【作者】 张维丽；

【导师】 陈如泉；

【作者基本信息】 湖北中医学院 ， 中医内科， 2009， 博士

【摘要】 目的本课题通过观察散结消瘿方及温肾健脾方对自身免疫性甲状腺炎大鼠模型进行干预、治疗,深入探讨散结消瘿方及温肾健脾方对自身免疫性甲状腺炎的治疗作用,从细胞凋亡及免疫应答、细胞炎性因子方面进一步明确其发病机制,并从中医“痰瘀”及“脾肾亏虚”的角度来探讨自身免疫性甲状腺炎的中医病机,进一步加深对自身免疫性甲状腺炎免疫学发病机理及中医病机的认识,为其治疗提供新的思路。方法将健康的雌性Wistar大鼠90只,体重约120±20g,购回后适应性饲养1周,随机分为6组:正常组(G1):15只;模型组(G2):15只;雷公藤组(G3):15只;散结消瘿方组(G4):15只;温肾健脾方组(G5):15只;散结消瘿方及温肾健脾方联合治疗组(G6):15只。G1组给予普通饲料饲养;G2—G6组造模:将甲状腺球蛋白100mg完全溶解于双蒸水50ml中,取10ml与等体积完全弗化剂充分混合成油包水状,取0.5ml在背、腹腔、腿、颈全身等多点皮下注射作为初次免疫,第2周取甲状腺球蛋白10ml与不完全弗化剂10ml充分混合后作为加强免疫,以后每周一次,至第5周,期间配合高碘水(1L水中加入0.64g碘化钠配制)饮水喂养;第6周开始,G2组给予普通饲料及饮水饲养至9周;G3组给予雷公藤片1.08mg/kg.d灌胃至10周;G4组给予散结消瘿汤20g/kg.d灌胃至10周;G5组给予温肾健脾方组20g/kg.d灌胃至10周;G6组给予散结消瘿汤及温肾健脾联合方20g/kg.d灌胃至10周。第11周将大鼠处死,取血并取出甲状腺组织,做HE染色光镜下观察;TGAb、TpoAb、FT3、FT4、TSH按照试剂盒要求检测;肝功能、肾功能、血液分析做生化及计数分析;TNF-α、IL-6用ELISA结果1.模型组血清TGAb和TPOAb较正常组升高,具有显著性差异(P<0.01)。雷公藤治疗组TGAb和TPOAb较模型组降低,有显著性差异(P<0.05);散结消瘿方组及联合方治疗组TGAb和TPOAb较模型组降低,有显著性差异(P<0.01);散结消瘿方组及联合方治疗组TGAb和TPOAb较雷公藤治疗组比较无统计学差异(P>0.05);温肾健脾方组TGAb和TPOAb较模型组差异无统计学意义(P>0.05);散结消瘿方及联合方治疗组组间TGAb和TPOAb比较无统计学意义(P>0.05)。光镜结构:正常组见完整滤泡上皮细胞,背景胶质丰富,呈浓稠片7状分布,滤泡间血管丰富,未见巨噬细胞和淋巴细胞,上皮细胞未见嗜酸性变。模型组甲状腺组织间质内可见广泛的淋巴细胞浸润,甲状腺上皮细胞呈明显的嗜酸性改变,并见淋巴组织分布于小叶内及小叶周边,形成具有生发中心的淋巴滤泡。雷公藤组、散结消瘿方组、温肾健脾方组及联合方治疗组甲状腺组织间质内淋巴浸润较模型组明显减少,小叶内及小叶周边形成具有生发中心的淋巴滤泡较模型组也明显减少。淋巴细胞主要浸润范围及程度较模型组也有所减少,散结消瘿方组及联合方治疗组淋巴细胞浸润程度较雷公藤组及温肾健脾方组明显减少,范围也明显变小,滤泡上皮细胞嗜酸性变较雷公藤组及温肾健脾方组变浅,未见明显淋巴细胞形成的生发中心。2.模型组淋巴细胞浸润程度较正常组明显增多,具有显著性差异(P<0.05)。雷公藤治疗组、温肾健脾方组淋巴细胞浸润程度较模型组显著降低,范围明显缩小,有显著性差异(P<0.05);散结消瘿方组及联合方治疗组淋巴细胞浸润程度较模型组显著降低,范围明显缩小,有显著性差异(P<0.01);散结消瘿方组、联合方治疗组较雷公藤治疗组淋巴细胞浸润程度有所减少,有显著性差异(P<0.05)。温肾健脾方组治疗组淋巴细胞浸润程度较雷公藤治疗组差异无统计学意义(P>0.05)。3.甲状腺功能:TSH含量正常组、模型组、雷公藤治疗组、散结消瘿方组、温肾健脾方组及联合方治疗组间无显著性差异(P>0.05);FT3、FT4含量正常组、模型组、雷公藤治疗组、散结消瘿方组间无差异(P>0.05);温肾健脾方组及联合方治疗组FT3、FT4较模型组有显著性差异(P<0.05)。4.造模前两周模型组与正常组体重、进食量及排泄量无明显差异,从第三周起,模型组大鼠体重增加较正常组明显减缓,进食量相对减少,活动减少,精神欠佳,大便干结,体重、进食量及排泄量明显减少,差异有统计学意义(P<0.05)。用药干预后可见温肾健脾方组较模型组、雷公藤治疗组、散结消瘿方组及联合方治疗组大鼠体重增幅明显增大,饮食量、大便量明显增多,活动较其他组频繁。可以看到雷公藤治疗组大鼠食欲减退、活动量少考虑与其胃肠系统副作用有关。模型组体温较正常组明显降低,具有显著性差异(P<0.01)。雷公藤治疗组、散结消瘿方组体温较模型组明无统计学差异(P>0.05);温肾健脾方组、联合方治疗组较其他组体温有所上升,差异有统计学意义(P<0.05)。同时我们可以明显观察到温肾健脾方组、散结消瘿方组及联合方治疗组大鼠活动及精神状态明显好于模型组。5.模型组与正常组肝功能、血白细胞、红细胞、血红蛋白及血小板、肌酐、尿素氮无统计学差异(P>0.05);雷公藤治疗组AST、肾功能、血白细胞、红细胞较正常组有显著性差异(P<0.05);散结消瘿方组、温肾健脾方组及联合方治疗组AST、肾功能、血白细胞、红细胞较雷公藤治疗组有显著性差异(P<0.05);散结消瘿方组、温肾健脾方组及联合方治疗组间无统计学差异(P>0.05)。6.正常组甲状腺组织Fas和FasL免疫阳性物呈浅棕色,在甲状腺上皮细胞表达很少,范围很小,颜色较浅;模型组甲状腺上皮细胞表达Fas和FasL免疫阳性物呈深棕色,表达范围广,颜色深,其OD值与正常组相比有统计学差异(P<0.01)。散节消瘿方组、联合方治疗组较雷公藤治疗组甲状腺上皮细胞表达Fas和FasL颜色显著变浅,OD值有显著性差异(P<0.05)。温肾健脾方组较雷公藤治疗组OD值无统计学差异(P>0.05);散结消瘿方组、联合方治疗组间甲状腺上皮细胞表达Fas和FasL差异无统计学意义(P>0.05)。7.正常组甲状腺上皮细胞表达CD40免疫阳性物呈浅棕红色,表达范围很小;模型组甲状腺上皮细胞表达CD40免疫阳性物呈棕红色,表达范围广,颜色深,其OD值与正常组相比有统计学差异(P<0.01);散节消瘿方组、联合方治疗组甲状腺上皮细胞表达CD40较雷公藤治疗组颜色明显变浅,OD值有显著性差异(P<0.05)。温肾健脾方组较雷公藤治疗组甲状腺上皮细胞表达CD40无统计学差异(P>0.05)。散结消瘿方组、联合方治疗组间甲状腺上皮细胞表达CD40差异无统计学意义(P>0.05)。8.模型组血清TNF-α含量较正常组升高,具有显著性差异(P<0.01)。雷公藤治疗组、散结消瘿方组、联合方治疗组及温肾健脾方组血清TNF-α较模型组降低,有显著性差异(P<0.01);散结消瘿方组及联合方治疗组血清TNF-α含量较雷公藤治疗组及温肾健脾方组降低,具有显著性差异(P<0.01);散结消瘿方组、联合方治疗组间血清TNF-α,差异无统计学意义(P>0.05)。9.模型组血清IL-6含量较正常组升高,具有显著性差异(P<0.01)。雷公藤治疗组、散结消瘿方组、联合方治疗组及温肾健脾方组血清IL-6较模型组降低,有显著性差异(P<0.01);散结消瘿方组及联合方治疗组血清IL-6含量较雷公藤治疗组及温肾健脾方组降低,具有显著性差异(P<0.01);散结消瘿方组、联合方治疗组间血清IL-6,差异无统计学意义(P>0.05)。结论1.散结消瘿方可以减少自身免疫性甲状腺炎自身抗体TGAb和TPOAb的形成,减少自身抗体对甲状腺组织的损伤。2.散结消瘿方及温肾健脾方都能减少自身免疫性甲状腺炎淋巴细胞的浸润,减轻其炎性反应,可以缩小肿大的甲状腺,但散结消瘿方疗效尤其显著,优于雷公藤及温肾健脾方。3.温肾健脾方可以增加大鼠食欲,进食量及排泄量增大,升高大鼠基础体温,增强甲状腺功能,并使自身免疫性甲状腺炎大鼠基础代谢增加,温肾健脾方与雷公藤相比较,可避免其胃肠反应,有效改善桥本氏甲状腺炎的临床症状。对改善自身免疫性甲状腺炎表现出的甲状腺功能减退症状及调节甲状腺功能效果明显。治疗效果优于雷公藤片及散结消瘿方。4.散结消瘿方、温肾健脾方对大鼠肝、肾功能及血液系统有保护作用,优于雷公藤。5.散结消瘿方、温肾健脾方都能抑制TNF-α、IL-6过度分泌,减轻炎性因子对甲状腺的损伤,但散结消瘿方效果更为明显,优于雷公藤片及温肾健脾方。6.作为第二活化信号的共刺激分子CD40在自身免疫性甲状腺炎中有异常表达,说明CD40与自身免疫性甲状腺炎的发病有关,可以通过Th2途径致病,同时散结消瘿方、温肾健脾方通过减少CD40的表达,减少T细胞的过度活化,减轻免疫应答。7.甲状腺上皮细胞凋亡机制是引起自身免疫性甲状腺炎发病的机制之一。散结消瘿方、温肾健脾方能有效抑制甲状腺上皮细胞的凋亡,逆转甲状腺炎的发生。更多还原

【Abstract】 ObjectiveTo further study the therapeutic effects of eliminating goiter recipe and formular wenshenjianpi and formular sanjie-xiaoying by using them for intervening and curing model rats of autoimmune thyroiditis and to define its pathogenesis from the aspects of apoptosis,immunoreaction and cytokines,and to discuss the traditional Chinese medicincal pathogenesis of autoimmune thyroiditis from traditional Chinese medicincal view of phlegm-blood stasis and deficiency of spleen and kidney with the expectations of deepening the knowledge of the pathogenesis and traditional Chinese medical pathogenesis of autoimmune thyroiditis immunology and pioneering new thinking for it.Methods:90 healthy female Wistar rats fed for one week（weight:120±20g） were randomly divided into 6 groups:normal group（G1） 15;model group（G2）:15;tripterygiumforrestii and thyroid tablets treatment group（G3）:15;formular sanjiexiaoying group（G4）:15;formular wenshenjianpi group（G5）:15;combined group of formular sanjiexiaoying and formular wenshenjianpi （G6）:15.G1 with common feed;G2 - G6 treatment:100mg pig’ s TG was completely dissolved in 50ml double distilled water, and 10ml was thoroughly mixed with same volume isovolumetic complete freund’s adjuvant into water in oil type,and gave subcutaneous injection 0.5ml as primary immunization on back, cavum abdominis,legs and neck of rats.Two weeks later,we thoroughly mixed pig’ s TG 10ml with incomplete freund’ s adjuvant 10ml as boosting immunization once a week combined with sodium iodide to feed the rats with water until the fifth week（1L distilled water with 0.64g sodium iodide）.From the sixth week,to feed G2 with common feed and water until the tenth week;to feed G3 with 2.75g/kg.d thyroid tablets and tripterygiumforrestii tablets by gastric perfusion until the tenth week;to feed G4 using 20g/kg.d formular sanjiexiaoying by gastric perfusion until the tenth week;to feed G6 20g/kg.d formular sanjiexiaoying and formular wenshenjianpi by gastric perfusion until the tenth week.In eleventh week,the rats were killed and blood and the thyroid tissues were taken out with HE colouring and observed the structure of rat’s thyroid tissues with microscope.Hepatic、kidney function and blood-routine with reagent box’s show. TGAb,TpoAb,FT₃,FT₄,TSHwere tested by reagent box’ s show,TNF-α, IL-6were tested by ELISA,thyroid cells CD40,Fas,FasL were tested by immunohistochemistry.Results:1.Blood serum TGAb and TpoAb of model group increased and had significant difference（P < 0.01） compared with the normal group.Blood serum TGAb and TpoAb of tripterygiumforrestii group reduced and had significant statistical difference（P < 0.05 ） compared with model group;Blood serum TGAb and TpoAb of formular sanjiexiaoying group and combined treatment group reduced and had significant statistical difference（P < 0.01） compared with model group;Blood serum TGAb and TpoAb of formular sanjiexiaoying group and combined treatment group reduced and had not significant statistical difference（P> 0.05） compared with tripterygiumforrestii group;Blood serum TGAb and TpoAb between the formular sanjiexiaoying group and combined treamtent group,their differences had no statistical significance（P> 0.05）.Blood serum TGAb and TpoAb between the formular wenshenjianpi group and model group,their differences had no statistical significance（P> 0.05）.In normal group complete endolymph was soaked backgroud’s colloidwas abundant,their distributing is concentrated and flaking,among follicle blood vessel was plenty.follicular epithelium was not acidophilic changed.Folial structure of model group was kept;interstitial endolymph was soaked; follicular epithelium acidophilic changed;epithelial cells enlarged as columnar;cell volume was big;boundary was clear; caryon was big.We found the lymph tissue distributing in and around folial and formed folliculi lymphaticus having germinal center.normal group,model group,tripterygiumforrestii and thyroid tablets treatment group,formular sanjiexiaoying group, formular wenshenjianpi group,combined group of formular sanjiexiaoying and formular wenshenjianp,the soaked inters -titial endolymph was letter than model group.The degree and bound of lymph cell soaked was reduce significant.formular sanjiexiaoying group and combined group the soaked interstitial endolymph was letter than tripterygiumforrestii and thyroid tablets treatment group,formular wenshenjianpi group. And follicular epithelium acidophilic changed was more lighten than tripterygiumforrestii and thyroid tablets treatment group、formular wenshenjianpi group.We foundn’ t the lymph tissue distributing in and around folial and formed folliculi lymphaticus having germinal center.2.Lymphocyte soakage degree of model group obviously increased and had significant statistical difference（P< 0.05 ） comparing with normal group.Lymphocyte soakage degree of tripterygiumforrestii group,formular wenshenjianpi group decreased and had significant statistical difference（P< 0.05 ） compared with model group.Lymphocyte soakage degree of formular sanjiexiaoying group and combined treatment group decreased and had significant statistical difference（P< 0.01） compared with model group.Lymphocyte soakage degree of formular sanjiexiaoying group and combined treatment group reduced and had significant statistical difference（P<0.05） compared with tripterygiumforrestii group.The differences between the lymphocyte soakage degree of formular wenshenjianpi group and tripterygiumforrestii group had no statistical significance（P > 0.05 ）.3.Blood serum TSH content of normal group、model group、tripterygiumforrestii group,formular sanjiexiaoying group, formular wenshenjianpi group,combined group were no significant statistical diference（P> 0.05 ）.Blood serum FT3, FT4 content of normal group,model group,tripterygiumforrestii treatment group,formular sanjiexiaoying were no significant statistical diference（P > 0.05 ）.Blood serum FT3,FT4 content of formular wenshenjianpi group and combined group obviously decreased and had significant statistical statistical difference（P < 0.05） compared with model group.4.Before the two weeks of madding mode,the model group compaired with normal,the growth rate of rats’ weight, Appetite and excreation had no statistical significance.From the third week,the growth rate of rats’ weight,appetite and excreation of the model group’s rat had decreased compaired normal group and had significant difference（P < 0.05 ）,their activity amount were less.After intervened by medicine,compared with model group, formular wenshenjianpi group,formular sanjiexiaoying group and combined treatment group,the growth rate of rats’ weight of formular wenshenjianpi group was obviously increased,rats’ appetite and excreation also increased and their activity amount were frequent comparing with other groups.We found the rats appetite of tripterygiumforrestii group decreased and their activity amount were less might relate with the side effect of their gastrointestinal system.The rats’ tempreture of model group obviously reduced and had significant difference（P < 0.01） compared with normal group.There were no statistical differences（P > 0.05 ） of the rats’ tempreture between tripterygiumforrestii group,formular sanjiexiaoying group and model group.The animal heat of formular wenshenjianpi group and combined treatment group increased comparing with other groups,their differences had statistical significance（P < 0.05 ）.Also we found the rats’ activity and mental state of formular wenshenjianpi group, formular sanjiexiaoying group and combined treatment group better than model group.5.The hepatic function between model group and normal group had no statistical differences（P> 0.05）.AST,BUN,Cr,WBC, RBC of tripterygiumforrestii group had significant difference （P< 0.05） compared with normal group.AST,BUN,Cr,WBC,RBC of formular wenshenjianpi group,formular sanjiexiaoying group and combined cure group had significant difference（P < 0.05） compared with tripterygiumforrestii group.Among f formular wenshenjianpi group,formular sanjiexiaoying group and combined cure group,AST,BUN,Cr,WBC,RBC had no statistical differences （P>0.05）.6.Fas/FasL immune positive substance of normal group presented light brown-red color,the expression ranges were very small.Fas/FasL immune positive of model group presented brown-red color,high concentration and the expression ranges were extended,compared with normal group and OD value had significant differences（P<0.01）The color of tripterygiumforrestii group,formular sanjiexiaoying group,formular wenshenjianpi group and combined treatment group obviously faded and the expression ranges of them became smaller.The color of formular sanjiexiaoying group and combined treatment group faded the most observably compared with tripterygiumforrestii group and OD value had significant differences（P <0.05）.OD value between formular wenshenjianpi group and tripterygiumforrestii group had no statistical differences（P > 0.05）.OD value between formular sanjiexiaoying group and combined treatment group had no significant statistical difference（P>0.05）.7.CD40 immune positive substance of normal group presented light brown-red color,the expression spectum was very small. CD40 immune positive substance of model group presented brown-red color,high concentration and the expression spectum was extended,compared with tripterygiumforrestii group and OD value had significant differences（P < 0.01）.The color of tripterygiumforrestii group,formular sanjiexiaoying group,for-mularwenshenjianpi group and combined treatment group obviously faded and the expression spectum of them became smaller.The color of formular sanjiexiaoying group and combined treatment group faded the most observably compared with tripterygium-forrestii group and OD value had significant differences（P <0.05）.OD value between formular wenshenjianpi group and tripterygiumforrestii group had no statistical differences（P > 0.05）.OD value between formular sanjiexiaoying group and combined treatment group had no significant statistical difference（P> 0.05）.8.We found blood serum TNF-αcontent of normal group was very small,blood serum TNF-αcontent of model group were more increased than normal group and had significant statistical differences（P < 0.01）.After treatment,blood serum TNF-α、IL-6 content of formular sanjiexiaoying group,combined treatment group,tripterygiumforrestii group,formular wenshenjianpi,were more decreased than model group and had significant statistical differences（P< 0.01）;among which, blood serum TNF-αcontent of formular sanjiexiaoying group and combined group were more decreased than tripterygiumforrestii group and formular wenshenjianpi and had significant statistical differences（P < 0.01）;blood serum TNF-αcontent of formular sanjiexiaoying group,combined treatment group had no significant statistical difference（P> 0.05）.9.We found blood serum IL-6 content of normal group was very small.blood serum IL-6 content of model group were more increased than normal group and had significant statistical differences（P < 0.01）.After treatment,blood serum IL-6 content of formular sanjiexiaoying group,combined treatment group,tripterygiumforrestii group,formular wenshenjianpi, were more decreased than model group and had significant statistical differences（P<0.01）;among which,blood serum IL-6 content of formular sanjiexiaoying group and combined group were more decreased than tripterygiumforrestii group and formular wenshenjianpi and had significant statistical differences（P < 0.01）;blood serum IL-6 content of formular sanjiexiaoying group,combined treatment group had no significant statistical difference（P> 0.05）.Conclusions:1.formular sanjiexiaoying and formular wenshenjianpi can reduce the formation of autoantibody TGAb and TPOAb of autoimmune thyroiditis,and adjust immune response.2.Dissipating and eliminating goiter recipe and formular wenshenjianpi can reduce lymphocyte soakage and lighten its’ inflammatory response.At the same time,formular sanjiexiaoying has an obviously efficacy for shortening swelled thyroid which is better than formular wenshenjianpi.Formular wenshenjianpi can increase the appetite and the excretion.The basal body temperature and basal metabolism of rats were improved.Comparing with tripterygiumforrestii,formular wenshenjianpi can avoidrats’ gastrointestinal reaction and improve the clinical symptoms of HT effectively.It has an obviously symptom of function decrease for improving rats autoimmune thyroiditis.The efficacy is better than formular sanjiexiaoying and tripterygiumforrestii.4.formular sanjiexiaoying and formular wenshenjianpi can protect rats’ hepatorenal function and hematological system which is better than tripterygiumforrestii.5.formular sanjiexiaoying and formular wenshenjianpi both can restrain TNF-α,IL-6 over secreting,it indicates that dissipating& eliminating goiter recipe is better than triptery -giumforrestii.6 The over expression of T cell commom stimulated molecule CD40 as the second activated signal was relevant ofmechanism of autoimmune thyroiditis.Autoimmune thyroiditis can be arisen through Th2 route.Formular sanjiexiaoying and formular wenshenjianpi can effectively restrain the espression of CD40, decrease T cell over activation and reverse the thyroiditis.7.The apoptosis mechanism of thyroid epithelial cells is one of reasons that cause the pathogenesis of autoimmune thyroiditis.formular sanjiexiaoying and formular wenshenjianpi can effectively restrain the apoptosis thyroid epithelial cells,decrease over activation and reverse the thyroiditis.更多还原