【作者】 徐敏；

【导师】 纪岩文； 张理涛；

【作者基本信息】 天津医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 目的:探讨检测T细胞受体基因重排用于蕈样肉芽肿的辅助诊断。蕈样肉芽肿(Mycosis Fungoides,MF)又名蕈样霉菌病,为原发于皮肤的T淋巴细胞瘤,大多疾病呈慢性经过。临床上典型的皮损依病程分3期:红斑期、斑块期和肿瘤期。但组织学上的改变直至斑块期才有诊断价值,出现浸润细胞亲表皮现象、Pautrier微脓疡和MF细胞。肿瘤期由于真皮大量异型性MF细胞浸润,诊断相对容易。红斑期皮损临床表现复杂,可呈副银屑病、银屑病、玫瑰糠疹、特应性皮炎、鱼鳞病、扁平苔藓和硬化样改变。红斑期组织学改变和临床一样,大多为非特异性。由于早期诊断的MF病例得到及时治疗有良好的预后,因此早期诊断是一个非常重要的临床课题。免疫组织化学技术检测MF皮损中的T淋巴细胞分化抗原虽可作为辅助手段,但多难以确定皮损的良恶性。恶性肿瘤的一个显著特点是增生的单克隆性,在淋巴瘤表现为淋巴细胞抗原受体分子重排的单克隆:B细胞淋巴瘤为免疫球蛋白基因单克隆性重排,T细胞淋巴瘤为T细胞受体(TCR)单克隆重排。根据这一特点人们应用PCR技术对TCR-γ基因重排进行研究,发现在皮肤T细胞淋巴瘤有很高的特异性,结合临床表现和组织病理,使阳性率明显提高。方法:采用“一步法”提取10%福尔马林固定,石蜡包埋处理的皮肤活检组织的DNA作为模板,应用聚合酶链反应/琼脂糖凝胶电泳方法,检测实验组及对照组的TCR基因重排。其中实验组包括61例蕈样肉芽肿,对照组包括9例亚急性皮炎、8例扁平苔癣、7例点滴状副银屑病、7例急性痘疮样苔癣状糠疹及20例来源于整形外科切取的外观正常皮肤组织。结果:经统计分析后发现2号及3号引物的x~2值分别为0.000及0.127,P值分别为1.000和0.722,P值均大于0.05,无统计学意义,故在进行最后的统计中去除这两组。对1号,4号引物的分析结果如下:其中实验组1号阳性例数为34例,4号阳性例数为12例,两者同时阳性例数为4例。故蕈样肉芽肿组的阳性例数为50例(82.0%),阴性例数为11例;对照组的阳性例数为13例(41.9%),阴性例数为18例。蕈样肉芽肿组与对照组进行统计分析x~2=15.260,P=0.000,有统计学意义。结论:1.用“一步法”提取MF石蜡包埋DNA作为模板可用于PCR/琼脂糖凝胶电泳技术检测TCR基因重排。且本方法省时省力,最大程度的避免在提取过程中DNA的损失。2.红斑期蕈样肉芽肿出现典型病理特征的比例较少,仅靠病理组织学诊断红斑期蕈样肉芽肿较为困难。斑块期和肿瘤期蕈样肉芽肿病理特征突出,诊断相对容易。3.研究结果显示61例蕈样肉芽肿患者中82.0%发生TCR-γ基因重排,其阳性率较高。4.对照组中亚急性皮炎、急性痘疮样糠疹和点滴型副银屑病中也都有阳性克隆,尤其是亚急性皮炎的阳性率较高,提示有出现假阳性的可能。5.应用PCR技术检测MF石蜡包埋组织的TCR-γ基因重排,并结合临床表现,病理和免疫组化等可用于MF的诊断。更多还原

【Abstract】 Objective:To detect the rearrangement of T cell receptor(TCR)gene for the diagnosis of mycosis fungoides(MF).Mycosis Fungoides is a low maliganent cutaneous T cell lymphoma,which has a variety of manifestation.It can be divided into 3 stages,including patche stage,plaque stage,and tumor stage.It can not be diagnosed easily by Histopathology until plaque stage.The presence of intraepidermal collections of atypical cells(Pautrier microabscesses)is a highly characteristic feature.In tumor stage,the dermal infiltrates become more diffuse. The tumor cells increase in number and size,showing variable proportions of small, medium-sized,to large cerebriform cells,blast cells with prominent nuclei,and intermediate forms.The manifestations of mycosis fungoides in its early stage may mimic clinically and histologically those of many benign inflammatory dermatoses.Therefore,the diagnosis of mycosis fungoides remains a major challenge for dermatologists and dermatopathologists.Although immunochemistory is an assistant method to detect the T-cell differentiation antigen,weather the lesion is benign or maliganent is hard to determine.The main character of the maliganent lymphocytes proliferation is clonal,which is manifested by clonal rearrangement of T cell receptor in T cell lymphoma.PCR is an available and simple method for detecting gene rearrangement because TCR gene rearrangement structure is simple and is almost present in all T lymphoma.Methods:We used one-step method to extract DNA from formalin-fixed(10%)and parafin-embedded tissue blocks.61 cases of mycosis fungeides(MF),9 of subacute dermatitis,8 of lichen planus,7 of guttate parapsoriasis,7 of parapsoriasis varioliformis and 20 of normal skin were included in this stady.The TCR gene rearrangement was detected with polymerase chain reaction and agrose electrophorcsis.Results:The second primer and the third primer can not be used,x~2 is 0.000(p＞0.05)and 0.127(p＞0.05)respectively.34 cases were positive in the first primer,12 cases were positive in the fourth primer,4 cases were positive in the first and second primer at the same time.So clear clonal TCR gene rearrangements were detected in 50 of 61 MF cases(82.0%)and 13 of 31 cases control groups(41.9%)by PCR(x~2=15.260,P=0.000).Conclusion:1.The DNA extracted from parafin-embedded specimens with one-step method should be used as template to detect TCR rearrangement with PCR.2.The histopathologic features are not diagnostic in erythema stage.It’s easier to diagnosis plaque stage and tumor stage MF by pathology.3.Clear clonal TCR gene rearrangements were detected in 50 of 61 MF cases (82.0%)by PCR,the positive rate is high.4.There are some positive cases in control group,especially it is so high in subacute dermatitis.So it is possible to have some false positive cases.5.This method should be an assistant way to diagnosis mycosis fungoids,but it should be combine with clinical manifestation and histopathology.更多还原