EB病毒及人类T细胞白血病病毒与蕈样肉芽肿发病机制的相关性研究

【作者】 孙青；

【导师】 徐从高；

【作者基本信息】 山东大学 ， 内科学， 2004， 博士

【摘要】 目的:皮肤T细胞淋巴瘤是一种发病机制不明的非霍奇金淋巴瘤,包括蕈样肉芽肿(Mycosis fungoides,MF)、Sezary综合征(sezary syndrome,SS)、MF相关毛囊黏蛋白病、湿疹样癌样网状细胞增生病、肉芽肿性松弛皮肤病、原发性皮肤T细胞性大细胞淋巴瘤、原发性皮肤CD30阳性大细胞淋巴瘤、皮下组织脂膜炎样T细胞淋巴瘤等。近几年有研究提示逆转录病毒及人类疱疹病毒在CTCL的发病中起作用,但关于原发性皮肤T细胞淋巴瘤与EB病毒感染关系的研究不多,研究结果也不相一致。部分研究发现在CTCL的PBMC或石蜡包埋的切片中可检测到EB病毒的DNA或致病基因,但部分研究得出相反的结论。蕈样肉芽肿又称蕈样霉菌病,是一种原发于皮肤的低度恶性外周辅助T细胞性淋巴瘤,组织病理表现为表皮内有大量的淋巴瘤细胞浸润,是CTCL的一种临床亚型和具有代表性的CTCL。该病在美国、日本的发病率较高,是CTCL的主要类型。我国MF少见,但近几年有增长趋势,该病病程可持续二三十年。MF从发现至今已190多年,尽管进行了大量的研究,但其发病原因仍不清楚。有感染、癌基因、细胞因子、职业或环境因素等学说,认为病毒感染、机体易感性、异常淋巴细胞、巨噬细胞、朗格汉斯细胞等相互作用,或异常抗原持续性刺激可导致蕈样肉芽肿的发生。目前关于MF与爱泼斯坦—巴尔病毒(Epstein-Barr virus EB病毒)感染的关系已引起重视,国外已进行了一些关于蕈样肉芽肿与该病毒感染关系的研究,但实验结果不一致,仍存着较大的争议。 EB病毒是一种嗜人类淋巴细胞的γ-DNA疱疹病毒,即疱疹病毒4型,包括亚型1和亚型2两个亚型。1964年Epstein-Barr首先从Burkitt淋巴瘤更多还原

【Abstract】 Objective: Cutaneous T-cell lymphoma is a non-Hodgkin lymphoma, thepathogenesis remains unknown. It consists of Sezary syndrome, MF-related follicular mucinosis, eczematoid carcinoma reticulosis granunulomatous slack skin, primary cutaneous large T-cell lymphoma, primary cutaneous CD30 positive large cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma and so on. Recently some studies suggested that retrovirus and herpes virus might play an improtant role in the pathogenesis of CTCL. But there are only few literature refered to the correlation between EB virus and primary cutaneous T-cell lymphoma and the conclusions were controversial. Some investigators detected DNA of EB virus or other abnormal gene positive in PBMNC of CTCL, while others got opposite findings. Mycosis fungoides are also called mycosis disease. It is a primary low-grade cutaneous peripheral helper T-cell lymphoma. Histophathology revealed that there were abundant lymphoma cells in the epidermis. It is a major subtype of CTCL. The incidence of MF in the Uuited States and Japan are higher than that of China. But there is an increasing trend. The course of the disease may last more than thirty years. MF has a history of 190 years. Numerous studies have been conducted, but the etiology has not been clear. However recent observations suggest that virus infection, oncogene, cytokine, occupational and environmental factors may be involved. The interaction of viral infection, host susceptiblility and immune cells, and persistant antigen stimulation may potentially causity in the development of MF. Recent observations revealed an important role of EBV in the pathogenesis of MF. An increasing body of data on on the correlation between MF and viral infection hasbeen acuumulated, but consensus has not been reached.EBV is a human lymphotrophic Y -DNA herpes virus., also refered to as herpesvirus type 4. It has two subtypes: I and II. In 1964, Dr. Epstein Barr firstestablished two lymphoma cell lines from Burkitt lymphoma. The electron-microscopic morphology of EB virus are similar to.other herpes viruses. The virus infects both lymphocytes and epithelial cells. It causes several clinical syndromes, such as infectious mononucleosis and B cell and nasopharyneal carcinoma. It had been previoulsy considered that EBV were only involved in the development of B-cell lymphoma, but more recently increasing evidences suggested that T-cell lymphoma is also related to EBV, while the exact mechanism is still unknown. Now it is clear that LMP-1 and BARF-1 are the virus oncogenes of EBV. LMP-1 is important for B lymphocyte immortalization, while LMP-1 can transform 3T3 cells in vitro, and form tumor in nude mice. In 1988, EBV-DNA was first detected in T-cell lymphoma with molecular-biology technique. After that, it has been confrimed by several recent studies that EBV positive T-cell lymphoma is far more than EBV positive B cell lymphoma in NHL except Burkitt lymphoma. The EBV detection rate of T-cell NHL is 63.6%. About 10% T-cell lymphoma is EBV related, and this subpopulation is more prevalent in Asia.How EBV infects T lymphocytes has not been made clear. Some studies suggested that T cells express EBV/c3d receptor (CD21) in their certain developmental stages, so EBV may infect T cells directly binding to c3d receptor. Studied found that it is difficult to infect normal T cells Ex vivo with EBV to establish immortalized cell lines and in vivo infection of normal T cells by the virus can hardly occur. All mentioned above implicate that T cell and B cell infection by EBV and the fate of EBV after infection may be defferent ,and the viral infection may exert various impacts on the host. T cell is the recepient of EBV. The DNA of EBV maintains linear molecular structure and self-circling does not occur inside T cell. The low circling of viral DNA in T cell results in low transformation of T cell induced by the virus. This indicates a possible pathogenesis of T-cell lymphoma induced by EBV and T cell proliferation.The EBV polymerase accessory protein, BMRF1, is an essential component ofthe viral DNA polymerase and is required for lytic EBV replication. It is a specific CTL epitope of EBV antigen. The sequ ence is 268-276. It is HLA restricted. The EBV specificity of BMRF1 is undertermined. The study by Qin demonstrated that BMRF1 is a trransactivator, inducing expression of the essential oriLyt promotor, BHLF1. Replication from oriLyt requires the products of six viral genes: BALF1, BMRF1, BSLF1, BBLF1 and BBLF2/3. The BMRF1 gene product is the major early phosphopotein induced druin EBV lytic replication and is a double-stranded DNA-binding protein that is essential for processive DNA synthesis by the viralpolymerase.Our study aimed to determine whether there were infection and replication ofEBV in MF patients, so to investigate the relationship of EBV infection and the onset of MF. We detected the expression of EBV DNA and BMRF1 gene from the peripheral blood mononuclear cells of seven MF patients with polymerase chin reaction.Method: To detect EBV DNA in PBMC of seven MFs and five normal controls. Amplify EBV BMRF1 gene in PBMC from EBV positive samples, using EBV cell line as positive control and deionized water as negative control. The specific primer sequences for EBV DNA amplification are as following: 5’ -CCAGACAGCAGCCAATTGTC-31 and 5-GGTAGAAGACCCCCTCTTAC-3’, which located in BamH-I-W region. The amplified product is 129bp. The specific primer sequences for EBV BMRF1 gene amplification are followed: 5’-CAGGCTTCCCTGCAATTTTA-CAAGCGC-3\ and5’-CCCAGAAGTATACGTGGTGACGTAGA-3\ The size of amplification product is 288bp. Electrophoresed in 1.5% agarose gel and stained with ethidium bromide, the PCR products band were visualized with an ultraviolet transilluminatior. Results: 1. Detection of EBV DNA in PBMCAfter amplify the extracted DNA of PBMC of MF patients using EBV DNA specific primers, the electrophoresis in 1% agarose gel showed that 2/7 MF samples were positive. Two clear electrophoresis bands were seen around 129bp, 5/7 samples were negative. The positive rate was 28.5%. For the two positive samples, one was in plaque stage and the other in tumor stage. The PCR products of five normal samples were all negative.更多还原