【作者】 何慧珍；

【导师】 金桂兰；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2012， 硕士

【摘要】 目的：利用免疫诱导法建立干燥综合征(Sjogren’s syndrome SS)动物模型,动态观察增液布津汤对SS模型小鼠一般指标及免疫功能的干预调节作用,并从分子细胞生物学角度观察增液布津汤对SS模型小鼠肿瘤坏死因子α (TNF-α)、白介素-6(IL-6)及颌下腺水通道蛋白-5(AQP5)的影响,为探讨增液布津汤治疗SS的作用机制提供基础依据,明确其治疗靶点,同时为更好地运用于临床提供可靠的实验依据。方法：1.理论研究方法：检索古今中外文献对干燥综合征的中医临床辨证分型、治法方药及实验研究方面的内容进行分析总结。2.实验研究方法：雌性BALB/c小鼠60只,随机分为正常组对照组、模型组、增液布津汤低剂量组、中剂量组、高剂量组和环戊硫酮组。除正常对照组外,其余5组均利用免疫诱导法(注射白百破疫苗+完全弗氏佐剂+SD大鼠颌下腺抗原)建立SS小鼠模型,造模后当天正常对照组及模型组用生理盐水20mL.kg-1ig,中药低、中、高剂量组用增液布津汤(相当于生药6.8、13.6、27.3g.kg-1)ig,阳性对照组用环戊硫酮12.5mg.kg-1ig。首次免疫后第3,7天,用乳化抗原对模型组和用药组加强免疫2次,剂量为0.2mL,多点背部sc。以后每隔14天用乳化抗原加强免疫1次,至实验结束共加强免疫2次,剂量、方法同前两次,正常对照组不作任何处理。在实验过程中,动态观察增液布津汤对SS模型小鼠摄食量、饮水量、体质量及唾液流量的影响。实验结束第50天,摘取各组小鼠颌下腺、胸腺、脾脏,计算颌下腺指数、胸腺指数、脾指数,同时镜下观察颌下腺病理变化,并用ELISA法,测定小鼠血清TNF-α、IL-6含量,用Western印迹法,测定颌下腺细胞AQP5蛋白的表达。结果：1.理论研究结果：在文献资料分析的基础上,我们认为：中医各家虽对该病从不同角度进行了论治,但均是围绕其本虚标实的病理特点进行辨证论治的。本虚即为诸脏腑气血阴阳亏虚,其中主要为阴液亏损，津枯液涸,脏腑不荣，津液输布失常；而标实主要是燥毒、瘀毒互结。故根据气、血、津液理论，拟用养阴益气、宣肺布津、活血通络的增液布津汤治疗本病。2.实验研究结果：2.1一般症状表现：与正常对照组相比,模型组小鼠唾液流量减少、饮水量增加、摄食量减少、体重减轻。此外,正常对照组小鼠精神状态良好,活动自如,皮毛光滑,无脱毛现象；模型鼠精神萎靡、嗜睡、懒动、活动度下降,皮毛光泽度下降,轻度脱毛,皮毛明显疏松,部分小鼠尾部破溃结痛,而增液布津汤能不同程度改善模型小鼠气阴两虚的症状,小鼠轻度脱毛,皮毛疏松不明显。2.2镜下观察颌下腺形态结构变化：模型鼠颌下腺组织出现不同程度的淋巴细胞浸润、腺泡萎缩或消失、导管扩张等病理变化,且颌下腺萎缩,颌下腺指数明显减小,而增液布津汤能明显减轻淋巴细胞浸润并抑制颌下腺萎缩。2.3在免疫功能方面：与正常对照组相比,模型鼠胸腺指数、脾指数均增加(P<O.01,P<0.05),表明机体内发生r较强的免疫反应,而给予药物治疗后,均能显著减小模型鼠的脾指数,胸腺指数,故推测增液布津汤可以抑制机体内增强的免疫反应。2.4对细胞因子TNF-α、IL-6的影响：与正常对照组相比,模型组TNF-α及IL一6含量明显增高(P<O.01)；与模型组相比,增液布津汤各剂量组TNF-α及IL一6含量均由不同程度降低(P<0.01)。2.5Western blot测定结果示：与正常对照组相比,模型组AQP5表达水平下降了27.90%(p<0.01)；与模型组相比,增液布津汤高、中、低剂量组AQP5表达水平分别升高了32.84%(p<0.01)、12.54%(p<0.05)、4.51%,环戊硫酮组AQP5表达水平升高了20.40%(p<0.01)。结论：增液布津汤可能通过抑制细胞因子TNF-a、IL-6的分泌,间接地抑制了辅助性T细胞(Th)业群增殖和分化,从而减轻免疫炎症反应,缓解SS模型鼠颌下腺淋巴细胞浸润,腺泡、导管结构及免疫器,l==j.的破坏,保护腺体的功能,促进AQP5表达,增加唾液分泌,进一步说明细胞因子及AQP5在SS发病过样中发挥着重要作用,同时,也表明养阴益气、宣肺布津、活血通络法的整体调节作用可有效缓解SS模型鼠气阴两虚的症状。创新点：从气阴两虚的角度，研究养阴益气、宣肺布津、活血通络法对SS小鼠干预作用及其机理,得出该治法通过抑制细胞因子的分泌,减轻免疫炎症反应,保护腺体功能,促进AQP5表达,增加唾液分泌,从而改善干燥症状,进一步说明中医药治疗SS的作用机制是多途径、多环节、多靶点。更多还原

【Abstract】 Objetctives:To establish experimental Sjogren’s syndrome (SS)model by induced with immune inducement method and observe dynamicly the effect of Zengye Bujin Decoction on symptoms and immune function of reguLating of mice model, from the point of molecuLar cell biology to explore the influence of TNF-a, IL-6, aquaporin-5with Zengye Bujin Decoction, to provide basic evidence for its mechanism, make its therapeutic targets clearly, at the same time provide the experiment evidence for better use it to the clinic.Method:1. Method of theoretical researeh:By retrieving literature at home and abroad,from Ancient to present,summarized the traditional Chinese medicine clinical syndrome differentiation, therapeutic methods and drugs and experimental study of SS.2. Method of experimental research:BALB/c rats were divided into normal group, model group, Zengye Bujin Decoction low dose group, Zengye Bujin Decoction middle dose group, Zengye Bujin Decoction high dose group, Anethol Trithione group.In addition to normal group, other five groups were induced with immune inducement method(injection of Pertussis vaccine, complete Freund’s adjuvant and mice glanduLa anguLaris antigen).Then the normal group, model group was given normal saline gavage, Zengye Bujin Decoction low dose (6.8g. kg-1), middle dose(12.6g. kg-1), haigh dose(27.3g. kg-1) was given treatment group, and Anethol Trithione(12.5mg.kg-1)was given positive group. After first immune, to reinforce immune with emuLsifier antigens in third, seventh days, the dose is0.2mL, sc on back with more points. At intervals of14days, to reinforce immune with emuLsifier antigens first, the dose and position as same as the previous twice, total of two times to reinforce immune to the end of experiment. Normal group has not deal anything. In50days of experiment, to observe the effect of Zengye Bujin Decoction on general indicators(Weight, water intake, food intake, saliva flow rate).When experiment was finished, to take Submaxillary gland. Thymus gland, spleen gland, calcul.ate Submaxillary gland index. Thymus gland index, spleen gland index, to observe pathological changes of Submaxillary. To take blood from eyes, serum from centrifugal, cytokine TNF-α and IL-6in the mice with SS was shown by ELISA analysis, to detect the AQP5expression changes of the submaxillary gland cells in mice through Western blots.ResuLts:1. ResuLts of theoretical research:Based on the analysis of literature, we think of various doctors of Chinese medicine make diagnosis and treatment from different point of view, but revolves around the pathological character of difficiency in origin and excess in superficiality to treatment based on differential diagnosis. Dificiency in origin referred to Qi-blood and Yin-Yang lost of every Zang-fu organ, the most important of it is that fluid exhaustion, Zang-fu organ malnutrition, disturbance of fluid in transportation, while excess in superficiality referred to cementation of toxin and blood stasis. So according to the theory of Qi-blood-fluid, draft the Zengye Bujin Decoction to invigorating Qi and nourishing Yin, distributing fluid and free collaterals, promoting blood circulation to remove blood stasis to treat SS.2. Experiment research resuLts2.1General state performance:Compared with the normal group, model group salivary flow rate decreased, water intake increased, food intake and weight reduced. In addition to, normal group in good shape, activities freely, fur smooth, not mouLt; model mices Spirits are drooping, sleepiness, lazy, mobility decreased, the fur glossiness droupped, loosen indistinctly and tail uLceration and crust of some mices, while each dose group of Zengye Bujin Decoetion can improve the symptoms of deficiency Qi-Yin, mouLt slightly and fur loosen not obvious.2.2Observe form and structure changes of submandibuLar gland by microscope: submandibuLar organization have massively lymphocytic infiltration, acinar atrophy or disappear, duct expansion etc, submandibuLar atrophy, submandibuLar index decreased, while Zengye Bujin Decoction can reduced lymphocytic infiltration, inhibit it atrophy.2.3Immunological function:Compared with the normal group,Thymus gland index of model group increased, spleen gland index increased(P<0.01, P<0.05), explain strong immune response happened in body, so infer that Zengye Bujin Decoction couLd suppress the immune response.2.4Changes of cytokincTNF-α. II-6:Compared with the normal group, TNF-a and IL-6of model group raised(P<0.01):compared with the modle group, different dosage of Zengye Bujin Decoetion couLd reduced TNF-α, IL-6(P<0.01).2.5ResuLt of Western blot:Compared with the normal group, the expression of AQP5declined27.90%(P<0.01); compared with the model group, the high, middle, low dose group of Zengye Bujin Decoction couLd raised the expression of AQP532.84%(P<0.01),12.54%(P<0.05),4.51%, Anethol Trithione group raised20.40%(P<0.01).Conclusion:Zengye Bujin Decoction couLd inhibit secretion of cytokineTNF-a, IL-6, indirectly to suppress the helper T cells (Th) proliferation and differentiation to reduce the immune inflammation reaction, relive the Iymphocytic infiltration on submaxillary gland, structure of acinus, ductal and immune organ damage, protection saliva secretion of submandibuLar, promote AQP5expression, show that cytokine and AQP5are play an important role in the course of SS furtherly, at the same time, prove the global reguLatory role of invigorating Qi and nourishing Yin, distribute fluid and free collaterals, promoting blood circuLation to remove blood stasis make contribute to relieve the symptom of Qi-Yin defieiency.Creative Points:According to Qi-Yin defieiency, to explored into the effect and mechanism of invigorating Qi and nourishing Yin, distributing fluid and free collaterals, promoting blood circuLation to remove blood stasis, show that it couLd inhibit secretion of cytokineTNF-a, IL-6, reduce the immune inflammation reaction, protect fonction of saliva, increase AQP5expression, promote saliva secretion, improve symptom of dry, it further illustrated the treatment of Chinese medicine was muLti-channel, muLti-link and muLti-arget.更多还原