【作者】 刘浩；

【导师】 黄仰模；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2008， 博士

【摘要】 类风湿关节炎(Rheumatoid Arthritis,RA)是一种病因未明,以慢性、对称性、多关节炎为特点,可累及全身其它各器官组织,包括皮肤、肌肉、血管、神经、胸膜、心肌和淋巴结等的全身性、自身免疫性疾病。在疾病发展过程中,性质类似肿瘤,侵袭破坏骨关节,最终导致关节强直、畸形、功能丧失等不同程度的残疾。RA是临床常见疾病,致残率高,对人民生活质量影响很大。各地医务人员都在积极寻求其致病根源及有效治疗方案,以减轻劳动力丧失和致残,最终根治本病,造福人类。祖国医学中无类风湿关节炎的病名,据其临床表现,可归之于“痹病”、“历节病”、“风湿”、“鹤膝风”、“白虎历节”等范畴。中医认为“风寒湿三气杂至合而为痹”是基本病机。《金匮要略》创立“桂枝芍药知母汤"“乌头汤”两方用以治疗历节病,充分体现出中_医药治疗本病的优势。通痹合剂2号以南蛇藤和川续断组成,方中南蛇藤和川续断一君一臣,相须相使,共达温经络散寒,祛风除湿,补益肝肾,通络止痛之功。本方作为治疗RA的手段或工具,既是民族的也是世界的,有必要深入探讨其治疗RA的机理。在RA的发病机制中,T淋巴细胞活化,滑膜细胞增殖,炎性细胞浸润,炎症因子异常分泌,致关节滑膜过度增殖形成血管翳。滑膜炎症状持久存在,出现关节肿胀、疼痛、关节软骨破坏等等一系列临床症状,甚至多系统受累。自身抗体和一些炎症性细胞因子与RA的发生、发展密切相关,B7/CD28分子的共刺激作用参与了对自身抗体和炎性细胞因子的合成和表达调控,使其成为自身免疫性疾病发生、发展中的关键信号分子,预示着B7/CD28分子将可能成为治疗自身免疫性疾病的理想靶目标。本课题研究通痹合剂2号对胶原诱导关节炎(Collagen Induced Arthritis,CIA)动物模型共刺激分子、细胞因子的影响、通痹合剂2号对滑膜细胞、关节软骨破坏的影响以及观察其对RA患者的疗效等方面,从分子生物学角度揭示传统中医药的作用靶点和机制,系统分析通痹合剂2号治疗RA的机理,为系统化和规范化使用本方,开发治疗该病的新药,提供可靠依据。研究方法与内容一、实验部分40只雄性无特殊病原体(Specific-pathogen Free,SPF)级Wistar大鼠,体重约100g,将大鼠随机分为5组,为通痹合剂2号组、甲氨蝶呤(Methotrexate,MTX)组、雷公藤多甙(Tripterygium Wilfordii Polyglycoside,TWP)组、造模组和正常对照组,每组均为8只。适应喂养1-2天。除正常组外,每只大鼠尾根部皮下多点注射胶原蛋白0.2mg,建立CIA大鼠动物模型,造模当天开始分组给药。分别予通痹合剂2号、MTX、TWP以及生理盐水治疗36天。1、通痹合剂2号对B7/CD28作用的研究。各组大鼠腹主动脉采血,流式细胞仪检测外周血CD28/CD152表达。2、通痹合剂2号对肿瘤坏死因子α(Tumornecrosis Factor,TN-F-α)作用的研究。各组大鼠腹主动脉无菌采血2 ml,分离血清,每组均设2个复孔,采用双抗体夹心ELISA法检测。反应终止后,用酶标仪在450 nm波长处测定光密度,通过绘制标准曲线来计算标本浓度。3、切取大鼠双后足关节立即作后肢关节X光照片,摄片条件为60 kV,曝光时间0.04ms,电流7 mA,与放射科医生一起阅片,采用美国风湿病学会推荐的放射学评分标准评分,进行影像学检查和分析。4、大鼠关节X线摄片后,将其滑膜组织经4%多聚甲醛溶液固定后,石蜡包埋,切片厚度为4um,苏木素-伊红(Hematoxylin and Eosin Staining,HE)染色,光学显微镜下进行病理学检查。二、临床部分1、诊断标准:类风湿关节炎的诊断参照1987年美国风湿病学学会(AmericanCollege of Rheumatology,ACR)分类标准。2、实验用药:通痹合剂2号由广州中医药大学第一附属医院制剂室煎煮,0.625g生药/ml。南蛇藤45g,先煎3.5h,川断15g,同煎0.5h,分两次服用。3、入选的病例全部为2004年4月至2008年1月,广州中医药大学风湿免疫科门诊及住院患者60人,其中治疗组40例,对照组20例。治疗组服用通痹合剂2号水煎液一天2次,每次100ml,治疗30天;对照组口服MTXlorag,每周1次,治疗30天。记录各项观察指标,观察通痹合剂2号对类风湿关节炎患者的疗效指标的影响。4、临床疗效的观察指标:(1)生物学指标:包括性别、年龄、身高、体重。(2)安全性指标:血常规,尿常规,肝功能,肾功能。(3)疗效性指标:主要症状体征:压痛关节、肿胀关节数、晨僵(min)、双手平均握力(mmHg)、20米步行时间、健康评估问卷(Health Assessment Questionnaire,HAQ)、关节疼痛及医患病情评价10cm视觉模拟评分(Visual Analogue Scale,VAS)测试。理化检查:类风湿因子(RheumatoidFactor,RF)定量,血沉(Erythrocyte Sedimentation Rate,ESR),C反应蛋白(C-reactiveProtein,CRP)。研究结果实验部分:通痹合剂2号组、MTX组、TWP组大鼠外周血CD28表达量较正常对照组高(P＜0.01),均低于造模组(P＜0.01),通痹合剂2号、TWP组和MTX组组间无差异(P＞0.05)。通痹合剂2号组大鼠外周血CD152表达量较MTX组、TWP组低(P＜0.05),和正常对照组组间无差异(P＞0.05)。通痹合剂2号组、MTX组、TWP组及正常对照组大鼠外周血TNF-α含量组间差异无统计学意义(P＞0.05),但均低于造模组(P＜0.05)。关节滑膜、软骨病理及影像学,通痹合剂2号组、MTX组、TWP组与造模组有差异(P＜0.008),通痹合剂2号、TWP组和MTX组组间差异无显著性意义(P＞0.05)。临床部分:治疗组总有效率80%,对照组70%。两组治疗前后关节肿胀、压痛数目、晨僵时间、双手平均握力、20米步行时间、VAS评分、HAQ等均有改善,经统计学检验有显著性差异(P＜0.001或P＜0.05)。治疗组与对照组相比,除关节压痛数外,治疗组各项指标的改善与对照组相比无明显差异(P＞0.05)。治疗组治疗前后相比ESR、CRP、RF均有明显下降,经统计学处理,治疗前后有显著差异(P＜0.001或P＜0.05)。对照组治疗前后相比ESR、CRP均有明显下降,经统计学处理,治疗前后有显著差异(P＜0.001),但对于RF,则治疗前后无显著差异(P＞0.05)。治疗组与对照组相比,治疗组ESR、CRP、RF的改善与对照组相比无明显差异(P＞0.05)。结论1、通痹合剂2号对能明显改善RA患者压痛关节、肿胀关节数、晨僵、双手平均握力、20米步行时间、HAQ、VAS等症状体征,降低RA患者ESR、CRP、RF的含量,提示中药通痹合剂2号对RA活动期有良好的治疗作用。2、通痹合剂2号对RA患者的关节滑膜组织炎症病变有着良好的治疗作用。其作用机理,可能是通痹合剂2号可以下调CD28,抑制T细胞活化,调节TNF-α至生理水平,从而减少其他炎性细胞因子的生成,减轻滑膜炎症和血管翳的生成,减少纤维组织增生和关节破坏,促进组织修复。这是通痹合剂2号治疗RA的主要机理之一。3、对RA病因病机与通痹合剂2号遣方用药进行了探讨,认为RA的病机为风寒湿邪,内侵为害,痰浊瘀血,交阻骨骱,最后导致筋伤骨损。筋伤骨损是疾病发展的趋势,从而确立了RA的治法当为温经散寒,祛风除湿,补益肝肾,通络止痛。4、以祖国传统医学理论为基础,结合临床观察与实验研究,认为通痹合剂2号的配伍特点为祛风除湿,重在通络除痹;补益肝肾,以防筋伤骨损;配伍精当,效宏力专。更多还原

【Abstract】 Objective:Rheumatoid Arthritis(RA)is a disease characteristic with uncertain etiopathogenisis, chronic,symmetric and polyarthritis,which could involve in other organ and tissue,such as skin,muscle,blood vessel,nerve,membrana pleuralis,cardiac muscle and lymphoid node. RA invades and destroys the joints,which is similar to tumor,induces various arthrocleisis, anisotrophy and functional incapacitation ultimately during the development of disease.RA is a common disease with high mutilation rate.It has severe influence on quality of life. The doctors seek its source of pathopoiesis and effective therapeutic regimen actively in order to lessen labor force loss and mutilation,radical cure the RA and benefit for mankind. There is not such a disease named RA in traditional Chinese medicine.RA can be category into "Bi Bing","Lijie Bing","Fengshi","Hexifeng","Baihu Lijie" according to its clinical manifestations.It’s considered that Bi Bing was caused by wind,cold and dampness invaded into body together by the theory of traditional Chinese medicine.The Golden Cabinet created the decoction of Guizhi Shaoyao Zhimu and the decoction of Wutou to cure Lijie which sufficiently reflected dominance of treat the RA with Chinese medicine.Tongbi 2 mixture is composed by Nansheteng and Chuanxuduan which promote therapeutic effect mutually.It can warm meridians and disperse coldness,expel wind-evil and remove wetness,tonify liver and kidney,activating meridians to ease pain.As a means to treat RA, the mixture is not only belongs to China but also to the world.Thus,it’s necessary to discuss the mechanism on treat RA of Tongbi 2 mixture.T lymphocyte activation,synovial cell multiplication,inflammatory cell infiltrate, inflammatory factor anomal secretion causes synovial multiplication and forms pannus in the pathogenesy of RA.A series of clinical manifestations,such as arthrocele,arthrodynia and arthrodial cartilage damage,occur and even multisystem is involved in.Autoantibody and some inflammatory eytokine are correlated with occurrence and development of RA. B7/CD28 molecule becomes the key signal in occurrence and development of autoimmune disease since it participate in the composition and expressing regulation of autoantibody and inflammatory cytokine with its costimulation.It prognosticates B7/CD28 might be ideal target in treating autoimmune disease.The topic,which reveals effective target and mechanism of traditional Chinese medicine from the view of molecular biology,systemically analyzes mechanism of Tongbi 2 mixture on treating RA via studying its influence on co-stimulating molecule,cytokine,synovial cell,arthrodial cartilage damage and clinical effect.It offers reliable data for developing new medicine to treating RA and applying the mixture systemically and normally.Methods:Part of experimental studyingForty specific-pathogen free rats were divided randomly into normal control group,Tongbi 2 mixture-treated group,MTX-treated group,TWP-treated group and untreated group,8 rats in every group.Except the normal control group(injection with normal saline at the same dose),the other groups were raised collogen induced arthritis(CIA)model through subcutaneous injection with collagen proteinⅡ.The groups were treated respectively with normal saline、Tongbi 2 mixture、MTX tablets and TWP tablets for 36 days.1.All of rats get blood from abdominal aorta.The expression of CD28 and CD152 was detected by flow cytometer,2.All of rats get 2ml blood from abdominal aorta and separate serum.Every sample set up 2 ambi-hole.Tumor necrosis factor-αwas analyzed by enzyme linked immunosorbent assay.3.Hind limb was token X-ray photo with 60kV voltage,0.04ms expose time,7mA current. Checked and analyzed imaging with score of radiology standardization recommended by American College of Rheumatology(ACR).4.Synovium was retained by 4%polyoxymethylene,imbedded by paraffin,4um slice, stained with hematoxylin and eosin and detected pathology with optical microscope.Part of clinical research1.Diagnostic criteria.Reference the separate criteria of ACR 1987.2.Experimental medication.Tongbi 2 mixture was decocted at 0.625g dried medicinal herb per mini-liter by manufacturing laboratory of first affiliated hospital,Guangzhou University of TCM.45g Nansheteng was decocted in advance for 3.5h while with Chuanxuduan for 0.5h more.Take medicine twice a day.3.All 60 selected cases,which was separated 40 cases for treat group and 20 cases for control group,were the patients in the department of rheumatism and immunization,first affiliated hospital,Guangzhou University from Apr,2004 to Jan,2008.The treat group was treated with Tongbi 2 mixture 100ml twice a day while control group was treated with MTX 10mg once a week correspondingly for 30 days.Recorded observed index.4.Observed index of clinical effect.(1)Biological parameter include in gender,age,height and weight.(2)Safe parameter contains blood,urine routine,hepatic function and renal function.(3)Effective index.Cardinal symptoms and physical sign:number of tender and swelling joints,morning stiffness,average grasp force of both hand,walking time for 20 m, health assessment questionnaire(HAQ)and visual analogue scale(VAS).Laboratory test: rheumatoid factor(RF),erythrocyte sedimentation rate(ESR),C-reactive protein(CRP).ResultsThe expression of CD28 among drug treated groups is same(P＞0.05),while higher than normal control group(P＜0.01)and lower than untreated group(P＜0.01).The expression of CD152 on Tongbi 2 mixture treated group is lower than MTX and TWP treated group (P＜0.050),but is same to normal control group(P＞0.05).It’s same on expression of TNF-αbetween drug treated and normal control groups(P＞0.05),lower than untreated group coincidently.Synovium of joint,cartilage pathology and imaging of joint among drug treated groups is same(P＞0.05),while different from normal control group and untreated group(P＜0.008).Total effective rate of treated group is 80%,meanwhile 70%of control group.It’s significant difference with pretherapy in number of tender and swelling joints,moming stiffness,average grasp force of both hand,walking time for 20 m,HAQ and VAS (P＜0.001 or P＜0.05).Compared with control group,physical signs are same except the number of tender joints.ESR,CRP and RF are obviously lower than that of pretherapy of treated group(P＜0.001 or P＜0.05).ESR and CRP are obviously lower than that of pretherapy of control group(P＜0.001)except RF has no significant difference(P＞0.05).It has no significant difference in improving ESR,CRP and RF compared with control group (p＞0.05).Conclusion1.It cues that Tongbi 2 mixture has favorable therapeutic effect on RA since it can improve the number of tender and swelling joints,morning stiffness,average grasp force of both hand,walking time for 20 m,HAQ and VAS of RA patients.2.Tongbi 2 mixture has satisfied therapeutic effect on treating inflammatory of synovial tissue of joint.It can inhibit activation and differentiation of T lymphocytes through regulating expression of CD28/CD152,and regulate expression of TNF-αdown to physiological level,thus decrease expression of other inflammatory cytokine.It can reduce production of pannus and arthromeningitis,lessen osteolysis,promote tissue repair.That may be the major mechanisms in curing RA.3.It’s considered that the pathogenesis of RA is wind,cold and dampness which invading the body to cause phlegm and stagnant blood hinder into tendon and bones mutually,results in tendon injury and bone damage ultimately.Tendon injury and bone damage is tendency of RA.Thus,therapeutic method of RA is to warm meridians and disperse coldness,expel wind-evil and remove wetness,tonify liver and kidney,activating meridians to ease pain.4.Based on the theory of traditional Chinese medicine,it’s regarded the compatibility feature of Tongbi 2 mixture is expelling wind and remove wetness,deoppilation meridians to ease the arthralgia spasm;tonify liver and kidney to prevent tendon injury and bone damage;refined compatibility and great clinical effect.更多还原