目的 探讨恶性胸/腹水结膜浸润淋巴细胞 (TILs) 的体外扩增培养方法，鉴定扩增细胞的功能和分子表型。方法 在无菌条件下提取胸/腹水液，并通过密度梯度离心法分离淋巴细胞。然后根据结合 IFN-γ、OKT3 和 IL-2 的方案对 TILs 进行扩增，记录其细胞形态和生长速率。通过流式细胞术分析扩增淋巴细胞的分子表型，并通过 CCK-8 实验检测其对肿瘤细胞的杀伤能力。结果 在这个培养方案中，TILs 在第 26 天之前处于良好状态，细胞增殖率自第 30 天开始下降。随着细胞培养时间的延长，CD4-CD8+ 和 CD8+CD56+ T 细胞的比例逐渐增加，而 CD4+CD25+ T 细胞的比例逐渐下降。与扩增前比较不同的是，T 淋巴细胞亚群 SLAMF7、CD45RO、PD-1 和粒酶 B 阳性细胞的比例显著增加，同时，疲劳 T 细胞标记 CD57 的表达也逐渐增加。TILs 中 CD8+ T 细胞的细胞毒性明显强于 PBMC 中的 T 细胞，细胞毒性在效应靶比为 10:1 时达到峰值，在不同的肿瘤细胞类型之间也有显著差异。结论 建立了一种从癌性胸/腹水中扩增 TILs 的培养方案，该方法简单高效。效应细胞主要是具有活性表型的 CD8+ T 细胞。

Objective To explore the culture method of mass amplification for tumor-infiltrating lymphocytes (TILs) from malignant pleural/ascites in vitro, and identify the function and molecular phenotype of these amplified cells. Methods The pleural/ascites fluid was extracted under aseptic conditions, and lymphocytes were isolated by density gradient centrifugation. Then TILs were amplified by the program based on combined IFN-γ, OKT3 and IL-2, and the cell morphology and growth rate were recorded. The molecular phenotypes of the amplified lymphocytes were analyzed by Flow cytometry, and the killing ability against tumor cells was detected by CCK-8 assay. Results In this culture program, TILs remained in good condition until the 26th day, and the proliferation rate began to decrease on the 30th day. The proportions of CD4-CD8+ and CD8+CD56+ T cells gradually increased as cell culture time extended while the proportions of CD4+CD25+ T cells decreased gradually. Unlike the proportions prior to amplification, the proportions of SLAMF7, CD45RO, PD-1 and granzyme B positive cells in T lymphocyte subpopulation were significantly increased, meanwhile, the expression of exhausted T-cell marker CD57 was also gradually increased. The cytotoxicity of amplified CD8+ T cells from TILs was significantly stronger than that from PBMC, and the cytotoxicity reached the peak at the effect-target ratio of 10:1 and was significantly different among tumor cell types. Conclusion A culture program for TILs amplification from cancerous thoracic/ascites is established. The method is simple and efficient. The effector cells are mainly CD8+ T lymphocytes with active phenotype.