【作者】 金桂兰；

【导师】 汪悦；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2009， 博士

【摘要】 干燥综合征(Sjogren’s syndrome,SS)是一种侵犯外分泌腺体尤以唾液腺和泪腺为主的慢性自身免疫性疾病。导师汪悦教授根据长期的临床经验,运用养阴益气、布津通络法治疗SS,方用增液布津汤在临床取得良好效果,本文通过实验研究进一步探讨养阴益气、布津通络法治疗SS的作用机制,为临床工作提供充实的依据。目的:探讨中医学对原发性干燥综合征发病机理的认识,从基础研究角度探讨养阴益气、布津通络法对干燥综合征小鼠干预作用及免疫功能影响,并从组织和细胞学角度探讨增液布津汤对干燥综合征小鼠AQP5的表达影响,为阐明运用养阴益气、布津通络法治疗原发性干燥综合征小鼠的作用机制提供依据,从而在证实其确切疗效的同时明确其作用部位,为更好地运用于临床提供理论与实验依据。方法:1.理论研究方法:检索古今中外文献对干燥综合征认识,并进行分析比较。2.实验研究方法:实验研究部分用BALB/c小鼠颌下腺免疫造成SS小鼠模型,同时给予增液布津汤。在8周的实验中,每周测定唾液流量,每4天测定体重、摄食量、饮水量。取胸腺和颌下腺计算胸腺指数和颌下腺指数,并观察颌下腺病理变化。并用流式细胞仪测定外周血CD4+/CD8+,ELIST法测定血清IFN-γ、IL-1、IL-6含量,计算IFN-γ/IL-6。用免疫组化法检测小鼠颌下腺AQP5的分布表达变化。结果:1.理论研究结果:在文献资料分析的基础上,结合导师汪悦教授的临床经验,认为:由于燥热之邪导致阴津耗伤,津能载气,气随津耗,则气阴两虚是大多数pSS病人的共同转归,因此确立养阴益气、布津通络为治疗大法,根据气能生津、气能行津的原则,重用养阴益气药,运用增液布津汤治疗pSS,取得良好的效果。2.实验研究结果:2.1在症状表现上,与正常小鼠相比,模型小鼠唾液流量减少、体重减轻、饮水量增加。而增液布津汤大、中、小剂量组均能不同程度改善小鼠口渴症状。2.2在免疫功能方面,与正常小鼠相比,模型组颌下腺指数降低、胸腺指数增加,T、B淋巴细胞转化增殖能力比值增加、颌下腺出现淋巴细胞浸润等病理变化,CD4+/CD8+增加,而给予增液布津汤能改善这些症状并减轻病理改变。2.3细胞因子IFN-γ、IL-1、IL-6变化情况。结果显示:与模型组比较,细胞因子IFN-γ、IL-1、IL-6在增液布津汤作用下,IL-1、IFN-γ与模型组比较显著降低,其中小剂量组降低明显P＜0.01。给予药物后IL-6也有不同程度降低,各组间IL-6无显著性差异。2.4对AQP5的影响。我们又研究养阴益气、布津通络对小鼠颌下腺细胞AQP5的影响。通过比较研究,显示:增液布津汤能改善小鼠颌下腺细胞AQP5的表达,与国内外学者研究有类似之处,但也有相异之处。结论:1.增液布津汤具有抑制小鼠腺体淋巴细胞浸润、抑制T、B淋巴细胞增殖、改善CD4~+/CD8~+比值,能够纠正免疫紊乱。2.增液布津汤可能通过抑制细胞因子IL-6、IFN-γ蛋白合成及分泌,间接地抑制了Th细胞的分化与增殖,从而缓解SS小鼠颌下腺淋巴细胞浸润,通过调节Th1/Th2细胞因子失衡来保护SS小鼠,进一步说明细胞因子在SS唾液分泌过程中发挥重要作用。3.增液布津汤能改变小鼠颌下腺细胞AQP5分布表达,与唾液分泌功能有关,因此为增液布津汤治疗SS提供了依据。创新点:首次从气阴两虚的角度,研究养阴益气、布津通络法对SS小鼠干预作用及其机理,得出该治法通过调节T淋巴细胞亚群、Th1/Th2细胞因子失衡,改变颌下腺细胞AQP5分布表达,最终得到改善唾液分泌功能、减轻干燥症状的目的。进一步说明养阴益气、布津通络法对SS作用机制是多途径、多环节、多靶点的。更多还原

【Abstract】 Sjogren’s syndrome (SS) is a chronic autoimmune disease that invades the exocrine glands, especially salivary and lachrymal glands. According to Professor Wangyue’s long-term clinical experience, prescription of Zengye Bujin Decoction, which has obtained good results in clinic, was used to treat pSS by nourishing yin and invigorating qi as well as distributing fluid and freeing collaterals. This essay further studies the mechanism of Chinese medical therapy on pSS by experiments, which provides powerful evidences for clinical applications.Objectives:To explore the pathological mechanism of SS in TCM, effects of Chinese medical therapy on the interference and immune function in SS-mice from basic researches, influence of Zengye Bujin Tang on AQP5 expression in SS-mice based on histology and cytology. Therefore it could provide evidence for the explanation of the mechanism of Chinese medical therapy in SS-mice, the curative effects as well as the site of action and serves as the theoretical and experimental evidences for clinical applications.Method:1. Method of theoretical research: By retrieving literature at home and abroad, from ancient to present, information related to SS is analyzed and compared.2. Method of experimental research: we made mice-models of SS induced bysub maxillary gland immune in BALB/C mice, and then fed Zengye Bujin Tang to them. During the eight weeks, salivary flow was measured every week, weight, food and water intakes every four days. By removing Thymus Index and Submandibular Gland Index, They were counted and pathological changes of sub maxillary gland were observed. We still tested CD4f/CD8f in the peripheral blood by flow cytometry and the serum content of IFN-γ, IL-1 and IL-6 through ELIST, calculated IFN-γand IL-6,detected the AQP5 expression changes of the sub maxillary gland cells in mice through immunohistochemicalstaining.Results:1. Results of theoretical research: Based on the analysis of literature, combined with professor Wangyue’s long-term experience, a conclusion was drew. Due to body fluid carrying qi and loss of body fluid followed by exhaustion of qi, dryness-heat leads to loss of yin-fluid so that the developments were both deficiency of yin and qi for most SS patients. Therefore the therapy of nourishing yin for invigorating qi and distributing fluid for freeing collaterals was established according to mutual-action of qi and body fluid. Drugs for nourishing yin and invigorating qi were greatly used in Zengye Bujin Decoction to treat pSS and good curative effects have been obtained.2. Experimental research results: 2.1 Symptomatic manifestations Compared with the normal mice, model mice were with the signs of saliva flow reduction, weight loses and water intake. While the high, middle and low dosage groups of Zengye Bujin Decoction relieved the mice’s thirsty in various degrees.2.2 Immunological functions The model mice had some pathological changes, such as decreased sub-mandibular gland, and thymus index increased and proliferation ratio of T,B lymphocyte, lymphocytic infiltration of submandibular gland, and CD4+/CD8+ also increased. Zengye Bujin Decoction could improve these symptoms and relieve the pathological changes.2.3 Changes of cytokine(IFN-γ, IL-1, IL-6) The resuits showed that under the effect of Zengye Bujin Decoction IFN-γin serum cytokine(IFN-γ, IL-1, IL-6) were much lower than the model groups, among which low dosage group reduced significantly(p<0.01). After taking drugs, IL-6 also reduced in various degrees, but there was no significant difference among the groups.2.4 Influences on AQP5 Influences on aquaporin5(AQP5) of the mouse submandibular gland cells by nourishing yin and invigorating qi as well as distributing water and free collaterals were studied in the paper. Comparative studies revealed that Zengye Bujin Decoction could improve the expressions of AQP5 of the mouse submandibular gland cells, revealing similarities and differences with research done at home and abroad.Conclusion:1. Zengye bujin Decoction could inhibit the lymphocytic infiltration of submandibular gland in mouse, restrain the proliferation of T and B lymphocyte, and improve the ratio of CD4+/CD8+ and balance immune disorders.2. Zengye Bujin Decoction would inhibit the differentiation and proliferation of Th cells indirectly to relieve the lymphocytic infiltration on submandibular gland of SS mouse by means of inhibiting protein synthesis and secretion of cytokine(IL-6, IFN-γ). Through balanced Th1/Th2 cytokine to protect SS mice, the method deeply illustrated that cytokines played an important role in the process of saliva excretion on SS.3. Zengye Bujin Decoction could change the distribution and expression of AQP5 of the mouse submandibular gland cells which is related to saliva secretion, therefore it provided evidences for treating SS with Zengye Bujin Decoction.Creative points:According to yin and qi deficiency, we explored into the effect and mechanism of interventing SS mouse by the therapy of nourishing yin for invigorating qi and distributing fluid for freeing collaterals for the first time. The research showed that this therapy could change the distribution and expression of AQP5 of the mouse submandibular gland to improve the function of salivary secretion by regulating T-lymphocyte subsets and the imbalance of Th1/Th2 cytokine. It further illustrated that the therapy of nourishing yin for invigorating qi and distributing fluid for freeing collaterals was multi-channel, multi-link and multi-target.更多还原