【作者】 杜勇；

【导师】 张金廷；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的:口腔扁平苔藓(oral lichen planus ,OLP)是口腔粘膜一种常见的非感染性慢性炎症,目前被认为是细胞介导的自身免疫性疾病,于1869年被首次报道,人群发病率国内文献报道约为0.5%-1%,国外文献报道约为0.5%-2.0%。1910年Hallopean首次报道OLP癌变,其后国内外研究相继表明OLP具有癌变倾向,其癌前性质引起广泛关注。1996年WHO正式将OLP确定为癌前状态。研究表明,OLP癌变是多种因素共同作用的结果,在局部免疫制剂、病毒感染、致癌剂和细胞因子等因素的共同作用下,组织发生癌基因活化、染色体突变和抑癌基因的异常,最终导致发生癌变。Fascin蛋白是一种细胞骨架蛋白,与F-肌动蛋白结合后使肌动蛋白形成有序的束状结构,加强肌动蛋白细胞骨架,抵抗细胞迁移时由细胞突起的运动引起的细胞膜延伸,对细胞移动起着重要作用。Fascin蛋白的异常表达可引起肿瘤细胞运动性显著增强和细胞粘附能力的改变,使肿瘤细胞具有更强的侵袭性,进入淋巴间隙,淋巴管和血管,增加转移几率。研究发现fascin蛋白在多种肿瘤组织和癌前病变组织中呈现阳性表达,其阳性表达的肿瘤组织学分级或者侵袭性往往较高。进一步的研究发现fascin蛋白在口腔白斑和口腔鳞状细胞癌中呈现过高表达。E-钙粘蛋白(E-cadhernin,E-cad)是介导上皮细胞间粘连的一种钙依赖性的细胞粘附分子,编码基因定位于16q21,是一种重要的细胞粘附分子,参与组织形态发生、胚胎发育过程。在生理条件下调节细胞与细胞之间、细胞与基质之间的粘附反应,维持组织结构形态和完整性。研究发现E-钙粘蛋白是一种重要的抑癌分子,在肿瘤的发生、发展过程中发挥着重要的作用,主要通过加强细胞间的粘附和抑制细胞增殖来发挥抑癌功能。E-cad在多种肿瘤中呈现不表达或表达下降,与肿瘤的分化程度、侵袭能力、淋巴结转移、远处转移的发生和复发率密切相关,在预示肿瘤进展及患者预后方面发挥着重要的作用。本研究通过免疫组化的实验方法检测fascin蛋白和E-钙粘蛋白在口腔扁平苔癣中的表达,进一步探讨口腔扁平苔癣癌变的发病机制,为扁平苔藓癌变的早期诊断和治疗提供依据。方法:本研究使用52例口腔扁平苔藓标本蜡块和41例口腔正常粘膜标本蜡块,口腔扁平苔藓标本均经病理检查证实。运用SP免疫组织化学法,以fascin单克隆抗体和E-钙粘蛋白单克隆抗体作为一抗,对切片标本进行免疫组化染色。以磷酸盐缓冲液(PBS)代替一抗作为阴性对照,用已知的fascin蛋白阳性的口腔鳞癌标本和已知的E-钙粘蛋白阳性的口腔扁平苔癣标本作为阳性对照。观察阳性染色的分布及强度,对表达情况进行研究分析。Fascin蛋白以细胞膜和细胞浆显示弥漫性棕黄色颗粒为阳性染色,每张切片随机计数10个高倍视野的1000个细胞中阳性细胞所占百分比。将表达情况分为4级:(1)未见棕黄色颗粒为阴性;(2)阳性细胞数<25%为弱阳性(+);(3)阳性细胞数25%-75%为阳性(++);(4)阳性细胞数>75%为强阳性( +++)。E-钙粘蛋白以细胞膜上出现棕黄色膜性染色作为正常表达的阳性标记。而位于细胞浆中或胞膜与胞浆共同有连续线状或斑点状黄色至棕黄色颗粒沉积者为异常阳性表达,无染色者为阴性表达。每张切片随机计数10个高倍视野的1000个细胞中阳性细胞所占百分比。将表达情况分为4级:(1)阴性:无染色,阳性细胞数<25%;(2)弱阳性:胞浆染色浅,阳性细胞数26%-50%;(3)中度阳性:胞膜胞浆混合染色,阳性细胞数51%-75%;(4)强阳性:胞膜连线状均匀染色,阳性细胞数>75%。采用SPSS17.0软件进行统计学分析,单因素研究实验结果采用χ2检验,P<0.05有统计学意义。Fascin及E-cadherin表达的相关性采用相关分析法,P<0.05有统计学意义。结果:1、Fascin蛋白的表达:在41例正常口腔粘膜标本中均呈阴性表达,在52例口腔扁平苔藓标本中5例呈现弱阳性表达,其余均呈现阴性表达,阳性率为9.62%,阳性率高于正常口腔粘膜。经统计学分析χ2值为4.166,P<0.05,有统计学意义。2、E-钙粘蛋白的表达:在41例正常口腔粘膜标本中39例呈强阳性表达,2例呈中度阳性,异常阳性率为4.88%,在52例口腔扁平苔藓标本中25例呈强阳性表达,27例呈现异常阳性表达,异常阳性率为51.92%,经统计学分析χ2值为23.64,P<0.05,有统计学意义。3、经相关性分析,fascin蛋白和E-钙粘蛋白在口腔扁平苔藓中的表达无相关性。(r=0.078 P>0.05)结论:1、在口腔扁平苔藓标本中fascin蛋白阳性表率高于正常粘膜,经χ2检验具有统计学意义,提示口腔扁平苔藓潜在恶变的可能。2、在口腔扁平苔藓标本中E-钙粘蛋白的膜性表达率明显低于正常口腔粘膜,膜性表达的丧失提示口腔扁平苔藓潜在恶变的可能。3、Fascin蛋白和E-钙粘蛋白在口腔扁平苔藓中的表达无相关性。(r=0.078 P>0.05)更多还原

【Abstract】 Objectives: Oral lichen planus(OLP) is a commonplace non-infectious chronic inflammation of the oral mucosa. Initially reported in 1869, it is regarded as a cell-mediated autoimmune disease at present with the recorded incidence of 0.5%-1% in domestic resources and 0.5%-2.0% in foreign reference. In 1910 the carcinogen tendency of OLP was revealed for the first time by Hallopean and demonstrated by successive researches home and abroad. This characteristic of OLP has been in the spotlight since then. OLP was defined as precancerous state by WTO in 1996 officially. It has been found out that the carcinogen of OLP was the combined result of multiple factors. In the combined action of local immunologic agent, virus infection ,carcinogenic agent, as well as cytokine, the tissue goes through a series of changes namely gene activation , chromosomal mutation and anti-oncogene abnormality ,which ultimately lead to the occurrence of carcinogen.As a cell skeleton protein, Fascin protein is able to combine with the F-actin, resulting in a ordered sheaflike structure of F-actin which not only reinforces the cell skeleton of F-actin but also counteracts against the cell membrane extension caused by the movement of the membrane protuberances in the process of cell dislocation, which is of vital significance for cell dislocation. The abnormal expression of Fascin protein can directly bring about the prominent increase of the tumor cell mobility and the notable alteration of cell-cell adherence, leading to a greater invasiveness of the tumor cell into the lymph space, lymph vessels and blood vessels which raises the incidence of metastasis. It has been discovered that positive expression of Fascin protein are prevail in various tumor tissues and precancerous tissues, often with a higher pathological grade or invasiveness. Further studies have affirmed that Fascin protein presents an over=expression in oral leukoplakia and oral squamous carcinoma, which is often the case.E-cadherni is a Calcium-dependent cell adhesion molecule mediating the epithelium attachment, with a code gene localized at 16q21. It is considered as a key cell adhesion molecule in terms of contributing to the tissue and embryonic development, regulating the cell=cell and cell-stoma attachment in physiological conditions, as well as maintaining the structure, form and the integrity of the tissue. Regarded as an essential cell adhesion molecule as well as a signal transductor, epithelial cadherin takes part in the processes of tissue morphogenesis and embryonic development. It possesses the functions of regulating the cell-cell and cell-matrix attachment, stabilizing the cell polarity, contributing to the modulation of cell differentiation as well as maintaining the organization structure form and integrity of the tissues. The absence or decrease of E-cad expression attach great relationship with the occurrence and recurrent of differentiation degree, invasiveness, lymphatic metastasis, distant metastasis of tumor, which plays an important role in predicting the carcinogenic progression as well as the patient prognosis.Our research is to further discuss the mechanism of OLP carcinogenesis and to provide evidence for an early diagnosis and treatment of OLP carcinogenesis by detecting the fascin protein and E-cadherin expression in OLP with immunohistochemical method.Method: Taking 52 cases of slice specimens containing OLP, which have been confirmed in pathological examination, as well as 41 normal mucous tissue, our research makes utilization of monoclonal antibodies of Fascin and E-cadherin as primary antibodies to dye the section specimen with SP immunohistochemical method. With phosphate buffer solution(PBS) as negative control while the known OSCC section with positive Fascin as well as the known OLP section with positive E-cad as positive controls, observations are made as to the distribution and intensity of positive stains as well as the stain changes on the cell membrane, cytoplasm and nucleus.The positive stain for Fascin protein is defined by the manifestation of diffuse brownish particles on the membrane and in the plasma. Take records of the ratio of positive cells out of 1000 cells under 10 randomly-chosen high power fields on each piece of section.4 grades can be classified according to the expression level:(1) negative is defined as no manifestation of brownish particles at all (2) mild positive is defined as <25% positive cells (+)(3)positive is defined as 25%-75%(++)(4)strong positive is of >75% (+++)E-cadherin presents itself as a normal positive remark in the form of brownish membranous stain on the surface. Alternatively, abnormal positive is identified by the expression of the accumulation of linear or dotted yellow to brownish particles in plasma and/or on membrane. No-stain is considered as negative expression. Again, take records of the ratio of positive cells out of 1000 cells under 10 randomly-chosen high power fields on each piece of specimen;There are 4 grades: (1)negative: cells with no stain<25%;positive cells>75%(2)mild positive: the plasma is tinted slightly;(3) medium positive: mixed stain of both the membrane and plasma; 51%~75% (4) strong positive: lineated homogeneous stain of the membrane.Results:1. The expression of Fascin protein: negative is seen in all the 41 normal oral mucous specimen, while mild positive is seen in 5 OLP cases out of the total 52 cases with the rest are all negative and a positive rate of 9.62%, higher than the normal cases.χ2 value is finalized as 4.166 in terms of statistic analysis. The result of P<0.05 makes statistic significance.2. The expression of E-cadherin: Strong positive is observed in 39 cases out of 41 normal oral mucous specimen, the other two medium positive, with an abnormal positive rate of 4.88%; Strong positive is seen in 25 cases out of 52 OLP cases, the rest 27 medium positive, with an abnormal positive rate of 51.92%.χ2 value is finalized as 23.64 in terms of statistic analysis. The result of P<0.05 makes statistic significance.3. There is no obvious pertinences between fascin protein and E-cadherin expression in either normal or OLP oral mucous by means of rank correlation analysis.Conclusion:1. The positive rate of Fascin expression stands higher in OLP mucous than normal ones, which makes statistic significance, indicating that OLP possesses the potential of malignant change.2. The positive rate of E-cadherin membranous expression stands evidently lower in all OLP mucous specimens than normal ones. The loss of membranous expression is suggesting that OLP possesses the potential of malignant change.3. There is no correlation between the expression of Fascin and that of E-cad in OLP. (r =0.078 P>0.05)更多还原