扶正化瘀方对NK92细胞活化和杀伤功能的影响

Effects of Fuzheng Huayu Decoction on the activation and cytotoxicity of NK92 cells

目的通过体外实验观察扶正化瘀方对人外周血NK细胞系NK92细胞活化和杀伤功能的影响。方法CCK8法检测扶正化瘀方对NK92细胞的最大无毒质量浓度,基于最大无毒质量浓度,选取不小于3个有效药物质量浓度对NK92细胞进行细胞功能和活性评价,Annexin V/7-AAD复染法检测NK92细胞凋亡,荧光定量PCR法检测NK92细胞表面激活型受体NKG2D、NKp30、NKp46的基因表达,流式细胞术检测并验证NK92细胞表面激活型受体的表达变化,ELISA法检测药物孵育后NK92细胞分泌因子IFN-γ和颗粒酶B的水平;以聚肌胞苷酸PolyI:C为阳性对照药物,在NK92细胞与K562细胞共培养杀伤体系中评价药物对NK92细胞杀伤功能的影响。结果扶正化瘀方在100μg/mL及以下质量浓度时对NK92细胞无明显毒性,较低质量浓度时扶正化瘀方对NK92细胞凋亡有保护作用;经不同质量浓度扶正化瘀方孵育的NK92细胞,可不同程度增强细胞表面激活型受体的表达以及促进的IFN-γ、颗粒酶B分泌;杀伤性实验评价显示扶正化瘀方在无毒质量浓度范围呈现剂量依赖性地提高NK92细胞对K562细胞的杀伤功能。结论扶正化瘀方可以促进NK92细胞活化,增强其杀伤功能,充当NK细胞正性调节剂。

AIM The effects of Fuzheng Huayu Decoction on the activation and cytotoxicity of natural killer cell line NK92 cells in human peripheral blood were observed through in vitro experiments.METHODS Based on the maximum non-toxic concentration of Fuzheng Huayu Decoction on NK92 cells determined by CCK8 assay,three effective drug concentrations were applied for the assessment of the cell function and cytotoxicity of NK92 cells.NK92 cells were subjected to apoptosis detection by Annexin V/7-AAD counterstain method,the detection of gene expression of NK92 cells surface activated receptors NKG2D,NKp30,NKp46 by RT-qPCR method,the determination of expression changes of cellular surface receptors by flow cytometry,and the levels of NK92 cells secreted factor IFN-γand Granzyme B after drug incubation by ELISA method.With PolyI:C as a positive control drug,the effects of drugs on the cytotoxicity of NK92 cells were evaluated in the NK92 cells and K562 cells co-culture system.RESULTS Fuzheng Huayu Decoction was not obviously toxic to NK92 cells at a concentration of 100μg/mL and lower levels,and was protective to NK92 cells apoptosis at a lower concentration.After incubation with different concentrations of Fuzheng Huayu Decoction,NK92 cells shared more activated receptors expression on the surface and enhanced secretion of IFN-γand granzyme B at varying degrees.The cytotoxic activity experiment revealed that Fuzheng Huayu Decoction dose-dependently increased the cytotoxic activity of NK92 cells against K562 cells within the non-toxic concentration range.CONCLUSION Fuzheng Huayu Decoction,a positive regulator to NK cells,can improve the activation and cytotoxicity of NK92 cells.