Establishment of Thymic Epitherial Cell Lines from a Human Thymoma and Its Immuno-biological Analysis.

我们用分离培养技术，从一位56岁女性胸腺瘤患者的胸腺中建立了人胸腺上皮细胞(Thm-1)，并用克隆培养方法建立了几种亚型细胞系。克隆细胞系的生长特征大致可分为两种类型：多角型和纺锤型。多角形亚系(3D)和纺锤体亚系(7A)的体外倍增时间分别为1.3天和7.4天。当这些细胞被注射到裸鼠体内时，被证明具有高度的致瘤性。在裸鼠体内生长的Tm-1肿瘤与没有淋巴细胞的原始肿瘤在组织学上是相同的。流式细胞仪免疫表型分析显示上皮性肿瘤细胞表达表面E-钙粘附素和CEA抗原。免疫组织化学显示细胞表面有细胞角蛋白表达。培养细胞的电子显微镜下可见桥粒、张力纤维和颗粒。Northern分析表明，E-钙粘素基因在这些细胞中表达。这些数据表明，该细胞系代表了外胚层谱系。采用微量板法对淋巴细胞-肿瘤细胞混合培养反应(MLTR)进行检测。将PBL细胞与Thm-1细胞、肺癌细胞株和巨噬细胞在添加200mU/mlIL-2的培养液中共培养，定期进行淋巴细胞亚群的流式细胞术分析。结果发现，在Thm-1细胞和巨噬细胞共培养的16天和31天，CD8阳性细胞数增加(16天后为58.7%，31天后为72.1%)，但CD4阳性细胞较少(16天后为9.7%，31天后为7.5%)。为进一步研究胸腺上皮细胞的生物学行为和胸腺瘤的体内免疫效应提供了一个很好的模型。

Using isolation culture techniques, we have been able to establish human thymic epitherial cells (Thm-1) from a thymoma of a 56 year-old female patient and have developed several subtype cell lines using cloning culture method. The growth characteristics of the cloning cell lines can be roughly divided into twotypes, polygonal and spindle types. Doubling time in vitro was approximately 1.3 day in polygonal Subline (3D) and 7.4 day in spindle Subline (7A). These cells were demonstrated to be highly tumorigenic when injected into nude mice. Thm-1 tumors, grown in nude mice, are histologically identical to the original tumor without lymphcytes. Immunophenotypic analysis of the cells using flow cytometry showed epitherial tumor cells expressing a surface E-cadherin and CEA antigen. Immunohistologically, the cells were demonstrated to express cytokeratin on the cell surface. Electron microscopy of the cultured cells revealed desmozome, tonofirament and granule particles. Nothern analysis demonstrated that E-cadherin mRNA was revealed in these cells. These data indicate that this cell line represents the epitherial lineage. To measure immunological reactivity, mixed lymphocyte-tumor cell culture reaction (MLTR) was studied using a microplate method. The PBL cells were cocultured with Thm-1 cells, lung cancer cell lines and macrophages in the medium supplemented with 200 mu/ml IL-2 and analyzed at periodic intervals for expanded lymph cell-subsets by flow cytometry.We found increased numbers of CD8 positive cells in 16-and 31-day cultures from the coculturing of Thm-1 cells and macrophages (58.7 after 16 days and 72.1% after 31 days of culture), but were small for CD4 positive cells (9.7% after 16 days and 7.5% after 31 days of culture).Establishment of this human thymoma cell line, Thm1, provides an excellent model to study further the biological behavior of thymic epitherial cells and the in vivo immunological effects of a thymoma.