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环磷酸鸟苷-腺苷合成酶-干扰素基因刺激因子（cGAS-STING）信号通路激活对γδT细胞杀伤肝癌细胞效应的调控作用
DOI: 10.12449/JCH251219
伦理学声明：本研究于2023年4月18日经由石河子大学第一附属医院科技伦理委员会审批，批号：KJ2023-163-01。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：胡帅负责撰写论文；王二强负责部分课题设计、实验指导；多小勇、徐志、张玉梦负责数据分析，论文修改；谢士伟、荣利华、王宇晨负责图表数据的可视化处理与优化；李江、张示杰负责课题设计，指导撰写文章并最后定稿。
Activation of the cyclic guanosine monophosphate-adenosine monophosphate adenosine synthetase-stimulator of interferon genes signaling pathway regulates the cytotoxicity of γδT cells against hepatoma cells
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摘要:
目的 通过体外实验，探究环磷酸鸟苷-腺苷合成酶-干扰素基因刺激因子（cGAS-STING）信号通路对γδT细胞杀伤肝细胞癌效应的调控作用，为提升基于γδT细胞的过继免疫疗法效果提供新思路。 方法 分离外周血单个核细胞并扩增γδT细胞，随后检测其纯度；将成熟的γδT细胞分为γδT组、γδT-G10组和γδT-H-151组，体外刺激24 h后，利用Western Blot检测cGAS-STING信号通路关键蛋白的表达水平；通过酶联免疫吸附试验检测干扰素-γ（IFN-γ）及肿瘤坏死因子-α（TNF-α）浓度；将各组细胞分别与肝癌细胞MHCC-97H、Huh-7共培养6 h，利用细胞计数试剂盒-8技术检测各组肝癌细胞的存活率。计量资料多组间比较采用单因素方差分析，进一步两两比较采用Dunnett’s T3多重比较检验和Tukey多重比较检验。 结果 流式细胞术结果显示，γδT细胞纯度达99%以上；Western Blot结果表明，与γδT组相比，γδT-G10组和γδT-H-151组细胞中cGAS表达无显著差异，γδT-G10组中STING、磷酸化STING（P-STING）、TANK结合激酶1、磷酸化TANK 结合激酶 1、干扰素调节因子3及磷酸化干扰素调节因子3的表达上调，而γδT-H-151组中上述蛋白表达均下调；酶联免疫吸附试验结果显示，与γδT组相比，γδT-G10组γδT细胞分泌的IFN-γ、TNF-α显著增加（P值分别为<0.01、<0.05），而γδT-H-151组γδT细胞分泌的IFN-γ、TNF-α显著减少（P值分别为<0.01、0.000 1）；细胞计数试剂盒-8检测结果显示，与γδT组相比，γδT-G10组MHCC-9H和Huh7细胞株肝癌细胞的存活率显著下降（P值均<0.000 1），γδT-H-151组则显著上升（P值均<0.000 1）。 结论 cGAS-STING信号通路可在体外调控γδT细胞对肝细胞癌的杀伤效应。 Abstract:Objective To investigate the regulatory effect of the cyclic guanosine monophosphate-adenosine monophosphate adenosine synthetase （cGAS）-stimulator of interferon genes （STING） signaling pathway on the cytotoxicity of γδT cells against hepatocellular carcinoma （HCC） through in vitro experiments， and to provide new ideas for improving the efficacy of adoptive immunotherapy based on γδT cells. Methods Peripheral blood mononuclear cells （PBMCs） were isolated and γδT cells were multiplied， and their purity was measured. Mature γδT cells were divided into γδT group， γδT-G10 group， and γδT-H-151 group. After 24 hours of in vitro stimulation， Western blot was used to measure the expression levels of key proteins in the cGAS-STING signaling pathway， and ELISA was used to measure the concentrations of interferon-γ （IFN-γ） and tumor necrosis factor-α （TNF-α）. The cells in each group were cocultured with MHCC-97H and Huh-7 cells for 6 hours， and then CCK8 assay was used to measure the survival rate of HCC cells in each group. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups， the Dunnett’s T3-test and the Tukey test were used for further comparision between two groups. Results Flow cytometry showed that the purity of γδT cells reached above 99%. Western blot showed that there was no significant difference in the expression of cGAS between the γδT group and the other two groups； compared with the γδT group， the γδT-G10 group had significant increases in the expression levels of STING， phosphorylated STING， TBK1， phosphorylated TBK1， interferon regulatory factor 3 （IRF3）， and phosphorylated IRF3， while the γδT-H-151 group had significant reductions in the expression of these proteins. ELISA showed that compared with the γδT group， the γδT-G10 group had significant increases in the secretion of IFN-γ and TNF-α by γδT cells （P<0.01 and P<0.05）， while the γδT-H-151 group had significant reductions in IFN-γ and TNF-α （P<0.01 and P<0.000 1）. CCK-8 assay showed that compared with the γδT group， the γδT-G10 group had significant reductions in the survival rates of the two HCC cell lines （P<0.000 1）， while the γδT-H-151 group showed significant increases （P<0.000 1）. Conclusion The cGAS-STING signaling pathway can regulate the cytotoxicity of γδT cells against HCC in vitro. -
注： a，从外周血分离出的PBMC中γδT细胞占比；b，培养7 d后PBMC中γδT细胞占比。
图 1 γδT细胞纯度检测结果
Figure 1. Purity detection of gamma delta T cells
注： a，不同浓度的G10及H-151刺激的γδT细胞光学显微镜图像（×100）；b，不同浓度的G10及H-151刺激的γδT细胞团直径；c，不同浓度的G10及H-151刺激的直径>70 μm的γδT细胞团数量。
图 2 激动剂及抑制剂对γδT细胞生长的影响
Figure 2. Effects of agonists and inhibitors on the growth of gamma delta T cells
图 3 cGAS-STING信号通路关键蛋白的Western Blot结果
Figure 3. Western Blot results of key proteins in the cGAS-STING signaling pathway
注： a，不同刺激的γδT细胞对MHCC-97H细胞株的杀伤结果；b，不同刺激的γδT细胞对Huh-7细胞株的杀伤结果。*P<0.000 1。
图 4 不同刺激的γδT细胞对不同肝癌细胞株的杀伤结果
Figure 4. The killing effect of different stimuli of gamma delta T cells on different HCC cell lines
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