伴发Castleman病的副肿瘤性天疱疮发病机制研究

The Pathogenesis of Paraneoplastic Pemphigus Associated with Castleman′s Disease

目的研究Castleman病在副肿瘤性天疱疮患者(PNP)发病机制中的作用。方法肿瘤组织常规免疫组化染色。RT-PCR扩增6例患者肿瘤组织中RNA,将PCR产物进行聚丙烯酰胺凝胶电泳及克隆测序;并以原位杂交法与6例无皮肤黏膜损害的Castleman病和3例淋巴结反应性增生患者肿瘤B细胞克隆相比较。以RNA印迹法了解重排基因的表达情况。结果6例PNP患者RT-PCR产物聚丙烯酰胺凝胶电泳皆获得1条约124bp的锐利条带;克隆测序得到2种高度同源的序列,分别为128bp,122bp,其中6例患者皆具有长序列,4例患者同时还有相同短序列。以128bp的重排基因为探针进行原位杂交,阳性信号仅见于PNP患者肿瘤组织切片的淋巴滤泡样结构中;RNA印迹示该探针与6例PNP患者肿瘤组织mRNA皆有较强的杂交信号。结论6例PNP患者肿瘤组织中主要B细胞克隆是相同的。这种B细胞克隆的免疫球蛋白重排基因在肿瘤组织中有丰富表达,所产生的免疫球蛋白可能直接与皮肤中的抗原发生作用,引起皮肤黏膜的免疫损伤。

Objective To investigate the role of Castleman′s disease in the pathogenesis of paraneoplastic pemphigus (PNP). Methods In six PNP patients associated with Castleman′s disease, routine immunohistochemistry was performed on tumor tissue. Reverse transcription - PCR, DNA sequencing of cloned PCR product and in situ hybridization (ISH) were used to estimate the clonality of the B-cells in the tumors. The expression of the specific tumor B-cell clones was evaluated by Northern blot. Six patients with Castleman′s disease without mucocutaneous lesion and 3 patients with reactive lymphadenopathy were used as the controls. Results Immunohistochemistry showed that CD20-positive B-cells in high density located in lymphoid follicles. The PCR produced one discrete band of about 128 bp in every paraneoplastic pemphigus patients. After sequencing the cloned PCR product, only two kinds of highly homologous sequences were found in all of the PNP patients. The 128 bp sequences were the major clones seen in all patients, and the 122 bp sequences were the relatively minor one seen in 4 patients. Anti-sense RNA probe transcribed from a clone of 128 bp was used in ISH. Signals of ISH located in cytoplasm of the cells in follicles of the tumors. Furthermore, this probe was also used for Northern blot and showed a strong signal in PNP patients. Conclusions Castleman′s disease associated with PNP share a major B-cell clone. The B-cell clone is expressed and maybe produces functional antibody initiating the mucocutaneous immune injury.