【作者】 杨平；

【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:采用血清型小肠结肠炎耶尔森氏菌免疫纯种Wistar大鼠建立GD模型,比较清肝泻火方不同剂量之间的疗效差异,探讨清肝泻火方的组方合理性,验证GD发病与甲状腺炎症反应引起的病理损伤密不可分的假说,初步阐明GD从肝火论治的机理。为GD病辨证施治提供新思路,进一步开发新药物奠定理论基础与实验依据。方法:采用血清型小肠结肠炎耶尔森氏菌免疫纯种Wistar大鼠建立GD模型,再将造模后GD大鼠随机分为五组:1模型组;2中药低剂量组;3中药中剂量组;4中药高剂量组;5甲巯咪唑组以及未造模的生理对照组。分别应用清肝泻火方大、中、小剂量组和西药组干预治疗,并运用放射免疫分析法检测甲状腺激素、免疫组化法检测核因子-κB、逆转录多聚酶链法（RT-PCR）等方法检测细胞因子、用ELISA法检测血清C-反应蛋白、细胞间黏附分子和肿瘤坏死因子。结果:1.GD大鼠甲状腺激素的实验结果:模型组大鼠的血清TSH明显降低,FT₃、FT₄明显升高并与正常组有显著性差异（P<0.01）,提示造模成功。经过治疗后,西药组和各中药治疗组TSH、FT₃、FT₄与造模组有显著性差异（P<0.01）,提示各治疗组治疗效果较好,具有明显抗甲状腺功能亢进的作用。小剂量、中剂量中药组与西药基本相当,而大剂量组明显好于其他各组,有显著性差异（P<0.01）。2.GD大鼠血清C-反应蛋白表达的实验结果:模型组大鼠血清CRP明显升高、与正常组相比有显著性差异（P<0.01）,提示GD的发生、发展与炎症密切相关。小剂量、中剂量、大剂量中药治疗组CRP含量与模型组有显著性差异（P<0.01）。中剂量中药组与小剂量、大剂量中药组也存在显著性差异（P<0.01）,中剂量组优于大剂量组和西药组。这一结果提示,中药中剂量组治疗效果较好,优于中药大剂量组和西药组。中药大、中、小剂量组的血清CRP含量变化与其相应的各组甲状腺激素水平的变化的不同步,提示CRP含量与甲状腺激素水平可能无相关性,CRP是否为GD病发生发展的独立因素还有待于进一步研究。3.GD大鼠细胞间黏附分子（ICAM-1）表达的实验结果模型组大鼠的血清ICAM-1含量明显升高,与正常组相比有显著性差异（P<0.01）。小剂量、中剂量、大剂量中药治疗组ICAM-1含量与造模组有显著性差异（P<0.01）。各中药治疗组之间无明显差异（P>0.05）,与西药组亦无明显差异（P>0.05）,提示各治疗组治疗效果无差异。4.GD大鼠甲状腺NF-κB表达的实验结果:模型组大鼠甲状腺上皮细胞NF-κB表达明显升高,与正常组有显著性差异（P<0.01）。病理结果显示模型组大鼠甲状腺细胞光密度显著增强,多数阳性信号同时出现于胞浆和胞核中,细胞胞浆内棕黄色颗粒堆积较多,呈片状分布。中药小剂量、中剂量、大剂量治疗组NF-κB含量与模型组有显著性差异（P<0.01）。各治疗组胞浆内棕黄色颗粒堆积较分散,呈点状分布,提示中药组各个剂量组治疗效果较明显。其大剂量中药组与小剂量、中剂量中药组存在显著性差异（P<0.01）,与西药组无差异（P>0.05）,大剂量组疗效优于小、中剂量组。5.GD大鼠血清IL-6mRNA,IL-10mRNA表达的实验结果:模型组大鼠血清IL-6mRNA表达明显升高、光带最亮,其平均光密度OD值与正常组有显著性差异（P<0.01）。各个治疗组的OD值均显著下降,与模型组有显著性差异（P<0.01）,中剂量组与西药组、小、大剂量组有显著性差异（P<0.01）,说明中剂量组疗效优于大剂量组和西药组。模型组大鼠血清IL-10mRNA光密度值显著降低,与正常组相比有显著性差异（P<0.01）,中药大、中、小剂量组与模型组相比有显著性差异（P<0.01）,中药大剂量组与小剂量组、中剂量组、西药组之间存在显著性差异（P<0.01）,中药大剂量组疗效最好。6.GD大鼠血清TNF-α表达的实验结果造模组大鼠血清TNF-α明显升高,与正常组有显著性差异（P<0.01）,小剂量、中剂量、大剂量中药治疗组TNF-α含量与模型组有显著性差异（P<0.01）。各治疗组之间无显著性差异（P>0.05）,这一结果直接证实TNF-α参与GD免疫反应过程,随着疾病的控制,其含量也显著性的降低。结论:1采用大鼠尾静脉注射小肠结肠炎耶尔森氏菌制作的GD模型表达出动物GD主要特征,用其进行药物及病理等方面的研究具有实用性。2中药清肝泻火方能够改善甲状腺激素水平,具有抗甲状腺功能亢进的作用。3 GD大鼠模型存在炎性因子和炎性蛋白的高表达,显示GD与炎症的密不可分性。4中药清肝泻火方具有调控和改善炎性因子和炎性蛋白表达的作用。更多还原

【Abstract】 Objective:Graves Disease（GD） model is established by immuning Wistar rats with Y.E.to draw analogy between the curative effect of the decoction that subdue the pathogenic fire in liver in different dosage,discuss whether the composition of the decoction that subdue the pathogenic fire in liver is reasonable,verify the hypothesis that the occuring of GD is closely related to the pathological injury caused by partial inflammation in the thyroid gland and expond the mechanism of treating GD through subduing the pathogenic fire in liver.So as to find new methods for the bianzhengshizhi of GD,and lay the theoretical foundation and experimental evidence for the further development of new drugs.Method:Take immuned Wistar rats with Y.E.as the experimental model, then divide the rats into 5 groups randomly:1.model group;2.group of small dosage of herbal medicine;3.group of medium dosage of herbal medicine;4.group of large dosage of herbal medicine;5.group of thiamazole;treat them with small,medium,large dosage of herbal medicine and western medicine respectively.And TH is examined by RIA,NF-κB is examined by Immunohistochemistry Technique,CK is examined by RT-PCR,CRP and ICAM-1 is examined by ELISA. Result:1.Result of TH of the GD rats:The Tsh of the rats in model group decreased obviously,and t he FT₃,FT₄ of the rats in model group rose significantly,and the re were obvious difference between the normal group and the mod el group,indicating that the model was successfully established （P<0.01）.It was of significant difference when the group of we stern and the groups of herbal medicine were compared with the model group（P<0.01）,which indicated that the curative effect was remarkable and played the role of anti-thytoglobulin.And th ere was no obvious difference between the group of western medi cine and the groups of small,medium dosage of herbal medicine（P >0.05）,and the large-dosage of hebal medicine was quite differe nt.Curative effect of small-dosage,medium-dosage of hebal med icine and western medicine group was quite basic and large-dosa ge was significantly better than the other groups（p<0.01）.2.Result of CRP of the GD rats:The CRP of the rats in model group rose obviously,which was significantly different from the model group（P<0.01）,indicating that the occurance and development of GD were closely related to the inflammation.There were obvious differences between the treating groups of small,medium and large dosage of herbal medicine and the model group（P<0.01）.And there were also obvious differences between the group of small,large dosage of herbal medicine and the group of mediun dosage of herbal medicine（P<0.01）.The group of medium dosage of herbal medicine was more superior than the group of large dosage of herbal medicine and the western medicine（P<0.01）. The result indicated that the curative effect of the group of medium dosage of herbal medicine was more remarkable than the group of large dosage of herbal medicine and the western medicine group.The fluctuation of TH of the rats in the groups of small,medium and high dosage of herbal medicine did not synchronise with the fluctuation of CRP,indicating the fluctuation of CRP beared no obvious relation to the fluctuation of TH.So,whether CRP is an independent factor of the occurrence and development of GD should be studied further more thoroughly.3.Result of ICAM-1 of the GD rats:The ICAM-1 of the rats in model group rose obviously,which was significantly different from the normal group（P<0.01）.There were obvious differences in ICAM-1 between the treating group of small,medium,large dosage of herbal medicine and the model group（P<0.01）.Apart from the above mentioned,there were no significant differences among the treating groups of herbal medicine（p>0.05）,and they were also different from the western medicine group（p>0.05）,indicating that the treatment groups have no different in treatment effect.4.Result of NF-kB of the GD rats:The NF-κB of the rats in model group rose obviously,which was significantly different from the normal group（P<0.01）.The pathological result showed that the density of TEC of the rats in model group increased obviously,the majority of positive signals existed in nucleus and cytoplasm at the same time.And there was lots of brown particles in the cytoplasm,ranging in slices, indicating that there were obvious differences in NF-κB between the treating group of small,medium,large dosage of herbal medicine and the model group（P<0.01）.The treatment group intracytoplasmic accumulation of brown granules scattered,suggesting the group of herbal medicine treatment in all dosage groups was more apparent. There was a significant difference between its large-dosage group and small-dosage group medicine in the dosage of herbal medicine, （P<0.01）,but no difference with the western medicine（p>0.05）.In treatment effect,the large-dosage group was better than the small and medium dosage group.5.Result of IL-6mRNA,IL-10mRNA of the GD rats:The IL-6mRNA of the rats in the model group rose obviously,with a brightest light band,and its average of OD was significantly different from the normal group（P<0.01）.ODs in each treating groups fell obviously,and was significantly different from the model group（P<0.01）.There were also of great difference among the group of medium dosage of herbal medicine,the group of western medicine and the group of large dosage of herbal medicine（P<0.01）,indicating that the curative effect of the group of medium dosage of herbal medicine was better than the group of large dosage of herbal medicine and western medicine.The IL-10mRNA of the rats in the model group decreased obviously, and was obviously different from the normal group（P<0.01）.There were significant differences between the treating groups and the model group（P<0.01）.There were significant differences between the large-dosage group and small-dosage group,medium-dosage group, the western medicine group.And the large-dosage group of herbal medicine had the best effect.6.Result of TNF-a of the GD rats:The TNF-αof the GD rats in the model group rose obviously, which was significantly different from the normal group（P<0.01）.There existed obvious differences between the treating groups of small,medium,large dosage of herbal medicine and the model group（P<0.01）.No significant difference was found in each treating group（P<0.01）.There was significant difference between the treating groups and the normal group（P<0.01）.This result directly verified that TNF- a took part in the immune response.With the controlling of the disease,the content of TNF-αfell obviously and its fluctuation was not synchronical with the ones of FT₃,FT₄,TSH.It indicated that TNF-αcould not be the independent factor of the cause of GD while the disease was the result of the pathogenic factor in many collaborative.Conclusion:1.The GD model established by injecting Y.E.into tail vain of rat shows the major character of GD in animal.This method is effective and advanced in researching of medicine and pathology.2.The decoction that subdue pathogenic fire in liver can improve TH level,and play the role of anti-thyroid.3.The high level of inflammatory protein and inflammatory factors in the rats in GD model group thoroughly proved that GD is closely related to inflammation.4.The decoction of subduing pathogenic fire in liver can control and improve the condition of disorder like the inspects saying above.更多还原