摘要
目的 研究松萝酸通过调节程序性死亡受体1(PD-1)/程序性死亡配体1(PD-L1)信号通路影响肺癌细胞增殖、凋亡和免疫逃逸。方法 分离培养人CD8~+T淋巴细胞,1∶1比例与人肺癌细胞A549共培养,并将其进行分组:对照组,低浓度松萝酸组、中浓度松萝酸组、高浓度松萝酸组,松萝酸+空载组,松萝酸+过表达PD-L1组;CCK8法测定A549细胞增殖;ELISA测定细胞上清液INF-γ和TNF-α水平;流式细胞术测定A549细胞凋亡;RT-PCR检测A549细胞TNF-α、INF-γ、PD-L1、CD8~+T细胞PD-1 mRNA水平;Western blot检测CD8~+T细胞PD-1、A549细胞PD-L1、cleaved-caspase 3、ki67蛋白水平。结果 与对照组相比,低、中、高浓度松萝酸组A549细胞A_(450)值、上清液TNF-α水平、A549细胞TNF-α、PD-L1、CD8~+T细胞PD-1 mRNA水平、CD8~+T细胞PD-1、A549细胞PD-L1、ki67蛋白水平均显著性降低,A549细胞凋亡率、上清液INF-γ水平、A549细胞INF-γ mRNA水平以及cleaved-caspase 3蛋白水平升高(P<0.05)。与松萝酸+空载组相比,松萝酸+过表达PD-L1组A549细胞A_(450)值、上清液TNF-α水平、A549细胞TNF-α、PD-L1、CD8~+T细胞PD-1 mRNA水平、CD8~+T细胞PD-1、A549细胞PD-L1、ki67蛋白水平均显著性升高,A549细胞凋亡率、上清液INF-γ水平、A549细胞INF-γ mRNA水平以及cleaved-caspase 3蛋白水平均显著性降低(P<0.05)。结论 松萝酸可能通过阻滞PD-1/PD-L1信号通路诱导A549细胞凋亡,并抑制免疫逃逸和增殖。
Objective This study aims to explore the effects of usinic acid on proliferation,apoptosis,and immune escape of lung cancer by regulating the programmed death receptor 1(PD-1)/programmed death ligand 1(PD-L1)signaling pathway.Methods Human CD8+T lymphocytes were isolated and cultured,which were then co-cultured with human lung cancer cells A549 at a ratio of 1∶1.The co-cultured cells were divided into six groups:control group,low concentration usinic acid group,medium concentration usinic acid group,high concentration usinic acid group,usinic acid+empty group,and usinic acid+overexpression PD-L1 group.CCK8 experiment was performed to detect the A549 cells proliferation;the level of INF-γand TNF-αin the supernatant of cells were detected by ELISA;flow cytometry was performed to detect the A549 cells apoptosis;RT-PCR was performed to detect the level of TNF-α,INF-γ,PD-1,and PD-L1 mRNA in activated CD8+T cells and A549 cells;Western blot was performed to detect the level of PD-1 in CD8+T cells,PD-L1 in A549 cells,cleaved-caspase 3,and ki67 protein expression.Results Compared with the control group,the A450 value of A549 cells,supernatant TNF-αlevel,A549 cells TNF-α,PD-L1,CD8+T cells PD-1 mRNA levels,CD8+T cells PD-1,A549 cells PD-L1,and ki67 protein levels in the low,medium,and high concentration usinic acid groups were significantly reduced,the apoptosis rate,supernatant INF-γlevel,INF-γmRNA level,and cleaved-caspase 3 protein level were increased(P<0.05).Compared with the usinic acid+empty group,the A450 value,supernatant TNF-αlevel,A549 cells TNF-α,PD-L1,CD8+T cells PD-1 mRNA levels,CD8+T cells PD-1,A549 cells PD-L1,and ki67 protein levels in the usinic acid+overexpression PD-L1 group were significantly increased,the apoptosis rate,supernatant INF-γlevel,INF-γmRNA level,and cleaved-caspase 3 protein level were significantly reduced(P<0.05).Conclusion Usinic acid may induce apoptosis of A549 cells,and inhibit immune escape and proliferation by blocking the PD-1/PD-L1 signaling pathway.
作者
陈莉
卢红
汪甜
CHEN Li;LU Hong;WANG Tian(Department of Geriatrics,Xianning Central Hospital,the First Affiliated Hospital of Hubei University of Science and Technology,Xianning 437100,Hubei,China;Hubei University of Science and Technology Medical Department Basic Medical College,Xianning 437100,Hubei,China)
出处
《医学研究与战创伤救治》
北大核心
2024年第11期1142-1147,共6页
Journal of Medical Research & Combat Trauma Care
