[摘 要] 目的：构建绿色荧光蛋白（GFP）/萤光素酶（Luc）双荧光标记的肿瘤细胞株，建立人链式激活免疫细胞制剂对非小细 胞肺癌细胞杀伤活性的检测方法，并进行初步验证。方法: 通过HLA高分辨分型筛选出HLA-A、HLA-B、HLA-C基因型别为纯 合子且相应等位基因的分布频率高于2.500%的非小细胞肺癌细胞系作为细胞模型，采用携带有GFP和Luc基因的重组慢病毒感 染原始细胞株而获得GFP、Luc稳定表达的细胞系作为靶细胞。通过效应细胞与靶细胞共培养，并优化靶细胞前处理步骤、共培 养时间、效靶比等参数，建立人链式激活免疫细胞制剂对非小细胞肺癌杀伤活性的检测方法，并进行专属性、精密度验证。结果: 通过HLA高分辨分型，成功筛选出高频等位基因HLA?A*11:01:01（20.893%）、HLA?B*52:01:01（2.991%）、HLA?C*12:02:02 （3.139%）的非小细胞肺癌（HCC827）细胞株作为细胞模型。通过慢病毒载体成功构建GFP/Luc双荧光标记的HCC827细胞株， GFP阳性率达 96%。重组慢病毒滴度为1.83 × 10? TU/mL。效靶比为5∶1、10∶1、15∶1、20∶1时，各组间杀伤活性差异显著 （P < 0.05），且杀伤活性随培养时间延长显著升高（P 0.05），说明方法重复性良好。结论: 成功建立了人链式激活免疫细胞制剂对非小细胞肺癌杀伤活性的检测方 法并验证成功，该方法有助于人链式激活免疫细胞制剂在细胞免疫治疗有效性评价中发挥重要作用。

[Abstract] Objective: To create a luciferase (Luc) / green fluorescent protein (GFP) dual-fluorescent labeled tumor cell line and establish a detection method for the cytotoxic activity of human cascade-primed immune cell injection against non-small cell lung cancer (NSCLC), and to conduct preliminary verification. Methods: NSCLC lines, which possessed homozygous HLA-A, HLA-B, and HLA-C genotypes and an allelic distribution frequency higher than 2.500% were screened through high-resolution HLA typing for use as cell models. The cell line stably expressing the green fluorescent protein (GFP) and luciferase (Luc) was obtained through infecting the original cell line with a recombinant lentivirus that carries the GFP gene and Luc gene. This cell line was then used as the target cell. Through co-culturing effector cells and target cells as well as optimizing parameters including the pretreatment steps of target cells, co-culture duration, and effector-to-target proportion, a detection method for the cytotoxic activity of human cascade-primed immune cell injection against NSCLC was established, Subsequently, both the specificity and precision of this method were thoroughly verified. Results: Through high-resolution HLA typing, the non-small cell lung cancer cell line HCC827 harboring high-frequency alleles HLA-A11:01:01 (20.893%), HLA-B52:01:01 (2.991%), and HLA-C*12:02:02 (3.139%) was successfully selected as the cellular model. The HCC827 cell line with GFP and Luc dual fluorescence labeling was successfully constructed using a lentiviral vector, with a 96% GFP-positive rate. The titer of the recombinant lentivirus was 1.83 × 10? TU/mL. Significant differences in the cytotoxic activity were observed among groups with effector-to-target (E∶T) ratios of 5∶1, 10∶1, 15∶1, and 20∶1 (P < 0.05), and the cytotoxic activity increased significantly with prolonged co-culture duration (P < 0.000 1). After comprehensive evaluation, the optimal parameters were determined as an effector-to-target (E∶T) ratio of 10:1 and a co-culture duration of 72 h. Methodological verification demonstrated that the established method exhibited strong specificity, with a coefficient of variation of 0.80% - 1.86% for repeatability and 1.00% - 1.58% for precision. Furthermore, no significant differences were observed via variance analysis (P > 0.05), confirming good repeatability of the method. Conclusion: A detection method for the cytotoxic activity of human cascade-primed immune cell injection against NSCLC has been successfully established and verified. This method might help human cascade-primed immune cell injection play an important role in the effectiveness evaluation of cellular immunotherapy.