【作者】 魏群；

【导师】 赵玉铭；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2004， 硕士

【摘要】 前言 皮肤是机体的重要免疫器官，具有多种免疫功能，构成机体抵制外界微生物侵犯的重要一线屏障。近年来越来越多的研究发现表皮朗格汉斯细胞(LC)及树突状表皮T细胞(DETC)与皮肤免疫系统(SIS)密切相关。而许多现象证明毛囊在SIS中占重要地位。以往很多文献报道了毛囊相关免疫系统包括黑素细胞、角朊细胞、神经因子、细胞因子等影响毛囊生长周期，进而影响SIS。我们也曾报道了MHC-Ⅱ及和Thy-1抗原在毛囊周期中的表达及作用。那么LC及DETC在SIS中的作用是否与毛囊周期具有相关性呢?本研究通过动态观察C57BL/6小鼠拔毛诱导的毛囊周期中毛囊间表皮LC及DETC数量和形态的变化，进一步探讨免疫细胞与毛囊周期及SIS的关系和作用。 材料和方法 1．动物：C57BL/6纯系小鼠15只，6-8周龄，体重15-20克。选择皮色粉红(毛发处于休止期)的小鼠。 2．试剂：所用单克隆抗体分别为：OX3、OX4及JIJ、22．1。ABC试剂盒(含生物素化马抗鼠IgG)。 3．方法：小鼠分3组，每组5只(3雌2雄)，将松香/蜡1：1混合，涂于小鼠背部以拔毛，隔天取皮肤标本，应用ABC法进行免疫组化染色。 4．观察指标：(1)每天观察小鼠皮色变化。(2)光镜下观察拔毛前、后不同时间内小鼠表皮免疫细胞的染色情况。 结果 1．小鼠皮色变化：拔毛后第1-4天内，小鼠皮肤呈粉红色；第5-6天鼠皮变黑色；第9-11天局部可见新毛长出；至17-19天鼠皮变灰色；第20天左右鼠皮恢复到原先的粉红色。 2.表皮LC及DETC变化。 (l)密度变化:拔毛后第2天开始二者密度较拔毛前升高(P<0.05)，，拔毛后第4一8天二者在表皮中密度最高(P<0.01)，以后有所下降，直至第16天，但仍较拔毛前升高(P<0.05);拔毛后第17一20天，表皮毛囊间LC及DETC密度基本恢复到拔毛前数值，二者之间的差异无统计学意义(P>0 .05)。 (2)形态变化:拔毛前、后3天内，多数LC及DETC胞体小、圆形，树突短小，1一2个;第4一8天，二者多数细胞体大，圆或不规则形，树突粗大，约3一5个，有的有分枝或次级突起;第9一16天，胞体大小不一，树突变细，2-4个;17一20天，多数胞体较小甚至消失，树突纤细，长短不一，约2一3个。讨论 小鼠毛发周期分为退行期、休止期及生长期。在对拔毛诱导C57 BU6小鼠毛发生长周期中，以往文献已报道了毛囊的显著形态学变化，即拔毛后第2一4天内，毛囊上皮细胞处于增生分裂状态，第5天达生长高峰，出现黑色素及新生毛发，鼠皮呈黑色(生长期);以后是增生平稳状态，至17一19天出现退化改变，黑素生成减少，鼠皮呈灰色(退行期)，直到第20天则停止生长，鼠皮亦恢复到原先的粉红色(休止期)。可根据皮肤颜色变化判断毛囊周期的变化。以上这些拔毛诱导的鼠毛发生长周期中毛囊的形态学变化与自然状态下的鼠毛发生长周期中毛囊的形态学变化基本一致。 表皮LC是骨髓来源的树突状细胞，主要位于毛囊及基底细胞之上的表皮层内，在常规HE染色的石蜡切片中为透明细胞而很难辨认，用氯化金及ATP酶等特殊染色可以表现为树突状细胞。LC是表皮细胞中唯一具有补体C3及Fc一IgG受体以及Ia抗原的细胞，因此可用免疫组化方法将其显示。许多与免疫有关的皮肤病有LC改变，特别是在迟发超敏反应(DTH)及覃样肉芽肿(MF)的发病机理中占重要地位，同时LC与皮肤移植物排斥有关。人表皮内此细胞约有460一1000个/~2，真皮内也偶可见到。LC具有迁移吞噬能力，能摄取、处理、携带并递呈抗原到局部淋巴结，激活淋巴细胞。但LC密度在毛囊周期中如何变化目前尚报道，此研究有利于进一步探讨免疫细胞与毛囊周期及515的关系和作用。 DETC是一种存在于小鼠表皮内的表达，盯细胞受体(TCR)的T细胞，此细胞具有Thy一1(CD90)、CD3及CD45，呈树突状外观。现已证实Thy一1+的DETC均匀分布于小鼠毛囊间表皮内，缺乏循环T细胞具有的apTCR、CD4或CDS，它可以被IL一2及Con一A激活而具有辅助T细胞作用，并能分泌IL一2及，一IFN。DETC是一种重要的表皮免疫细胞。半抗原化表皮细胞富含DETC时可使动物耐受。目前认为DETC是一个重要的表皮免疫效应细胞，DETC在不同品系小鼠及不同部位存在明显差异，这些均表明DETC与515关系密切。 本实验选用C57 BU6小鼠建立‘毛囊周期。自动发育的毛囊周期同步性差，拔毛可快速刺激表皮增生及毛发生长。本研究通过拔毛诱导小鼠毛囊进人生长期、退行期直至休止期的变化，动态观察了此三阶段变化过程中毛囊间表皮LC及DETC的密度及形态变化。结果显示毛囊间表皮LC及DETC于拔毛后第2天开始密度值升高，第4一8天出现最高值，以后逐渐下降，第17一20天密度逐渐恢复到拔毛前数值，且二者形态也有相应变化。表明毛囊间表皮内LC及DETC于毛囊生长期密度最高，树突多且有的有分枝;退行期下降，至休止期稳定，树突也变少。 拔毛破坏毛囊完整性的同时，必然对皮肤产生一定影响。表皮中LC及DETC的动态变化可能是作为一种抵御外界有害刺激的因素之一。引起此变化的机理还不清楚，我们考虑主要有两点:更多还原

【Abstract】 IntroductionSkin is one of the important immune organ, it has many kinds of immune functions; it is the first protective screen. Recently, more and more studies indicate that epidermal Langerhans cells ( LC) and dendritic epidermal T cells ( DETC) are tightly correlated with skin immune system (SIS) , in which the hair follicle ( HF) plays a significant role. HF correlated immune system including nerve factors ,cytokine,melanocyte,keratinocyte affects HF growth cycle and SIS mentioned in many previous literatures, furthermore, we reported the expression of MHC - II and Thy - 1 antigen in murine hair cycle. Our studies chiefly applied the immunohistochemical technique to observe the dynamic changes of the density and form of LC and DETC in epidermis among the hair follicles during HF cycle, so as to discuss the relation and interaction among immune cells ^follicle cycle and SIS.Materials and MethodsSkin of syngeneie C57BL/6 mice (9 female, 6 male. 6 -8 w old; 15 -20 g weight. ) were obtained from mice with all back skin HF either in telogen or in defined stages of the induced hair cycle. Anagen was induced by depilating hair shafts on the back of mice with all follicles in telogen when skin was identified by its uniformly pink color.Skin color was observed daily, expression of LC and DETC was observed every other day. The reagents were shown as.- McAb 0X3 and 0X4 (defending Ia antigen) , 22. 1 and JIJ (defending Thy -1 antigen) , ABC Reagent Box.Results1. Mice skin color change: within the day 4 after depilation, the mice skin color showed pink; on the day 5-6 showed black; on the day 9 -11 the local grew out new fair; by the day 17 - 19 the mice skin come into gray; on the day 20 the skin come back to former pink,.2. LC and DETC changes:(1) Density changes: There were not distinct changes of LC and DETC on the day 1 after epilation compared with that of before epilation. Their density began to rise from the day 2 (P < 0. 05) , and attained the top value on the day 4 -8(P<0.01) , and then descended; On the day 17-20, the density of LC and DETC resumed the value of before epilation.(2) Form changes: The most cell bodies of LC and DETC were small and round with 1-2 short dendrites on the day 1 after epilation; the most cell bodies of both were big and round or annomal with 3-5 crasstude dendrites and some had divarication or hypo - apophysis on the day 4 - 8; the cell bodies were different in size with thinner 2 -4 dendrites on the day 9-16; the most cell bodies were small even vanished with sligh and different length dendrites on the day 17-20.DiscussionThe mice follicle cycle is artificially devided into canagen ^telogen Nanagen. Previous scholars described the obvious form changes of hair follicle by depilating C57BL/6 mice telogen hair shafts; within the day 1 after depilation, the histology morphology of HF had no obvious difference from the telogen before depilation. Skin color also was pink (telogen). From the day 2 after depilation HF and epidermis both showed evident growing state. Nearly on the day 5, the proliferation changes come to the peak and the melanin and the new hair formation cound be seen, skin color also was black ( anagen) ; Later was the stable proliferation stage. After the day 17, the mophology of HF showed regressionchanges, the melanin formation depressed and disapeared on the day 20, the skin color turned from gray ( canagen) to original black (telogen).LC derives from marrow with dendritic appearance, and mainly is located in HF and epidermal base cells superior stratums. LC expresses complement c3,Fc - IgG receptor and la antigen, and can be showed by immunohistochemical technique. The function of LC includes two respects: one is taking part in the kerotosis progress of keratinocytes and controling it; the other is playing an important role in SIS. Especially LC is tight correlated with the pathogenesis of DTHUMF and skin graft repulsion. There are about 460 - 1000/mm LCs in human epidermis, which take and present antigens to local ly更多还原