在体外进行软骨细胞的培养,通过进一步的体外试验研究表明,IL-17诱导组与未诱导组的软骨细胞培养上清液中均可以检测到ADAMTS-4蛋白的表达,实时定量PCR分析结果表明IL-17诱导组软骨细胞ADAMTS-4的表达量明显升高,与未诱导组的差异显著,可以推测在RA患者体内IL-17除了可以通过促进炎症细胞因子TNF-α、IL-1β的分泌,间接促进炎症发展以外,它还可以在关节局部直接通过诱导软骨细胞分泌ADAMTS-4来降解软骨中的蛋白聚糖,破坏软骨基质,使RA患者关节局部发生畸形,甚至残疾,从而在RA病症进程中发挥间接和直接的重要作用,可以推测阻断IL-17的作用是RA治疗中的一个潜在作用靶点,有可能成为有效筛选治疗RA有效药物的方式之一。更多还原

【Abstract】 Rheumatoid arthritis (RA ) is a kind of chronic autoimmune disease charactered by astrosynovitis and destructive astropathy. There has been no specific therapeutics. The drug of geneticallyengineered antibody researched by the staff of biotechnology college in Northeast Agricultural University will make up a deficiency in the two-potency antibody drug treatment for the RA in China and reduce the difference in biotechnology drug between China and internation. The preclinical pharmacology research will provide the guarantee for the safety, effect and quality control of the biotechnology drug. The aim of this research is to prepare the animal disease models which reflect the features of RA and further to study its pathogenesis. This research provides the foundation for the preclinical pharmacological research of the orininal new drug.The action target of drug was the IL-1βand IL-17 which are the main proinflammatory factor in RA. The mechanism of IL-1βin RA has been clearly explained, that is mainly induced the synoviocyte and chondrocyte expression the collagenase and other protease and inhibition the synthesis of proteoglycan. IL-17, recent years finding in the synovium supernatant of RA patient, can significantly promote the secretion of TNF-αand IL-1β. The aggrecanase(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS) ADAMTS-4 can degradate the extracell matrix in cartilage. This research will culture the chondrocyte, and induce them with IL-17, then determine the ADAMTS-4 protein expression and mRAN change level. The aim of this research is to investigate the action mechanism of IL-17 in a local joint and further analyze the pathogeny of rheumatoid arthritis on the molecular level. This will supply a solid base for preclinical pharmacology research of the orinal drug.Firstly, the whole animl desease model was established. 45 male SD rat were divided into 3 groups: simply immune-induced group; immune-induced and cold wetness-stimulated group; control group. Collagen-induced arthritic rat were intradermally immunized at the base of the tail with 0.25 mL of the emulsion containing chicken typeⅡcollagen and Freund’s complete adjuvant. On day 7 after the initial collagen immunization, the rats were intraperitoneally boosted with 0.1mL emulsion. Rat in immune-induced and cold wetness-stimulated group put in ice-water 1h for 10 days based on the immune with emulsion.The same volume of vehicle (glacial acetic acid)was administered as a control.Weight body and engorgement of foot and calw measured each week. Serum collected on 21d and 42d after initial collagen immunization for the determination of antibody for collagenⅡ; TNF-α; IL-1βand IL-17. Rats were euthanized by cervical dislocation on day 42 after initial collagen immunization. Histopathological features of peripheral ankles were assessed in hematoxylin-stained formalin-fixed paraffin-embedded sections. 5 rats in each group on day 42 after immune were anestetized by ethylether for radiological examination.Chondrocytes were isolated from articular cartilage of rats and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum. Chondrocytes were serum-starved in DMEM for 24h and treated with IL-17for another 24h or 48h. RNA was extracted by the guanidinium isothiocyanate procedure and reverse-transcribed. The resulting cDNA was used as a template for qRT-PCR for primers designed using ADAMTS-4 gene sequence. Expression of ADAMTS-4 was analysed, as well as the house keeping gene GAPDH to normalize expresⅡon between different samples relative mRNA levels of ADAMTS-4 were determined using the fomula 2-△ΔCt. The conditioned media were concentrated to SDS–PAGE under reduction and immunoblotted with anti-ADAMTS-4 antibody.the development of the blots was carried out using the ECL plus chemiluminescence substrate.After initial immune, the behind paws and joint were redness and swelling on the afterday, and then fadeaway gradually. After the boosted immune the whole paws and joint were swere inflammation. The disease incidence in simply immune-induced group was 80%; in immune-induced and cold wetness-stimulated group 85%. The solvent control group had no sequence inflammation.the levels of anticollagen antibody TNF-α; IL-1βand IL-17 in rat serum increased, and the difference were significece compared with control group (P<0.01).Rat ankle joint cavity of tissue sections in control group can be seen clearly; proliferation of synovial cell and chondrocyte in treated group can be seen. Inflammatory cell infiltrated into the joint cavity and lymphocyte infiltrated into the synovium tissue. Radiographs demonstrate marginal erosion, blurred joint and cartilage damage.After primary chondrocytes inoculation, under microscope, a large number of round cells in suspension, and had strong refraction, adherent cell Adherent cell body were flat, triangle or polygon, monolayer and mixed together gradually.the passage chondrocyte adherented completely within 24h. After freeze and recovery, the rate of living chondrocyte was 87%.All chondrocyte was found to express mRNA for ADAMTS-4. ADAMTS-4 in 20h and 48h group showed a statistically significant increase (P<0.01) in mRNA expression levels following induction of IL-17, respectively, 6.37 and 8.62 folds. The protein of ADAMTS-4 in chondrocyte showed the positive result using the western blotting.This research has prepared succeedfully the rheumatoid arthritis disease model with the following evaluation target: clinical symptom; arthritis index; the level of anticollagen antibody; TNF-α; IL-1βand IL-17; histopathology and radiological examination. The further research indicated that the IL-17 can induce the expression increase of the destructive enzyme-ADAMTS-4 and play an important role in the approaching destruction of cartilage matrix in rheumatoid arthritis. IL-17 could be the theruapeutic target.In the animal desease model, the level of IL-1β、IL-17 increased significantly compared to the control group. Consideration the environment was complex in vivo desease model, chondrocytes were cultured and induced with IL-17 in vitro in order to avoid the interferers of many factor. The results indicated directly that IL-17 can induce the chondrocyte expression the ADAMTS-4, which degradate the extra cell matrix in cartilage of local joint and resultly the abnormity of joint and even crippledom happened. This illustrate that blocking the action of Il-17 in RA promptly will treat effectly the symptom of rheumatoid arthritis.更多还原