【作者】 吴莹；

【导师】 赵岩；

【作者基本信息】 中国协和医科大学 ， 内科学风湿免疫， 2008， 硕士

【摘要】 背景干燥综合征（Sjogren syndrome）常见于中年女性，是主要累及全身外分泌腺的慢性进展性自身免疫病。其外分泌腺受累以唾液腺和泪腺为主，此外还可累及呼吸、消化、肾脏、神经等多个系统。干燥综合征外分泌腺的损伤机制多样，包括淋巴细胞大量或灶性浸润导致正常腺体结构的破坏，以及某些抗体所介导的外分泌腺功能异常等。本研究通过检测抗乙酰胆碱3受体（M₃R）抗体在干燥综合征患者血清中的阳性率，分析其与临床表现的相关性，探讨其可能的临床应用价值。并利用动物模型研究抗M₃R抗体对唾液流率的影响，初步探讨该抗体的致病性。目的分别建立酶联免疫吸附法（ELISA）和免疫印迹法（Western blot）检测抗M₃R抗体在干燥综合征、系统性红斑狼疮和健康对照中的阳性率；分析抗M₃R抗体在干燥综合征中的临床意义；通过大鼠模型验证抗M₃R抗体抑制唾液腺分泌的作用。方法实验对象：自2005年-2007年于北京协和医院入组国际干燥综合征登记网络研究（SICCA）证实为肯定的干燥综合征患者共70例作为实验组；同期住院诊治的系统性红斑狼疮（SLE）患者且除外继发性干燥综合征共49例作为疾病对照组；并以同期健康献血者共76例作为健康对照组。三组间年龄、性别匹配。实验方法：以重组的M₃R受体为抗原通过ELISA法检测三组血清中抗M₃R抗体，以健康对照组吸光度的（?）+SD为阳性界定，计算ss组和SLE组的抗M₃R抗体阳性率；并以重组的的抗原用Western法进一步验证ELISA法检测的可靠性；分析SS组中抗M₃R抗体阳性患者较阴性患者在Schirmer试验、BUT、角膜染色、结膜染色、五分钟唾液流率、下唇小唇腺灶性淋巴细胞浸润指数、RF、IgG、抗SSA抗体阳性例数、抗SSB抗体阳性例数间的差异。用亲和层析法纯化抗M₃R抗体阳性患者血清中的IgG型抗M₃R抗体，以15mg／kg的剂量（7．6mg／ml）注射大鼠尾静脉观察大鼠唾液腺流率的改变，阴性对照用相同浓度和剂量的人IVlO，阳性对照用阿托品（0.125mg／kg）。结果（1）ELISA法检测76例健康组的（?）+SD的值为0.1710+0．0723，以此为阳性界定值计算出SS患者的抗M₃R抗体的阳性率为47．10％（33／70），SLE患者组中抗M₃R抗体的阳性率为4．02％（2／49），健康对照组中抗M₃R抗体的阳性率为14．47％（11／76）；抗M₃R抗体在SS患者中的阳性率明显高于SLE组（47．10％vs 4．02％，p<0．05）健康对照组（47．10％vs 14．47％，p<0．05），其间有统计学显著差异。而SLE组和健康对照组间的抗M₃R抗体阳性率无统计学差异（4．02％vs 14．47%,P>0．05）。（2）Western法检测出SS患者的抗M₃R抗体的阳性率为60．00％（42／70），SLE组巾的抗M3R抗体的阳性率则为12．20％（6／49），健康对照组中抗M₃R抗体的阳性率为15．79％（12／76）；抗M3R抗体在SS患者中的阳性率明显高于SLE组（60．00％vs12．20％，p<0．05）和健康对照组（60．00% vs 15．79％，p<0．05），有统计学显著差异。而SLE组和健康对照组间的抗M₃R抗体阳性率无统计学差异（12．20％vs15．79％，P>0．05）。Western法和ELISA法的符合率达74．9％。（3）SS组中抗M₃R抗体阳性患者IgG（39．97 vs 29．64，p<0．05）、RF值（42．23vs 27．92，p<0．05）及抗SSB抗体阳性例数（31 vs 9，p<0．05）较阴性患者明显增高，其间有统计学显著差异。而抗M₃R抗体阳性组与阴性组相比，唇腺活检阳性率、Schirmer试验、BUT、五分钟唾液流率、角膜染色和结膜染色间无显著统计学差异。但是抗M₃R抗体阳性组的患者唇腺活检中淋巴细胞灶性浸润（?）3的患者例数较阴性组明显为高（27 vs 10），p<0．05。（4）以亲和层析柱纯化的含抗M₃R抗体的人血清IgG（实验组，剂量按15mg／kg，浓度为7．6mg／ml）注射大鼠尾静脉后可减少大鼠的唾液流率（0．0938g vs 0．0748g，P>0．05），阿托品组（阳性对照组）用药后唾液流率较用药前显著减少（0．09327g vs0．02198g，P<0．05），实验组与阳性对照组相比，减少的差值无显著统计学差异（-0．02227g vs -0．07128g，P>0．05）。正常人IVIG组（阴性对照组）用药后唾液流率较用药前显著增加（0．08230g vs 0．43425g，P<0．05）。比较实验组与阴性对照组用药后与用药前唾液流率改变的差值，两组间差异具有统计学意义（-0．02227gvs0.35195g,P<0．05）。结论（1）ELISA和Western法检测SS患者抗M₃R抗体阳性率明显高于SLE患者和健康对照者。（2）SS患者中抗M₃R抗体阳性组较阴性组IgG值、RF值、抗SSB抗体阳性例数以及下唇小唇腺淋巴细胞浸润为高。（3）纯化的含抗M₃R抗体的人血清IgG可部分阻断匹罗卡品刺激大鼠唾液腺分泌的功能。更多还原

【Abstract】 BackgroundSjogren syndrome （SS） is a chronic autoimmune disease which occurs mainly in the fourth and fifthdecades and characterized by histologic and functional alterations of exocrine glands with progressive loss of salivary and lacrimal gland secretion. Respiratory, digesting, renal and nervous system are as well involved in SS. There are many mechanisms of loss of exocrine glands such as destruction of the acinar cells by lymphocytes infiltration and the antibody-mediated destruction of the exocrine glands. It has been recently proposed that pathology underlying the glandular hypofunction contributes inhibitory autoantibodies especially the autoantibodies directed against mucarinic recptors. The aim of this work is to test the positive rate of the anti-M₃ receptor antibody in SS , the relationship between the antibody and other clinical manifestations and the salivary flow rate decreased by the affinity-purified antibody from SS patients in mice.ObjectivesTo detect anti-M₃ receptor antibodies in Sjogren’ s Syndrome （SS） patients, SLE patients and the donating controls by methods of Enzyme linked immunosorbent assay and Western blotting. And to explore its association with clinical manifestation. At last detect the affinity-purified antibody’ s pathogenic mechanism in animal experiments. Materials and MethodsSpecimens:70 pieces serum of Sjogren syndrome patients from the SICCAobject in PUMCH from 2005-2007. 49 pieces serum of patients of SLE in-patients in PUMCH at the same time. 76 pieces serum of healthy donators in PUMCH. And the three groups match in sex and age.Methods: With the muscarinic 3 receptor（from sigma） as the antigen ,to detect anti-muscarinic 3 receptor antibody in SS, SLE and donator controlsusing methods of ELISA. The positive cut-off is （?）+SD in donator controls.And test the positive rate of anti-muscarinic 3 receptor antibody in SS, SLE by means of western blotting. Analyze the differences of the antibody-positive patients and antibody-negative patients in SS in aspects of Schirmer test,BUT,PEE on the corneas,the conjunctival epithelial defects, whole saliva flow rate in 5 minutes,FI,RF,IgG,anti-SSA and anti-SSB.Affinity- purified of human polyclonal IgG antibodies （15mg/kg）by Protein A chromatography was passive transfered to mice and detect the differences of the salivary secrection in the groups of IVIG （15mg/kg）which is the negative controls, atropine（0.125mg/kg） which is the positive controls and the human IgG.Results1.Using ELISA the positive rate of anti-M₃ antibody in SS is 47. 10%（33/70）, in SLE is 4.02%（2/49） and in donator controls is 14.47%（11/76）.The rate in SS is much higher than in SLE（47.10% vs 4.02%,P<0.05）. Compared with patients in donator control the antibody is more frequently found in patients with SS（47.10% vs 14.47%, P<0.05）.2. Using Western the positive rate of anti-M₃ antibody in SS is 60.0%, in SLE is 12.2% and in normal controls is 15.79%. Compared with patients with SLE and donator controls, the anti-M₃ antibody in SS is more frequently found,P<0.05.3. The incidence of IgG,rheumatoid factor, anti-SSB and FI>3 are higher in positive group than in negative group using ELISA. And the differences are significant,P<0.05. But the differences between the two goups in Schirmer test,BUT,PEE on the corneas,the conjunctival epithelial defects, whole saliva flow rate in 5 minutes and anti-SSA are not significant.4. The data of passive transfer to mice indicates that the recipient mice of purified human IgG from anti-M₃ antibody positive SS（15mg/kg） develop glandular hypofunction which is similar to those recipient mice of atropine. But that is not similar to those recipient mice of IVIG.And the differences between them are significant.P<0.05Conclusions1. The positive rate of anti-M₃ antibody in SS is much higher than that in SLE and in normal controls.2. The IgG, RF, anti-SSB and FI in SS patients with positive anti-muscarinic 3 receptor antibody are much higher than in patients with negativeanti-muscarinic 3 receptor antibody.3. The data of WS mice experiments indicates that the anti-muscarinic 3 antibody may decrease the salivary secretion.更多还原