中国肿瘤生物治疗杂志2025，Vol.32Issue(8)：862-869,8.DOI:10.3872/j.issn.1007-385x.2025.08.010
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建立及验证
Establishment and verification of a detection method for the cytotoxic activity of human cascade-primed immune cell injection against non-small cell lung cancer
周巧 1李美玲 1陈志刚 2王壮 1尚孙玉麟 1徐秋玲 1朱艳萍 1张益丽1
作者信息
- 1. 云南精准检验有限公司 检验部,云南 昆明 650000
- 2. 赛德特生物制药有限公司 临床样品制备部,云南 昆明 650000
- 折叠
摘要
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 1.83×10⁷ 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.关键词
人链式激活免疫细胞制剂/非小细胞肺癌/萤光素酶/绿色荧光蛋白/杀伤活性/方法学建立/方法学验证Key words
human cascade-primed immune cell injection/non-small cell lung cancer(NSCLC)/luciferase/green fluorescent protein(GFP)/cytotoxic activity/establishment of method/verification of method分类
医药卫生引用本文复制引用
周巧,李美玲,陈志刚,王壮,尚孙玉麟,徐秋玲,朱艳萍,张益丽..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建立及验证[J].中国肿瘤生物治疗杂志,2025,32(8):862-869,8.
