蕈样肉芽肿候选肿瘤标记物 PRDX1、PPIA、MnSOD和FABP5的初步研究

发布时间：2018-11-26 07:51

【摘要】：研究背景：蕈样肉芽肿(mycosis fungoides, MF)是最常见的原发性皮肤T细胞淋巴瘤,以小到中等程度大的具有脑回状核的T淋巴细胞亲表皮性增生为特征。由于与多种良性淋巴细胞增生性疾病和反应性皮肤疾病相似,MF不易确诊,特别是在疾病早期。MF目前无特异性的肿瘤标记物,可用于疗效观察和判断预后的生物学标记物十分有限。近年来,蛋白质组学已初步开始用于MF肿瘤标记物的研究。本课题组前期通过双向凝胶电泳结合MALDI质谱对MF患者皮损组织和正常人皮肤组织进行比较分析,鉴定出31种差异表达蛋白质,其中锰超氧化物歧化酶(MnSOD)、脂肪酸结合蛋白5(FABP5)和S100A8这3种蛋白质经多组比较反复在MF皮损组织表达增高。之后提取MF皮损组织和正常人皮肤组织总蛋白,应用免疫印迹法(Western blot, WB)证实上述3种蛋白在MF皮损组织高表达。目的： (1)用相应抗体验证另外两种质谱鉴定出的差异表达蛋白即过氧化氢酶1(peroxiredoxin 1, PRDX1)和肽酰-脯氨酰-顺反式异构酶A(peptidyl-prolyl isomerase Cyclophilin A, PPIA)在MF患者皮损组织和正常人皮肤组织中的差异表达； (2)定位PRDX1、PPIA、MnSOD和FABP5在MF患者皮损组织和正常人皮肤组织中的差异表达部位； (3)找到MF皮损淋巴细胞内差异表达的相关蛋白,研究其在人皮肤T细胞淋巴瘤细胞系(Hut-78)中的表达。方法： (1)提取正常皮肤组织和MF皮损组织总蛋白,用WB法检测PRDX1和PPIA的表达； (2)用免疫组化法定位PRDX1、PPIA、MnSOD和FABP5这4种蛋白在MF组织中的表达； (3)采用正常PBMCs和Hut-78细胞,WB法和免疫细胞化学法检测MF皮损组织淋巴细胞内出现差异表达的相关蛋白。结果： (1) PRDX1蛋白在MF皮损组织淋巴细胞中高表达,PPIA蛋白在MF皮损组织的表达与正常对照相比无明显差异,MnSOD蛋白在MF皮损组织的表皮和真皮淋巴细胞中高表达,FABP5蛋白在MF皮损组织表皮内高表达； (2)与正常PBMCs相比,PRDX1蛋白在Hut-78细胞中高表达,MnSOD蛋白在Hut-78细胞中表达降低。结论： (1) PRDX1和MnSOD蛋白可能为MF潜在的肿瘤标记物； (2) PPIA和FABP5蛋白不适于做为MF的肿瘤标记物； (3)体外培养环境对皮肤恶性T淋巴细胞的MnSOD蛋白表达可能存在影响。
[Abstract]:Background: fungoid granuloma (mycosis fungoides, MF) is the most common primary cutaneous T-cell lymphoma characterized by small to medium degree epidermogenic proliferation of T lymphocytes with gyrus nucleus. MF is not easy to diagnose because it is similar to many benign lymphoproliferative diseases and reactive skin diseases, especially in the early stage of the disease. MF has no specific tumor markers at present. Biological markers for therapeutic observation and prognosis are limited. In recent years, proteomics has been applied to the study of MF tumor markers. We compared and analyzed the lesions of MF patients and normal skin tissues by two-dimensional gel electrophoresis and MALDI mass spectrometry, and identified 31 differentially expressed proteins, including manganese superoxide dismutase (MnSOD),). Fatty acid binding protein 5 (FABP5) and S100A8 were repeatedly expressed in the lesions of MF. The total proteins were extracted from the lesions of MF and normal human skin, and the overexpression of the three proteins in the lesions of MF was confirmed by Western blot (Western blot, WB). Objective: (1) to identify two differentially expressed proteins identified by mass spectrometry, peroxiredoxin 1 (PRDX1) and peptidyl-prolyl isomerase Cyclophilin A, (peptidyl-proline cis-trans isomerase A). The differential expression of PPIA in the lesions of MF patients and normal skin tissues; (2) location of differential expression of PRDX1,PPIA,MnSOD and FABP5 in lesions of MF patients and normal skin tissues; (3) to find the differentially expressed proteins in lymphocytes of MF and to study their expression in human cutaneous T-cell lymphoma cell line (Hut-78). Methods: (1) the total protein of normal skin and MF was extracted and the expression of PRDX1 and PPIA was detected by WB method. (2) the expression of PRDX1,PPIA,MnSOD and FABP5 in MF was detected by immunohistochemical method. (3) using normal PBMCs and Hut-78 cells, WB and immunocytochemistry were used to detect the differentially expressed proteins in the lymphocytes of MF lesions. Results: (1) the expression of PRDX1 protein in lymphocytes of MF lesions was higher than that of normal controls. The expression of PPIA protein in MF tissues was not significantly different from that in normal controls. The expression of MnSOD protein was highly expressed in the epidermis and dermis lymphocytes of MF lesions. FABP5 protein was highly expressed in the epidermis of MF lesions. (2) compared with normal PBMCs, PRDX1 protein was highly expressed in Hut-78 cells and MnSOD protein was decreased in Hut-78 cells. Conclusion: (1) PRDX1 and MnSOD proteins may be potential tumor markers for MF, (2) PPIA and FABP5 proteins are not suitable for MF tumor markers. (3) the expression of MnSOD protein in skin malignant T lymphocytes may be affected by the culture environment in vitro.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5

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