【作者】 贾波；

【导师】 张丽；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2002， 硕士

【摘要】 干燥综合征(Sj(?)gren’s Syndrome，SS)是一种侵犯外分泌腺体尤以唾液腺和泪腺病变为主的慢性自身免疫病。其病理学的典型改变是腺体有大量的淋巴细胞浸润，造成腺体破坏，导致腺体萎缩，腺细胞功能减退或丧失，出现口眼干燥的症状。SS在中医文献中无相似的病名记载，根据其临床表现，多数学者将其归属中医“燥证”范畴。在诊治时也采用西医病名，称“干燥综合征”，进行辨证施治。由于主要受损组织是唾液腺，且唾液腺标本容易获取，所以对唾液腺的研究成为SS的重点。到目前为止，SS的发病机理尚未阐明。而动物模型的缺乏正是限制SS机理研究的一个重要的因素。因此，本研究根据免疫学原理，通过四种粗制抗原、两种纯化抗原的筛选，并通过选择不同的抗原剂量、免疫加强次数以及佐剂种类和用量等方法，建立了模拟SS颌下腺典型病变的SS小鼠模型，并在此模型基础上，对SS模型小鼠的T、B细胞功能，SS模型小鼠与气阴虚的关系进行了研究；同时复制了SS大鼠模型，探讨了SS模型大鼠与血瘀的关系。为SS的机理研究及中西医结合治疗提供有力的工具。 1．建立SS小鼠模型 1．1粗制抗原诱发C57bl／6j小鼠SS样病变的实验研究 在国内常用的动物品系中选择四个品系C57bl／6j小鼠、Balb／c小鼠、SD大鼠和Lewis大鼠作为抗原提供鼠。用上述鼠的粗制唾液腺匀浆离心10分钟(4℃，3000rpm)，取上清和弗氏完全佐剂(CFA)乳化，得到抗原浓度为2mg／ml，内含BCG原浆3mg／ml的乳化抗原，分别免疫C57bl／6j小鼠。每隔两周加强免疫1次，经过6次加强免疫、建模时间为12周。 模型小鼠颌下腺的组织切片HE染色结果显示：四种不同来源的粗制抗原，经过7次加强免疫后，都能诱发不同程度的SS样病理改变，其中Lewis抗原和C57抗原诱发的靶器官的病理学改变重于SD抗原和Balb／c抗原，表现为模型小鼠颌下腺间质中有少量散在的淋巴细胞浸润，腺体导管稍有扩张，但均未见淋巴细胞浸润灶及腺体破坏、纤维化等典型的病理改变。Balb／c组和SD组间质中仅有极少量的淋巴细胞浸润，其病理学表现与正常组基本相同。佐剂组无明显改变，与正常组病理表现基本相同。提示在上述实验条件下，粗制抗原不能有效地诱导C57bl／6j小鼠颌下腺出现典型的SS样病理改变。 双向琼脂扩散结果显示：各模型组均出现融合型抗原-抗体沉淀线，表明四种粗制抗原均能诱导C57小鼠产生抗体。但四种抗原诱导的抗体效价亦不等，Lewis组和SD组抗体效价高于C57bl／6j组和Balb／c组。 1．2纯化抗原诱发C57bl／6j小鼠SS样病变的实验研究 采用差速离心法提纯Lewis大鼠、C57bl／6j小鼠的颌下腺抗原，分离并选择．2－干燥综合征小鼠模型的建立及病机探讨PS抗原组分作为免疫用抗原。PS抗原组分设两个抗原浓度（1刀mg／ml和0．smg／ml人采用多点注射法免疫C57 ／J’鼠。CFA、百日咳杆菌（BPL纯化抗原联合使用，加强免疫。每隔两周多点注射法加强免疫1次，并设立不加强组；加强1次组；加强2次组；加强3次组。各加强组于末次免疫后每10天取材1次。 经组织学检查，比较不同试验条件时颌下腺的免疫性损伤情况，动态观察不同时问段病变，结果显示：PSH3组首次免疫后60天时，间质淋巴细胞中度浸润，腺泡轻度萎缩，小导管增生，导管壁淋巴细胞中度浸润，轻度纤维增生。70天时，间质淋巴细胞中度浸润，腺泡大小不等，部分已破坏、消失，导管壁淋巴细胞中度浸润，纤维组织增生。80天时，间质淋巴细胞重度浸润，其中可见散在嗜酸性变的上皮细胞。腺泡大小不等，部分已破坏、消失。导管壁中有中度淋巴细胞浸润，管腔轻度扩张，纤维增生。90天时，间质中有灶状淋巴细胞浸润，形成滤泡。腺泡大小不等，破坏乃至消失，上皮细胞嗜酸性变。血管周围灶性淋巴细胞浸润。导管增生、扩张。在低倍镜下易见多个淋巴细胞浸润灶。 实验中，同时检测了模型动物特异性抗体的水平。结果显示：＊5＊3组模型小鼠的抗体滴度在首次免疫60天，已明显增高，此后仍持续增高，并一宜维持在较高水平。 PSH3组模型小鼠的唾液分泌量逐渐减少；且在病理出现典型病变之前，唾液分泌量就已明显低于正常组和佐剂组。表明模型小鼠的唾液腺功能变化是先于病理改变的。 结果表明：经 PS抗原大浓度门刀 mg／Inl）免疫，并经 3次加强免疫的 PSH3组模型小鼠颌下腺的病理学改变最为明显。提示我们采用纯化抗原经过3次加强免疫，首次在C57bl／6j ／J’鼠成功诱导了 SS病理改变，建立了 SS ／J＼鼠模型。2．机理探讨2二探讨SS的免疫学机理 复制巳建立的SS小鼠模型，采用MTT法测定脾T、B细胞的增殖功能，并观察SS模型小鼠对二硝基氟苯（DNFB）诱发的DTH反应的程度。结果显示SS模型小鼠的DTH反应明显高于正常组，B细胞功能增强，但脾T细胞功能受抑。表明SS模型小鼠具有与临床SS患者相似的免疫调节紊乱。2．2 SS小鼠模型与气阴虚的关系探讨 复制已建立的SS小鼠模型，观察模型鼠表现出的气阴虚症状。同时采用游泳劳损实验进一步探讨SS与气阴虚的关系。 结果显示：SS模型小鼠出现体更多还原

【Abstract】 Sjogren’s Syndrome (SS) in humans is considered a chronic autoimmune disease characterized by diffuse lymphoid cell infiltration into the salivary and lacrimal glands. The consequences of immune cell activation in these exocrine glands are symptoms of dry mouth (xerostomia) and dry eye (keratoconjunctivitis sicca) due to insufficient secretion. SS has no name in traditional Chinese medicine, acceding to its clinic manifestation; it belongs to syndrome caused by dryness-evil. But on diagnosis and therapy, it also adopts the name SS, conducting diagnosis and treatment based on overall analysis of symptoms and signs. As is one of the major damaged organs, and the biopsy is available, salivary gland becomes the focus of SS. By now, the clear pathogenesis of SS is unknown. The lack of appropriate animal models is one of the main causes. In this study, four crude and two pure antigens were used. Through different antigen doses, immune times, sorts and quantities of adjuvant, an mouse model of SS were set up. the sialoadenitis of which was similar to SS in human. On the basis of this model, we investigated the function of T and B cell, researching the relationship between model mice and deficiency of qi and yin. At the same time, we reproduced a rat model, making an inquiry into the blood stasis of model rats. Supplying a powerful tool for the research and therapy of SS through combination of Chinese traditional and Western medicine. 1. Establish mice models of SS1.1 Experimental study on inducing SS-like disease in C57bl/6j mice with crude antigens.Antigen donor animals were four murine strains as follows: C57bl/6j mice, Balb/c mice, SD rats and Lewis rats. The submandibular glands homogenates of above animals were centrifuged at 3000rpm for ISmin.The supernates were mixed with complete Freund’s adjuvant (CFA). Then we get the immunogen emulsions with the protein concentration 2 mg/ml (including Bacillus Calmette Guerin (BCG) 3 mg/ml) and injected mice with them, one time every two weeks, total is 6 times and 12 weeks.The results of HE stain showed that antigens from Lewis rats and C57bl/6j mice induced only mild pathologic changes with few lymphocytes infiltrated in submandibular glands (SMG) of C57bl/6j mice, and gentle changes happened in mice with antigens from Balb/c mice and SD rats. The results of double agar diffusion showed that every group presented antibodies to individual antigens, but the groups of-5-Balb/c and SD have higher liters than the other two groups. Nearly no changes wereobserved in group of adjuvant. These changes reflect above experimental conditionscouldn’t induce typical pathologic changes in SMG of C57bl/6j mice.1.2 Experimental study on inducing SS-like disease in C57bl/6j mice with pureantigens.Purify the SMG of Lewis rats and C57bl/6j mice by differential centrifugation. Select supernatesS (P5) with two concentrations (l.Omg/ml and 0.5mg/ml). Sensitize C57bl/6j mice on multiple spots with SMG hi CFA plus Bordetella pertussis. Mice were divided into groups from no immunoenhancement to 3 times.The results of HE stain showed that group of P5H3 at 60 days had moderate lymphocytes infiltration in substantial. By day 70, intense lymphocytes infiltration presented in substantial. Generalized infiltration with moderate parenchyma destruction was observed at 80 days. Several, generalized foci of periductal infiltrate with severe parenchyma destruction presented at 90 days. Antibodies increased steady from 60 days to 90 days. The salivary secretion decreased before pathologic changes happened.Above changes reflect we successfully establish a mouse model for SS. 2. The study on pathogenic mechanisms of SS2.1 ImmunopathogenesisInvestigate the function of spleen T and B lymphocyte stimulated by ConA and LPS, and observe the delayed type hypersensitivity (DTK) induced by DNFB. Function of model mice spleen T is decreased and B lymphocyte is increased, DTH is also increased. It reflects the immunoregulation of model mice were disturbance.2.2 Relationship更多还原