【作者】 高昱；

【导师】 杨森； 张学军；

【作者基本信息】 安徽医科大学 ， 皮肤病学与性病学， 2002， 硕士

【摘要】 狼疮带试验(LBT)是系统性红斑狼疮(SLE)临床辅助诊断的重要方法之一。一般认为LBT是免疫复合物(IC)沉积于皮肤基底膜带(BMZ)所致，然而这些IC是循环IC在BMZ上的沉积还是循环自身抗体与BMZ原位自身抗原的局部结合尚不清楚。近十年的研究表明SLE血清中存在基底膜带抗体(BMZ-Ab)，但这些自身抗体是否参与狼疮带的形成，是否还有其他抗原抗体的参与还不明确。本课题旨在初步探索SLE狼疮带的形成机制，研究SLE皮肤BMZ-Ab以及dsDNA抗原或dsDNA-抗dsDNA抗体免疫复合物是否参与狼疮带的形成。我们采用直接和间接免疫荧光(IF)、免疫印迹(IB)及包埋后金标记免疫电镜(gold-IIEM)技术对15例活动期SLE患者(LBT阳性者10例，阴性者5例)进行研究。盐裂皮肤直接免疫荧光(DIF)(取其中10例SLE皮肤标本，LBT阳性者7例，阴性者3例)显示9／10例SLE忠者皮损有表皮或真皮侧荧光。盐裂皮肤间接免疫荧光(IIF)显示14／15(93． 3％)例SLE患者在BMZ有荧光沉积，并全部位于表皮侧。IB表明，15例SLE血清全部存在多种IgG或／和IgA类自身抗体与表皮或／和真皮提取物抗原反应，结合230kD、200kD、180kD、130kD、97kD等表皮提取物抗原，180kD、160kD、130kD、116kD、97kD、75kD等真皮提取物抗原。直接及间接gold-IEM下，金颗粒在BMZ的沉积部位相同，结合于BMZ各个层次。ds-DNA抗体与SLE患者及正常人皮肤反应结果相同，IF研究显示除表皮角质形成细胞核周边型荧光外还可见细胞膜弱荧光，未见BMZ荧光：IEM研究见BMZ基底细胞膜内少量金颗粒沉积；IB法示抗ds-DNA抗体与热分离真、表皮提取物抗原反应皆为阴性。本研究进一步证实了SLE血清中BMZ-Ab的存在，它可以结合多种表皮或真皮抗原，呈明显异质性；SLE BMZ-Ab的组织结合部位与狼疮带中免 — — 疫复合物的沉积部位一致，提示其参与了狼疮带的形成。ds－DNA抗体与SLE 患者皮肤BMZ呈阴性反应，而与患者及正常人表皮角质形成细胞膜有弱的 结合反应，提示 尔－DNA抗原或其免疫复合物可能不参与狼疮带的形成， ds－DNA抗体与表皮角质形成细胞膜间可能存在交叉反应。更多还原

【Abstract】 Lupus band test (LET) is one of the important assistant methods in diagnosing systemic lupus erythematosus (SLE), but the mechanisms of it’s formation is still unknown. It is well known that lupus band results from the deposition of immunocomplex. It is unclear the immunocomplex depositing at basement membrane zone (BMZ) is from blood circulation or is composed of circulating antibodies and BMZ autoantigiens in situ. Recent studies show thatthere are cutaneous basement membrane zone autoantibodies (BMZ-Ab) in seraof SLE patients. But it remains ambiguous if there are BMZ-Ab or otherautoantigens and antibodies in lupus band. This study was investigated to detectif BMZ-Ab and if DNA participate in the formation of lupus band in SLE.Immunofluorescence (IF), immunoblotting (IB) and gold-labeled indirectimmunoelectron microscope (gold-IIEM) techniques were utilized to localizeantigen recognized by BMZ-Ab and anti-dsDNA antibodies in 15 SLE cases (LBT are positive in 10 cases and negative in 5 cases). Direct immunofluorescence (DIF) staining showed that 9/10 cases of specimens hadfluorescence on epidermal or dermal side of salt-split skin. Indirectimmunofluorescence (IIF) staining revealed that 14/15 (93.3%) cases of SLE serashowed fluorescence along the roof of epidermal of salt-split skin. By IB analysis,all the 15 SLE sera have several kinds of IgG and/or IgA type autoantibodies which reacted with epidermal and/or dermal extracts., such as 230kD. 200kD, 180kD, 130kD, 97kD epidermal extracts and 180kD, 160kD, 130kD, 116kD, 97kD dermal antigens. By gold-DIEM and HEM analysis, gold particlesdeposited at about the same area of BMZ. Gold particles directed to every region of BMZ of either SLE patients skin or normal human skin. It got the same resultthat anti-dsDNA antibodies reacted with skin of both SLE patients and normal human. IF staining showed no fluorescence along BMZ, but revealed pemphigus-like plasmalemma hypofluorescence besides perinuclear stain of keratinocytes. By IBM analysis, a few gold particles deposited at basal plasmalemma of BMZ. By IB analysis, anti-dsDNA antibodies showed reactivity with neither epidermal nor dermal antigens.This study further comfirms the existence of BMZ-Ab in SLE sera. These antibodies show obvious heterogeneity for their reaction with many kinds of dermal or epidermal antigens. The tissue-binding deposion of BMZ-Ab is identical with that of immunocomplex in lupus band, which suggests BMZ-Ab participate in the formation of lupus band. We didn’t detect any positive reactions along dermoepidermal junction using nti-dsDNA antibody reacted with skin of SLE patients, but found a weak reaction on plasmalemma of keratinocytes whether use SLE skin or normal human skin as substrates. These results hint that dsDNA or the immunocomplex of dsDNA and anti-dsDNA antibodies might not take part in the formation of lupus band and there may be a cross-reaction between anti-dsDNA antibody and plamalemma of keratinocytes.更多还原