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Research for a retrovirus in myasthenic thymus and study for the pathogenesis

肌无力胸腺逆转录病毒的研究及发病机制研究

基本信息

批准号：
02454248
负责人：
ONO Akira
金额：
$ 2.24万
依托单位：
The Kitasato Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454248/
关键词：
myasthenia gravis thymus thymoma retrovirus 遺伝子クロ-ニングレトロウィルス

项目摘要

By electron microscopy, we have demonstrated in retrovirus particles in cultured thymus cells (thymomas and thymus hyperplasias) of patients with myasthenia gravis. In most cases, these particles were detected in thymus epithelial cells when cultured with mitomycin C-treated human B-cells. For further characterization of these particles, virus-producer cell lines were established by co-culturing human T-lymphoblastoid cells with B-cell-stimulated human thymus cells. Mn^<++>-dependent RNA- directed DNA polymerase activity was detected at a density of 1.15-1.17 g/cm^3 on sucrose density gradients in the culture fluid of these co-cultured T-cells. We molecularly cloned the cDNA prepared by the detergent-activated endogenous polymerase reaction in the culture fluid of KK-ll cells, one of the virus-producer T-cell lines. The ^<32>p-labelled cloned cDNA hybridized to DNAs from freshly prepared hyman thymoma cells, in addition to KK-ll cell DNA. We sequenced about 400-1200 bp of seven cDNA clones, and the sequence of each cDNA was compared with that of all genes with known sequences present in EMBL data base. No striking similarity was observed when compared to other sequences. The nucleotide sequence of seven cloned cDNAs constantly revealed high homology (50-70%) with that of retroviruses such as HIV-l and equine infectious anemia virus (EIAV). These results indicate that cDNAs obtained here are new viral genes, possibly retroviral genes, derived from human thymus cells.

通过电子显微镜，我们已经证明在逆转录病毒颗粒培养的胸腺细胞（胸腺瘤和胸腺增生）的重症肌无力患者。在大多数情况下，当与丝裂霉素C处理的人B细胞培养时，在胸腺上皮细胞中检测到这些颗粒。为了进一步表征这些颗粒，通过将人T淋巴母细胞样细胞与B细胞刺激的人胸腺细胞共培养来建立病毒生产细胞系。在这些共培养的T细胞的培养液中，在蔗糖密度梯度为1.15-1.17 g/cm ^3时，检测到Mn^<++>依赖性RNA指导的DNA聚合酶活性。我们在病毒生产T细胞系之一的KK-11细胞的培养液中通过去污剂激活的内源性聚合酶反应制备了cDNA，并对其进行了分子克隆。除了<32>KK-11细胞DNA外，13 P标记的克隆cDNA还与来自新鲜制备的hyman胸腺瘤细胞的DNA杂交。我们对7个cDNA克隆进行了约400-1200 bp的测序，并将每个cDNA的序列与EMBL数据库中所有已知序列的基因的序列进行了比较。与其他序列相比，没有观察到惊人的相似性。7个克隆的cDNA序列与HIV-1和马传染性贫血病毒（EIAV）等逆转录病毒的核苷酸序列同源性较高（50-70%）。这些结果表明，获得的cDNA是新的病毒基因，可能是逆转录病毒基因，来自人胸腺细胞。