【作者】 于淑萍；

【导师】 王玉坤；

【作者基本信息】 山东大学 ， 皮肤病与性病学， 2009， 硕士

【摘要】 研究背景:蕈样肉芽肿（Mycosis Fungoides,MF）是原发于皮肤的亲表皮性皮肤T细胞淋巴瘤（cutaneous T cell lymphoma,CTCL）,系一种最常见的低度恶性皮肤非霍奇金淋巴瘤。其早期局限于皮肤,随着病情的缓慢发展,晚期可侵犯淋巴结及内脏系统。MF的发病机制目前仍不清楚,讫今无满意的治疗方法。免疫学异常与其发病密切相关,表现为患者皮损中树突状细胞分布和功能异常,T细胞凋亡障碍,Fas表达下降和FasL表达增加,趋化因子及其受体、IL-4、IL-15、CTLA-4、HECA-452等免疫分子异常表达。其中T细胞的凋亡障碍导致肿瘤细胞的凋亡减少,从而促进MF的发展。亲表皮现象是MF组织病理学的特征之一,这提示表皮细胞与淋巴细胞的相互作用可能在MF的发病中发挥一定作用。角质形成细胞（keratinocyte,KC）是人体表皮中的主要细胞,具有一定的抗原递呈作用,主动参与皮损中T细胞的再活化。同时其分泌的细胞因子在皮肤免疫系统中也发挥重要作用,MF时角质形成细胞中某些免疫分子的表达存在着显著不同,如IL-15蛋白的高度表达是皮肤T细胞淋巴瘤的一个特征。PDCD4是一种抑癌基因,PDCD4蛋白是细胞凋亡所需要的新生的大分子复合物之一。其通过抑制肿瘤细胞的转录和翻译过程中起始复合物的形成抑制肿瘤细胞的生长。多种肿瘤细胞中PDCD4蛋白表达降低甚至缺失,并且随着肿瘤恶性程度的递增以及患者生存时间的减少,PDCD4的表达发生丢失情况也不断加重,证明PDCD4参与了多种肿瘤的发生和发展,在抑制肿瘤的形成中发挥了重要作用。目前已有PDCD4缺陷鼠自然发生淋巴瘤并且寿命显著缩短的报道,而且大多数肿瘤来源于B细胞,并不断转移到肝和肾。证明PDCD4在网状内皮系统肿瘤中也发挥了一定作用。关于皮肤T细胞淋巴瘤中该因子的表达及其影响的研究少见报道,表皮细胞在MF发病中的作用研究也较少。Ki67抗原简称Ki67,是一种细胞核抗原,仅在增殖细胞核中表达,其单克隆抗体能标记G₁后期、S期、G₂期和M期细胞核,而G₀期和G₁早期细胞核不被标记。Ki67是目前应用最广泛的增殖细胞指标之一,是较为敏感的细胞增生分化指标。Ki67的测定是评估人类各种组织增殖组分可靠且简单的方法。为此我们检测了PDCD4和Ki67蛋白在不同期蕈样肉芽肿的表皮及真皮中的表达,以正常皮肤为正常对照,以扁平苔藓为炎症对照,探讨PDCD4在MF发病中的作用,进而初步了解角质形成细胞在MF发病及病情进展中的作用。目的:检测并比较程序性细胞凋亡因子（PDCD4）、Ki67蛋白在正常皮肤、蕈样肉芽肿（MF）及扁平苔藓皮损中的表达,探讨程序性细胞凋亡因子在正常皮肤组织、炎症性皮肤病、不同期蕈样肉芽肿中的表达差异及临床意义,并比较两蛋白的相关性。材料和方法:1.收集齐鲁医院皮肤科近10年保存的经临床及病理确诊的27例MF完整蜡块,其中红斑/斑块期MF21例,肿瘤期MF6例;取门诊手术室及整形美容外科切除正常皮肤5例进行甲醛固定与石蜡包埋,作为正常对照组;另取皮肤科病理室保存的扁平苔藓石蜡标本5例,作为炎症对照组。2.采用免疫组化方法检测并比较PDCD4及Ki67在不同期MF、LP及正常对照组中的表达情况。3.应用JMT JFX简明统计分析软件10.34进行处理,统计方法采用成组设计多样本比较的秩和检验或方差分析;两蛋白的相关性用Spearman’S等级相关检验进行分析,P＜0.05为差异有统计学意义。结果:1.PDCD4在各组皮损中免疫组化染色位置及着色强度结果:·5例正常对照组的表皮细胞和真皮浸润的淋巴细胞中PDCD4染色均为强阳性,多数为棕褐色,细胞核着色;·5例扁平苔藓炎症对照组的表皮细胞和真皮浸润的淋巴细胞呈弥漫阳性,棕黄色,分布较密集,胞核染色;·21例红斑/斑块期MF的表皮阳性细胞数量较正常对照组和扁平苔藓组略减少,胞核、胞浆均有阳性染色,为棕黄色或棕褐色;真皮中阳性细胞较前正常对照组和扁平苔藓组明显减少,胞核、胞浆染色,为棕黄色或淡黄色;·6例肿瘤期MF表皮及真皮中阳性细胞均明显减少,散在分布,胞核或胞浆染色,部分真皮中PDCD4表达缺失。·移入表皮的淋巴细胞不染色,真皮浅层距离表皮较近的淋巴细胞染色较淡或不染色。2.各组PDCD4表达阳性等级的比较结果:（1）表皮中:①正常对照组、红斑/斑块期MF组到肿瘤期MF组,PDCD4表达从强阳性到阴性,呈梯度式减弱。红斑/斑块期MF组与正常对照组比较,差异无统计学意义（P＞0.05）,其余各组比较,差异有统计学意义（P＜0.05）;②扁平苔藓组、红斑/斑块期MF组到肿瘤期MF组,PDCD4表达从强阳性到阴性,呈梯度式减弱。其中,红斑/斑块期MF组与扁平苔藓组比较,差异无统计学意义（P＞0.05）,肿瘤期MF组与扁平苔藓组比较,差异有统计学意义（P＜0.05）。（2）真皮中:①正常对照组、红斑/斑块期MF组到肿瘤期MF组,PDCD4表达从强阳性到阴性,呈梯度式减弱。各组间差异均有统计学意义（P＜0.05）;②扁平苔藓组、红斑/斑块期MF组到肿瘤期MF组,PDCD4表达从强阳性到阴性,呈梯度式减弱。其中,红斑/斑块期MF组与扁平苔藓组相比较,差异无统计学意义（P＞0.05）,肿瘤期MF组与扁平苔藓组比较差异有统计学意义（P＜0.05）。3.Ki67蛋白在各组皮损中免疫组化染色位置及着色强度:均为细胞核染色·5例正常对照组皮肤部分基底细胞阳性表达,棕黄色;真皮中阴性表达;·5例扁平苔藓组表皮中基底层阳性细胞不连续性分布,基底细胞液化变性处表达减少,其余部分表达稍增多,均以基底层及其上细胞为主;真皮中阳性细胞较多,呈棕褐色或棕黄色,分布密集;·红斑/斑块期MF组基底层细胞阳性减少,表皮内稍增多,棕黄色;真皮中阳性细胞分布稀疏,棕黄色;·肿瘤期MF表皮阳性细胞增多不明显,棕褐色;真皮中阳性细胞分布较密集,细胞核大、异型者染色深。4.各组Ki67表达的阳性细胞数量比较结果:（1）表皮中:各组比较差异均无统计学意义;（2）真皮中:①正常对照组、红斑/斑块期MF组到肿瘤期MF组,Ki67的表达从阴性至强阳性,呈梯度式增强。各组间比较差异均有统计学意义（P＜0.05）;②扁平苔藓组分别与红斑/斑块期MF组和肿瘤期MF组比较差异均无统计学意义（P＞0.05）,5.PDCD4蛋白和Ki67蛋白的相关性分析:红斑/斑块期MF表皮中及真皮中PDCD4蛋白与Ki67蛋白的表达均无相关性（P＞0.05）;肿瘤期MF表皮及真皮中PDCD4蛋白与Ki67蛋白的表达均无相关性（P＞0.05）。结论:1、随着MF病期的进展,表皮角质形成细胞PDCD4的表达明显下调甚至缺如,提示角质形成细胞在MF的发病及病程进展中可能发挥了重要作用,具体作用尚需进一步研究。2、真皮中浸润淋巴细胞随着MF病期进展,PDCD4的表达明显下调甚至缺如,提示PDCD4与皮肤淋巴瘤MF的发生及病程进展密切相关,上调PDCD4的表达可能有助于抑制淋巴细胞的过度增殖,PDCD4有望成为皮肤T细胞淋巴瘤治疗的新靶点3、Ki67在正常皮肤、红斑/斑块期MF及肿瘤期MF真皮中的表达梯度上升,表明Ki67抗原与真皮淋巴细胞的增殖有关,但与扁平苔藓组相比差异并无显著性,提示单一Ki67抗原并不能作为判断细胞恶性增殖的指标。4、本实验的相关性研究并未发现各期MF皮损中PDCD4的表达与Ki67存在明显的相关性,提示与PDCD4相关的凋亡障碍与细胞的增殖不一定有直接联系。更多还原

【Abstract】 ObjectiveTo explore the expression of the Programmed Cell Death 4 and Ki67 antigen in the skin infiltrates of normal skin,skin lesions with mycosis fungoide (MF) in different stages and lichen planus(LP);And to explore the significance of the human programmed cell death 4 and Ki67 in MF.Methods1.27 cases diagnosed by clinic and pathology as granuloma fungoides in the last ten years in the department of Dermatology of QiLu Hospital were collected. As normal control,5 normal skin specimens obtained from clinic Operating Room and Plastic Surgery,were fixed by formaldehyde and embedded by paraffin.As inflammation control,5 lichen planus were obtained from Pathology Room of Dermatology.2.The expression of PDCD4 and Ki67 in epidermis and dermis was detected by immunohistochemistry technique.3.By using JMTJFX concise statistical analysis software 10.34,the expression of protein PDCD4 and Ki67 in epidermis and dermis was tested by rank-sum test of multisample by unitizing engineered or analysis of variance;The relevance of the two protein was analysed by Spearman’S rank correlation,the statistically significant difference is P＜0.05. Results1.Location and intensity of PDCD4 in immunohistochemical stainNormal control specimens were all strong positive,mostly with brownish palm color,and positive expression was located mainly in the nuclear.In LP,the keratinocyte and lymphocyte in dermis were widespreadly positive with buffy almost in nuclear,intense distribution.In erythema/plaque stage MF,slightly decreased positive cells with buffy or pallide-flavens in epidermis were observed and the positive expression was located in both nuclear and kytoplasm;obviously decreased positive cells in dermis with buffy or pallide-flavens were observed in both nuclear and kytoplasm.In tumor stage MF,significantly decreased positive cells in epidermis and dermis stained mostly in kytoplasm with diffused distribution,partly absent in dermis.2.Comparison of positive expression intensity among all groups(PDCD4).In epidermis,from normal control、erythema/plaque stage MF to tumor stage MF,or From LP、erythema/plaque stage MF to tumor stage MF,the expression of PDCD4 weakened gradiently.There was no significant difference in the number of PDCD4~+ cells between patch/plaque stage MF and normal control or LP(P＞0.05), and there was significant difference among the rest groups(P＜0.05).In dermis,from normal control、erythema/plaque stage MF to tumor stage MF,or From LP、erythema/plaque stage MF to tumor stage MF,the expression of PDCD4 weakened gradiently.There was no significant difference in the number of PDCD4~+ cells between patch/plaque stage MF and LP(P＞0.05),and there was significant difference in the rest groups comparison(P＜0.05).3.Location and intensity of Ki67:all in nuclearPositive basal cell with buffy in normal control was partially observed;and negative in dermis.In LP,the positive basal cells distributed discontinuously,the expression decreased where there were liquefaction of basal cells and increased in other place. Positive cells with buffy in dermis were more and intense. In erythema/plaque stage MF,positive cells with huffy decreased in basal layer and slightly increased in epidermis,while scattered in dermis.In tumor stage MF,positive cells with brown in epidermis scarcely increased while were intense in dermis,and cells obformed with karyomegaly anachromasis.4.Comparison of positive expression intensity among all groups(Ki67)In epidermis,there were no significant differences among all groups.In dermis,from normal control、erythema/plaque stage MF to tumor stage MF, the expression of Ki67 gradiently increased and there were significant difference among group comparison(P＜0.05).There were no significant difference between LP and erythema/plaque stage MF or LP and tumor stage MF(P＞0.05),while there was in latter two group comparison(P＜0.05).5.correlation analysis between PDCD4 and Ki67:There was on correlation of PDCD4 and Ki67 either in erythema/plaque stage MF or in tumor stage MF(P＞0.05).Conclusion1.The expression of PDCD4 in epidermis keratinocyte,with the progression of MF,obviously down regulated even absent,indicates that keratinocyte may play an important role in the pathopoiesis and progression of MF.But the detail role needs further investigation.2.The expression of PDCD4 in infiltrating lymphocyte of dermis,with the progression of MF,obviously down regulated even absent,indicates that PDCD4 intimately correlates with the pathopoiesis and progression of MF.It may contribute to inhibit excessly proliferation of lymphocyte by up-regulating the expression of PDCD4,and PDCD4 is expected to be a new target for treating CTCL.3.The expression of Ki67 in normal control、erythema/plaque stage MF and tumor stage MF rises up gradually,which suggests that Ki67 antigen relates to the proliferation of lymphocyte in dermis,but there is no significant difference with LP.So we consider that Ki67 alone can’t be the criteria to judge if cells proliferate malignantly. 4.No correlationship was observed between the expression of PDCD4 and Ki67, which suggests that apoptosis disturbance led by PDCD4 does not directly affect cells proliferation associated with Ki67.更多还原