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细胞毒检验是了解和评价机体免疫功能状态的重要方法,历来受免疫学家重视。51Cr释放法是上世纪60年代建立的细胞毒活力检验方法,随后又建立了多种检验方法,包括荧光标记流式细胞术方法,统称为传统细胞毒检验方法(TCA)。TCA方法用杀伤率表示细胞毒活力是概念应用错误,以效靶细胞比为条件检测的杀伤率是实验条件应用错误,违背药理学原理,不反映细胞毒活力,没有实用性。本文通过对TCA错误理念和方法条件的探讨分析,遵循药理学原理建立了一个比较实用的细胞毒活力检验方法。新方法以极限稀释分析方法为基础,以效应细胞密度为自变量,用效应细胞含有的细胞毒活性细胞频率(CCF)和100%杀伤率限定的效应细胞密度(ID100)做细胞毒活力指标,通过量-效曲线和条件标准化测定CCF和ID100;不标记细胞,用全或无计数方法定量靶细胞;而且可以预置实验误差和可信度。新方法用K562细胞作为靶细胞,在96孔细胞培养板上检测细胞毒活力,健康人外周血单核细胞(PBMC)的ID100为6.8×10~4/孔左右;CIK细胞ID100≤4.0×10~4/孔;人红细胞的克隆抑制率为0;同一个细胞样品,用红细胞裂解液处理,克隆抑制率随处理强度成比例变化;样本批间检测结果变异不大,表明该方法能真实反映细胞毒活力。
Cytotoxicity test is to understand and evaluate the status of the body’s immune function is an important method, has always been the focus of immunologists. The 51Cr release assay is a cytotoxicity assay developed in the 1960s. A variety of assays were subsequently developed, including fluorescence-labeled flow cytometry, collectively referred to as the traditional cytotoxicity assay (TCA). TCA method using cytotoxic activity expressed by the killing rate is the concept of application of error to the target cell ratio test conditions for the killing rate is the application of experimental conditions error, contrary to pharmacological principles, does not reflect the cytotoxic activity, there is no practicality. In this paper, TCA error concept and method conditions to explore and analyze, follow the principles of pharmacology to establish a more practical method of cytotoxic activity test. The new method was based on the limiting dilution method. Using the density of effector cells as an independent variable, the cytotoxic activity was determined by the cell viability (CCF) and the effector cell density (ID100) defined by 100% CCF and ID100 were normalized by volume-response curves and conditions; cells were not labeled, target cells were quantified by all-or-nothing counting methods; and experimental error and confidence were preset. The new method used K562 cells as the target cells and detected the cytotoxic activity in 96-well cell culture plates. The ID100 of peripheral blood mononuclear cells (PBMC) of healthy people was about 6.8 × 10 ~ 4 / well. The IDK of the CIK cells was 4.0 × 10 ~ 4 / well; the inhibition rate of human erythrocyte clone was 0; the same cell sample was treated with erythrocyte lysate, and the inhibition rate of clone changed proportionally with the treatment intensity; Cytotoxic activity.