【作者】 黄学应；

【导师】 陈飞虎；

【作者基本信息】 安徽医科大学 ， 药理学， 2008， 博士

【摘要】 类风湿性关节炎（rheumatoid arthritis,RA）是一种慢性自身免疫性疾病,在世界范围内平均患病率约1%,我国患病率约4‰;其突出的临床表现为关节肿、痛、僵硬和畸形;目前尚无有效的治疗方法,现行治疗的目标主要是减轻症状,预防关节破坏及功能丧失,提高生活质量。RA发病机制未明,其主要病理特征为成纤维样滑膜细胞（fibroblast-like synoviocytes,FLS）过度增殖、炎性细胞浸润及新生血管形成等;增生的滑膜组织构成血管翳侵蚀关节软骨和骨,造成关节进行性破坏、畸形和功能丧失等。因此,滑膜增生对类风湿性滑膜炎的发展起着重要作用。近期研究表明,细胞增殖与细胞死亡关系密切,RA FLS增殖与死亡也许存在不平衡,进而导致滑膜增生。因此,抑制RA FLS增殖,诱导其凋亡进而降低滑膜增生可成为治疗RA有效策略之一。弗氏完全佐剂（Freund’s complete adjuvant,FCA）诱导的佐剂性关节炎（adjuvantarthritis,AA）是一种实验性关节炎模型,其关节病理改变及细胞和体液免疫与RA相似,可用作评价RA治疗的测试系统。内抑素（endostatin）是一种强效内源性血管生成抑制因子,分子量为20kD左右,是胶原ⅩⅧ的降解产物,其抗血管生成活性机制可能是特异性抑制血管内皮细胞粘附、迁移和增殖,诱导其凋亡。近期研究证实,内抑素不仅可通过抑制内皮细胞功能（即抑制肿瘤新生血管形成）,而且还可通过直接抑制肿瘤细胞迁移、增殖及诱导其凋亡而发挥抗肿瘤效应。FLS是RA病理改变的最终靶细胞,其增殖活跃,具有肿瘤转化细胞的特征,RA滑膜组织类似于局限性侵袭生长的肿瘤,提示治疗肿瘤性疾病的途径可用于治疗RA,包括凋亡诱导因子和抗血管生成因子。目前有关endostatin的研究主要集中于抗肿瘤活性与机制等,而对其它病理性血管形成如慢性炎症性血管生成的影响国内外报道很少;以FLS为靶点,探讨endostatin对RA的作用及机制国内外尚未见报道。我们前期研究表明,腹部皮下注射重组人内抑素(recombinant human endostatin,rhEndostatin;(1.25,2.5,5.0 mg·kg^-1·d^-1,×7d)对大鼠AA有治疗作用,可明显减轻AA大鼠足爪肿胀程度,降低滑膜组织血管内皮（细胞）生长因子（vascular endothelial growth factor,VEGF）表达,减小滑膜微血管密度（microvessel density,MVD）。进一步研究证实,rhEndostatin（3.125,6.25,12.5,25,50μg/ml）可直接抑制AA FLS增殖并诱导其凋亡,但具体作用机制不清楚。本研究正是在此基础上进一步探讨rhEndostain对AA FLS作用的潜在机制,为RA治疗及寻找其治疗新靶点提供实验依据。目的:①观察rhEndostatin对AA FLS细胞周期的影响;②观察rhEndostatin对AA FLS细胞周期相关基因p53,p21,CDK₄,cyclinD₁,PCNAmRNA和CyclinD₁,PCNA蛋白表达的影响;③观察rhEndostatin对AA FLS凋亡相关基因fas,bcl-2,c-fos,c-jun mRNA及c-Jun,NFκB,Caspase-3蛋白表达的影响;④观察rhEndostatin对AA FLS胞质游离钙(ionized free calcium,Ca²⁺)的影响。探讨rhEndostatin抑制AA FLS增殖,促进其凋亡的分子及离子机制,为RA药物治疗及寻找其治疗新靶点提供实验依据。方法:①AA大鼠模型制备:雄性SD大鼠,体质量140-160g,安徽医科大学实验动物中心提供,后足趾皮内注射FCA制备AA大鼠模型。②滑膜组织准备:大鼠随机均分为正常组、AA模型组、rhEndostatin（2.5mg/kg）治疗组和甲氨蝶呤(methotrexate,MTX;（1mg/kg）治疗对照组;后三组造模后第10-16天,rhEndostatin治疗组大鼠腹部皮下注射rhEndostatin(2.5 mg·kg^-1·d^-1),MTX组大鼠腹部皮下注射MTX（1 mg/kg twice weekly）,AA模型组及正常组大鼠给予等剂量的注射用水;第28天处死大鼠,取出滑膜层组织。③FLS准备:应用正常和AA大鼠膝关节新鲜滑膜组织进行细胞培养,实验用传2代培养的滑膜细胞,通过流式细胞仪（flow cytometry,FCM）检测AA大鼠滑膜细胞血管细胞粘附分子-1（vascular cell adhesion molecule-1,VCAM-1）表达情况鉴定FLS。④FLS细胞周期检测:传1代培养的正常和AA大鼠滑膜细胞接种于6孔培养板中,AA大鼠滑膜细胞分设AA模型组、rhEndostatin（50μg/ml）治疗组、MTX（1μg/ml）治疗组,用无血清的RPMI-1640培养液培养24h,使细胞周期同步化,继之换上含20%血清的RPMI-1640培养液,并相应加入rhEndostatin（终浓度50μg/ml）和MTX（终浓度1μg/ml）,继续培养48h后收集细胞,FCM检测细胞周期。⑤细胞周期相关基因检测:分别提取各组大鼠滑膜组织中RNA、蛋白质;实时荧光定量PCR法检测p53,p21,cyclinD₁,PCNA,CDK₄ mRNA表达;Western Blot法检测PCNA,CyclinD₁蛋白表达。⑥凋亡相关基因检测:实时荧光定量PCR法检测各组大鼠滑膜组织中fas,bcl-2,c-fos,c-jun mRNA表达;Western Blot法检测c-Jun,NFκB,Caspase-3蛋白表达。⑦AA FLS胞质Ca²⁺检测:Fluo-3/AM作为Ca²⁺指示剂,终浓度为50μg/ml rh-Endostatin灌流AA FLS,同时用激光共聚焦显微镜（confocal laser scanningmicroscope,CLSM）动态观察、记录AA FLS在有Ca²⁺液和无Ca²⁺液中胞质Ca²⁺荧光强度（fluorescence intensity,FI）变化,检测rhEndostatin对AA FLS胞质Ca²⁺浓度(cytosolic free calcium concentration,[Ca²⁺]_i)的影响。结果:①rhEndostatin对大鼠FLS细胞周期的影响:FCM检测和分析结果显示,正常组大鼠FLS细胞周期主要停留在G₀/G₁期,比例为68.3%,S期和G₂/M期细胞较少,分别为23.65%,6.32%;与正常组相比,AA模型组大鼠FLS G₁期比例明显减少,约为10.0%,大部分细胞进入S期,为65.6%,G₂/M期细胞比例接近19.5%;加入rhEndostatin（50μg/ml）后,G₁期细胞由AA模型组10.0%增至74.1%,而S期和G₂/M期细胞比例显著减少,分别为12.6%和4.15%。②rhEndostatin对AA大鼠滑膜组织中细胞周期相关基因表达的影响:实时荧光定量PCR检测结果显示,rhEndostatin（2.5 mg/kg）能显著降低AA大鼠滑膜组织中p53,p21,cyclinD₁和PCNA mRNA表达水平（P＜0.01）,增加CDK₄ mRNA表达（P＜0.01）。Western Blot检测证实,rhEndostatin治疗组大鼠滑膜组织中PCNA和CyclinD₁蛋白表达较AA模型组显著降低（P＜0.01）。③rhEndostatin对AA大鼠滑膜组织中凋亡相关基因表达的影响:实时荧光定量PCR分析结果表明,rhEndostatin（2.5mg/kg）能明显提高AA大鼠滑膜组织中fas,bcl-2,c-fos和c-jun mRNA表达水平（P＜0.01）。Western Blot检测进一步证实,应用rhEndostatin治疗后,大鼠滑膜组织中43kD和48kD c-Jun蛋白和Caspase-3 p20表达水平较AA模型组显著提高（P＜0.01）;但NFκB p65表达较AA模型组增加不明显（P＞0.05）。④rhEndostatin对AA FLS[Ca²⁺]_i的影响:CLSM检测结果显示,当细胞处于无Ca²⁺液中时,rhEndostatin（50μg/ml）不能引起AA FLS胞质Ca²⁺ FI的改变。当细胞外缓冲液为有Ca²⁺液时,终浓度为50μg/ml rhEndostatin灌流AA FLS可使胞质Ca²⁺FI急剧增加达峰值,随即荧光开始减弱,FI随时间缓慢下降。结论:①rhEndostatin抑制AA FLS增殖与其引起AA FLS细胞周期G₁期阻滞有关,其分子生物学基础可能是rhEndostatin抑制AA FLS CyclinD₁和PCNA的表达,并非依赖P53-P21-CDK-Cyclin经典途径。②rhEndostatin促进AA FLS凋亡与其增加AA FLS fas,c-fos,c-jun,caspase-3基因表达与活化有关,该凋亡过程不依赖于bcl-2表达降低,也不能被bcl-2表达增加和NFkB活化所抑制。③rhEndostatin可引起AA FLS胞外Ca²⁺内流,钙稳态失衡是rhEndostatin诱发AAFLS损伤的一个早期的事件,细胞内钙超载可能作为AA FLS凋亡的一个重要始动环节。更多还原

【Abstract】 Rheumatoid arthritis（RA） is a chronic autoimmune disease affecting approximately 1% and 0.4%of the population worldwide and nationwide,respectively.RA presents clinically with joint swelling,pain,stiffness,deformity and is not healed.The major goals of treatment of RA are to reduce pain and discomfort,to prevent joint destruction and loss of joint function,and to maintain a productive and active lifestyle.Up to date, the mechanisms that contribute to the pathogenesis of RA are unknown.The affected RA joint is characterized by pronounced synovial hyperplasia composed of extensive proliferation of fibroblast-like synoviocytes（FLS） and inflammatory cell infiltration with neovascularization.Pannus tissue comprising the hyperplastic synovium leads to progressive joint destruction,deformity and functional incapacitation,which is manifested as bone erosion and cartilage destruction.Thus,the synovial hyperplasia has a critical role in the propagation of rheumatoid synovitis.It is generally accepted that there is a close relationship between cell proliferation and cell death,and that there may be an imbalance between the proliferation and death of FLS in RA,which leads to synovial hyperplasia.For this reason,the interruption of proliferation and induction of apoptosis in RA FLS have been proposed as one of the promising strategies for treating RA by way of reducing synovial hyperplasia in situ.Adjuvant arthritis（AA） is a model of experimental arthritis that is induced by injection of Freund’s complete adjuvant（FCA）.The similarities between the joint pathologies as well as the cellular and humoral immunities in AA and RA suggest that AA is a relevant animal model that acts as a useful test system for evaluating treatment of RA. Endostatin,a 20kD fragment of collagen XVIII,is a member of a group of powerfully endogenous antiangiogenic proteins that are activated by proteolytic processing.In fact, the antiangiogenic activity of endostatin is speculated to be specifically mediated by the inhibition of endothelial cell adhesion,migration and proliferation,and induction of apoptosis.Recently,endostatin has been studied not only for its inhibitory effects on vascular endothelial cell function but also its direct antitumor effects on cancer cell migration and proliferation,and induction of apoptosis.Fibroblast-like synoviocytes are the ultimate target cells of the pathologic changes in RA,also,they exhibit pre-neoplastic characteristic of transformed cells with active proliferation.The locally invasive tumourlike properties of RA synovial tissues suggest that approaches normally reserved for oncologic diseases might be useful in RA,including induction of apoptosis and antiangiogenesis agents.Studies on endostatin are mainly involved in its effects and mechanisms on anti-tumor now.There is few report on the effect of endostatin on upregulated angiogenesis in certain pathological conditions such as chronic inflammation. It is no reported that rhEndostatin has therapeutic effect on RA by targeting FLS. Previous studies have shown that subcutaneous administrations of recombinant human endostatin(rhEndostatin;(1.25,2.5,5.0 mg·kg^-1·d^-1,×7d) to the abdomen have therapeutic effect on AA rats,which is characterized by significantly inhibition of paw swelling, reduction of microvessel density（MVD） and the expression of vascular endothelial growth factor（VEGF） in the synovium of AA rats.Also,rhEndostatin（3.125,6.25,12.5, 25,50μg/ml） can directly have influence on AA FLS such as inhibition of proliferation and induction of apoptosis.However,the mechanisms of effects of rhEndostatin on AA FLS in rats remain unclear.The aim of the current study was to understand the potential mechanisms,and to provide a experimental data for the development of new therapeutic interventions and targets of RA.Objective:To clarify the molecular and ionic mechanisms of effect of rhEndostatin on AA FLS such as inhibition of proliferation and induction of apoptosis,and to provide a experimental data for the development of new therapeutic interventions and targets of RA,we investigated①the effect of rhEndostatin on cell cycle of AA FLS,②the effect of rhEndostatin on the expression of the cell cycle regulatory genes such as p53,p21, CDK4,cyclinD₁,PCNA mRNA and CyclinD₁,PCNA protein in AA FLS,③the effect of rhEndostatin on the expression of the apoptosis regulatory genes such as fas,bcl-2, c-fos,c-jun mRNA and c-Jun,NFr,B,Caspase-3 protein in AA FLS,and④the effect of rhEndostatin on the cytosolic ionized free calcium(Ca²⁺) in AA FLS.Methods:①Induction of the AA rat model:Male Sprague-Dawley（SD） rats weighing 140-160g were obtained from the Animal Center of Anhui Medical University.Rats were immunized on day 0 by intradermal injection of a 0.1-ml aliquot of FCA into the left hind paw.②Preparation of synovial tissues:The rats were randomly divided into normal,AA control,rhEndostatin（2.5mg/kg）-treated and methotrexate(MTX;（1mg/kg）-treated groups,and immunized to induce AA rat model in the groups except normal one. Subcutaneous administrations of rhEndostatin(2.5mg·kg^-1·d^-1) and MTX（1mg/kg twice weekly） to the abdomen were started on day 10 post-immunization and continued until day 16 post-immunization.Normal and AA control group received the same volumes of phosphate-buffered saline（PBS）.On day 28,the animals were sacrificed and the synovial tissues were promptly removed for subsequent experiments.③Preparation of FLS:Synoviocytes were isolated from knee joints of the normal and AA rats,and cultured under permissive conditions.The synoviocytes were used in experiments from the second subculturing ones.The cells obtained from AA rats were identified by their expressed vascular cell adhesion molecule-1（VCAM-1） as evaluated by flow cytometry（FCM） analysis.④Cell Cycle Analysis of FLS:The first subculturing synoviocytes obtained from normal and AA rats were plated in a 6-well plate.AA synoviocytes were divided into AA control,rhEndostatin（50μg/ml）-treated and MTX（1μg/ml）-treated groups.For synchronization,the cells were harvested in serum-free RPMI 1640 medium for 24h at 37℃.The cells were then placed in RPMI 1640 medium containing 20%（v/v） fetal calf serum（FCS）.For rhEndostatin-treated and MTX-treated groups,the cells were treated with the designated doses of agents for 48 h at 37℃.The cells were collected and analysed by FCM.⑤Detection of cell cycle regulatory genes:Total RNA and protein was extracted from each frozen synovial tissue sample obtained from rats in various groups.The expression of p53,p21,cyclinD₁,PCNA,CDK₄ mRNA was quantified by real-time fluorescent quantitative polymerase chain reaction（RT-FQ-PCR）.The expression of PCNA, CyclinD₁ proteins was detected by westem blot analysis.⑥Detection of apoptosis regulatory genes:The expression of fas,bcl-2,c-fos,c-jun mRNA in synovial tissue sample obtained from rats in various groups was quantified by RT-FQ-PCR.The expression of c-Jun,NFκB,Caspase-3 proteins was detected by westem blot analysis.⑦Measurements of the cytosolic Ca²⁺ in AA FLS:The cultured AA FLS were loaded with the calcium ion-sensitive fluorescent indicator fluo-3/AM,and perfused with rhEndostatin at the concentration of 50μg/ml.At the same time a confocal laser scanning microscope（CLSM） was applied to measure changes of cytosolic Ca²⁺ fluorescence intensity（FI） in AA FLS when cells were immersed in the buffer with or without Ca²⁺,and to investigate the effect of rhEndostatin on cytosolic free calcium concentration([Ca²⁺]_i) in AA FLS.Results:①Effect of rhEndostatin on cell cycle of FLS:Flow cytometric analysis indicated that the cell cycle progression of FLS obtained from normal rats was arrested at G1 phase.The percentages of cells in G₀/G₁ phase,S phase,G₂/M phase were 68.3%, 23.65%,6.32%,respectively.The rate of AA FLS in G1 phase（10.0%） was significantly lower than that in normal control group.The cell cycle progression of AA FLS was mostly in S phase.The percentages of cells in S phase,G₂/M phase were 65.6%,19.5%, respectively.Recombinant human endostatin（50μg/ml） significantly increased G₁ phase cells as compared with those of AA FLS（74.1%versus 10.0%）.The percentages of cells in S phase,G₂/M phase（12.6%,4.15%,） were significantly decreased compared with AA control group.②Effect of rhEndostatin on the expression of the cell cycle regulatory genes in synovial tissues obtained from AA rats:Analysis by RT-FQ-PCR showed that rhEndostatin（2.5mg/kg） could significantly decrease the expression of p53,p21, cyclinD₁,PCNA mRNA and increase the expression of CDK₄ mRNA in synovial tissues obtained from AA rats（P＜0.01）.Westem Blot analysis indicated that the expression of PCNA and CyclinD₁ protein in synovial tissues treated with rhEndostatin were significantly decreased than that in AA control group（P＜0.01）.③Effect of rhEndostatin on the expression of the apoptosis regulatory genes in synovial tissues obtained from AA rats:Real-time PCR analysis indicated that rhEndostatin（2.5mg/kg） could significantly increase the expression level of fas,bcl-2, c-fos and c-jun mRNA in synovial tissues obtained from AA rats（P＜0.01）.The results of Western Blot analysis indicated that the expression of c-Jun protein（43kD,48kD） and Caspase-3 p20 in synovial tissues treated with rhEndostatin were significantly increased compared with that in AA control group（P＜0.01）.But rhEndostatin could not significantly improve the expression of NFκB p65 in synovial tissues obtained from AA rats（P＞0.05）.④Effect of rhEndostatin on[Ca²⁺]_i in AA FLS:Analysis by CLSM showed that rhEndostatin（50μg/ml） did not change cytosolic Ca²⁺ FI in AA FLS when cells were immersed in non-calcium extracelluar solution.When cells were immersed in the buffer with Ca²⁺ and perfused with rhEndostatin at the concentration of 50μg/ml,the cytosolic Ca²⁺ FI in AA FLS was rapidly increased to maximum and then gradually declined.Conclution:①Suppression of CyclinD₁ and PCNA expression by rhEndostatin can produce a virtual inhibition of S-phase entry and arrest the cell cycle progression of AA FLS in G₁ phase,which induces inhibition of AA FLS proliferation and is not associated with the expression of P53,P21,CDK₄.②Apoptosis induced by rhEndostatin in AA FLS is strongly associated with the increased expression and activation of fas,c-fos,c-jun,caspase-3,but not bcl-2,NFκB in AA FLS.③Recombinant human endostatin can elicit the extracellular calcium influx in intact AA FLS.Imbalance of Ca²⁺ steady state induced by rhEndostatin is the very early events occur prior to AA FLS damage.Cytoplasmic Ca²⁺ overload may occur prior to any other molecular events of apoptosis in AA FLS.更多还原