摘要
目的探讨ATP诱导免疫原性细胞死亡在肝癌免疫应答中的作用及其机制。方法人肝癌细胞系HepG2购自于上海弘顺生物科技有限公司,经培养及传代后,随机分为对照组和ATP组。ATP组中采用6 mmol/L ATP的培养液孵育HepG2细胞24 h;对照组将处于对数期HepG2细胞常规培养。CCK8实验检测HepG2细胞毒性实验;流式细胞仪检测HepG2细胞的凋亡;免疫荧光技术检测HepG2细胞的钙网蛋白膜转位;酶联免疫吸附试验(ELISA)检测ATP和高迁移率族蛋白1(HMGB1)含量;蛋白质印迹法检测β2微球蛋白(β2m)、TAP相关糖蛋白(TAPBP)、抗原处理相关转运体(Tap)1、Tap2和鸟苷酸腺苷酸合成酶-干扰素基因刺激因子(cGAS-STING)通路蛋白表达;Real-time PCR检测PD-L1和ICAM-1 mRNA的表达。结果6 mmol/L的ATP的作用下,能使HepG2细胞的抑制率达到50%;ATP组人肝癌HepG2细胞凋亡率显著高于对照组,差异有统计学意义(P<0.05)。与对照组相比,ATP组中钙网蛋白明显发生膜转位;且ATP组中HepG2细胞释放ATP和HMGB1的表达量明显高于对照组,差异均有统计学意义(P<0.05)。ATP组中β2m、Tapbp、Tap1、Tap2蛋白表达明显高于对照组,差异均有统计学意义(P<0.05)。ATP组中PD-L1 mRNA表达显著低于对照组,ICAM-1 mRNA表达显著高于对照组,差异均有统计学意义(P<0.05)。ATP组中cGAS和STING蛋白表达明显高于对照组,差异均有统计学意义(P<0.05)。结论ATP可以通过激活cGAS-STING信号通路,诱导肝癌细胞免疫原性细胞死亡的发生,增强肝癌的免疫原性,激活抗肿瘤免疫。
Objective To investigate the role and mechanism of ATP induced immunogenic cell death in the immune response of liver cancer.Methods The human hepatoma cell line HepG2 was purchased from Shanghai Hongshun Biotechnology Co.,Ltd.After culture and passage,human hepatoma cell line HepG2 was randomly divided into control group and ATP group.In the control group,HepG2 cells in logarithmic phase were routinely cultured.In the ATP group,HepG2 cells were incubated with 6 mmol/L ATP medium for 24 h.CCK8 test was used to detect HepG2 cytotoxicity;the apoptosis of HepG2 cells was detected by flow cytometry;the calreticulin membrane translocation of HepG2 cells was detected by immunofluorescence technique;the contents of ATP and high mobility group protein 1(HMGB1)were detected by enzyme-linked immunosorbent assay(ELISA);the expression levels ofβ2-microglobulin(β2m),TAP-associated glycoprotein(TAPBP),antigen processing-related transporter gene(Tap)1,Tap2 and guanylate adenylate synthase-interferon gene stimulator(cGAS-STING)pathway protein were detected by Western blotting assay;the expression levels of PD-L1 and ICAM-1 mRNA were detected by real time PCR.Results Under the action of 6 mmol/L ATP,the inhibition rate of HepG2 cells reached 50%.The apoptosis rate of HepG2 cells in ATP group was significantly higher than that in control group,the difference was statistically significant(P<0.05).Compared with the control group,the membrane translocation of calreticulin occurred significantly in ATP group.The expression levels of ATP and HMGB1 released by HepG2 cells in the ATP group were significantly higher than those in the control group,and the differences were statistically significant(P<0.05).The expression levels ofβ2m,Tapbp,Tap1,Tap2 protein in ATP group were significantly higher than those in the control group,and the differences were statistically significant(P<0.05).The expression level of PD-L1 mRNA in ATP group was significantly lower than that in control group,and the expression of ICAM-1 mRNA in ATP group was sign
作者
李然
刘秀平
李新
穆晓艳
LI Ran;LIU Xiu-ping;LI Xin(Department of Medical Oncology,Qingdao University,Qingdao Shandong 266042,China;Department of Integrated Traditional Chinese and Western Medicine,Qingdao University,Qingdao Shandong 266042,China;Department of Medical,Affiliated Qingdao Central Hospital,Qingdao University,Qingdao Shandong 266042,China)
出处
《临床和实验医学杂志》
2022年第21期2241-2244,共4页
Journal of Clinical and Experimental Medicine
基金
国家自然科学基金面上项目支持项目(编号:82072927)
青岛市科技创新研究基金项目(编号:18-2-2-78-JCH)。
关键词
肝癌
ATP
免疫原性细胞死亡
免疫应答
Primary liver cancer
ATP
Immunogenic cell death
Immune response
