【作者】 华山；

【导师】 戚向敏；

【作者基本信息】 山东大学 ， 口腔临床医学， 2010， 硕士

【摘要】 口腔扁平苔藓(oral lichen planus, OLP),是口腔黏膜病中最常见的疾病之一,部分病损可发生恶变,世界卫生组织(WHO)将其列为可能的癌前状态。OLP的病因至今尚不十分清楚,目前认为是一种T细胞介导的免疫反应,其病程慢性迁延,治疗已成为临床上一个相当棘手的问题,目前还没有治愈OLP的方法。其典型病理特征为上皮不全角化,基底层液化变性,固有层密集的淋巴细胞浸润带。OLP淋巴细胞浸润带中虽以T细胞为主,但也有少量的B淋巴细胞,约6%。CD20是B淋巴细胞特有的表面标记,它出现于前B淋巴细胞,B淋巴细胞激活分化将导致CD20的高表达,B淋巴细胞分化至成熟的浆细胞时,CD20表达消失。单核细胞、静息或活化的T细胞、裸细胞及非淋巴细胞均不表达CD20分子。IL-12是一种p35与p40结合成的异二聚体分子,在B淋巴细胞、单核细胞、角质细胞以及DC中都有表达活性,其趋化作用主要由p40来完成。IL-12在Thl细胞介导的免疫反应和炎症性疾病中发挥着重要作用。目的：本实验通过免疫组化技术及荧光实时定量PCR技术检测正常口腔黏膜与OLP中B淋巴细胞及IL-12的表达情况,以观察B淋巴细胞及IL-12在两种不同组织中的变化规律,探讨其在口腔扁平苔藓发生过程中的作用及意义,为临床上治疗口腔扁平苔藓提供新的思路。方法：1.收集临床及病理确诊的口腔正常黏膜5例、OLP标本20例,将每个标本的连续切片分别进行HE染色、CD20及IL-12p40免疫组化分析和阴性对照,根据免疫组化结果从蛋白水平观察CD20、IL-12p40在不同组织中的表达情况。2.收集正常口腔粘膜组织和临床及病理确诊的OLP各6例,应用PCR技术检测标本中IL-12的表达,验证引物设计合成物无误后,进行荧光实时定量PCR,检测IL-12p40 mRNA在两组中的表达情况。结果：1.CD20免疫组化结果显示：在正常口腔黏膜中,CD20呈阴性表达。在OLP组织中,CD20在固有层淋巴细胞浸润带中呈阳性表达,且远离基底膜侧。2.IL-12p40免疫组化结果显示：在正常口腔粘膜中,IL-12p40呈阳性表达,主要位于棘层及颗粒层细胞以及结缔组织中,其染色略浅。在OLP组织中,IL-12p40呈强阳性表达,其表达范围与正常口腔黏膜中的相似,但其染色较正常口腔黏膜染色要深。3.IL-12p40荧光实时定量PCR结果显示：与正常口腔粘膜上皮及结缔组织相比,口腔扁平苔癣上皮及结缔组织中IL-12p40 mRNA的表达量增多,差异有统计学意义(P<0.05)。结论：CD20在正常口腔粘膜组没有表达,而在OLP组织有表达。IL-12p40 mRNA及蛋白在正常口腔粘膜组的上皮细胞和结缔组织中的表达分别低于其在OLP组织中的表达。提示B淋巴细胞在口腔扁平苔癣的发生发展过程中可能发挥一定的作用——不仅可能与基底膜区IgG、IgM:冗积有关,还可能通过分泌IL-12来促进Thl细胞分化进而参与细胞免疫,为临床上预防及治疗OLP提供新的思路。更多还原

【Abstract】 The prevalence of oral lichen planus (OLP) ranks second in oral mucosal diseases. Partial cases can transform from OLP into cancer. Accordingly, the World Health Organization(WHO) presume this disease a possible precancer. The etiology and pathogenesis are still obscure. There are extensive data to suggest that OLP is a T cell-mediated immune damage. Much effort has been spent on OLP therapy, but the outcome is still far from satisfaction. Histopathologic examination typically shows orthokeratotic hyperkeratosis, basal cell degeneration, and a dense well-defined infiltrate of lymphocytes in the superficial dermis. In OLP, there is a few B lymphocytes in the lymphocyte infiltration. CD20 is the surface marker of B cell especially. IL-12 is excreted by B cell, mononuclear cell, keratinocyte and DC. IL-12 play a unique role in promoting type 1 T helper cell responses and, thereby, cell-mediated immunity. IL-12 has been shown to exert striking therapeutic effects in a variety of infectious diseases.Objective:To detect the different expression of B cell, IL-12 in the tissue of normal oral mucosa and OLP at the level of protein by Immunohistochemistry and real time PCR respectively. And it is to be discussed about the function and mechanism involved with B cell and IL-12 in OLP. Different treatment means may be attempted.Methods:1. The study group consisted of 25 cases, included 20 cases of OLP and 5 cases with normal oral mucosa. These specimens were all defined by clinical and pathological diagnoses. Every specimen would be seriate cut for HE staining, Immunohistochemical staining of CD20 and IL-12p40, negative contrast,respectively. We studied the expression of CD20 and IL-12 in different tissues according to the results at the levels of protein by IH.2. OLP (n=6) and normal control (n=6) tissues were defined clinically and pathologically. To detect the expression of IL-12 using PCR technique and real-time PCR.3. To observe the distribution of CD20+B lymphocyte and IL-12 in OLP and normal oral tissue. To analyze the data of IL-12p40 mRNA using statistically system.Results:1. IH results of CD20:In normal mucosa, CD20 was negative.In OLP tissue, CD20 was positive in the lymphocytes infiltration.2. IH results of IL-12p40:In normal mucosa, IL-12p40 was positive in spinous layer cells, granulosa layer cells and connective tissues. In OLP, IL-12p40 had similar expression with normal mucosa in epithelium but with a stronger staining.3. Compared to normal oral mucosa, the expression of IL-12p40 mRNA is higher in OLP. Values of P<0.05 were considered statistically significant.Conclusion:CD20 in the tissue of OLP showed a strong expression, while none in the normal oral mucosa. IL-12p40 mRNA in the tissue of OLP shouwed a stronger expression than that in the tissue of normal oral mucosa. CD20+B cell may play a unique role in OLP development and progression by excreting IL-12.更多还原