溶瘤新城疫病毒激活NK细胞诱导结直肠癌细胞焦亡的作用机制  

Mechanism of pyroptosis induction in colorectal cancer cells by oncolytic Newcastle disease virus via NK cell activation

作　　者：孙桐 吴醒 李然 梁莹 樊晓晖 Sun Tong;Wu Xing;Li Ran;Liang Ying;Fan Xiaohui(Department of Microbiology,School of Basic Medical Sciences,Guangxi Medical University,Nanning 530021,China;Key Laboratory of Basic Research on Regional Diseases in Guangxi Universities,Nanning 530021,China)

机构地区：[1]广西医科大学基础医学院微生物学教研室,南宁530021 [2]广西高校区域性疾病基础研究重点实验室,南宁530021

出　　处：《国际生物制品学杂志》2025年第2期67-74,共8页International Journal of Biologicals

基　　金：国家自然科学基金(819605111);广西自然科学基金(2023GXNSFAA026280)。

摘　　要：目的探讨溶瘤新城疫病毒(Newcastle disease virus,NDV)激活的自然杀伤(natural killer,NK)细胞通过分泌颗粒酶A(granzyme A,GZMA)诱导结直肠癌细胞焦亡的作用机制,尤其是焦亡相关蛋白焦孔素B(gasdermin B,GSDMB)表达水平在其中的作用。方法以不同浓度的NDV(NDV1、2、3:0.001、0.010、0.100血凝单位/mL)与NK细胞共培养16 h,同时设空白对照组(仅培养基)和无NDV刺激的NK细胞对照组。各组分别与GSDMB高表达的SW837和低表达的SW480结直肠癌细胞共孵育后,通过定量PCR和ELISA分别检测NK细胞中效应分子的基因表达及蛋白质分泌水平,通过细胞增殖-毒性和乳酸脱氢酶释放检测评价2种肿瘤细胞株的存活率及焦亡情况,通过细胞形态学观察和GSDMB切割的蛋白质印迹检测验证NK细胞介导的焦亡效应。结果定量qPCR和ELISA结果显示,NDV感染上调了NK细胞中GZMA、IFN-γ和穿孔素的基因表达及蛋白质分泌,NDV3刺激组的GZMA(t=6.70,P=0.003)和IFN-γ(t=13.66,P<0.001)分泌水平最高,且与无刺激对照的差异均有统计学意义。相比于与未刺激的NK细胞共孵育者,与激活的NK细胞共孵育后SW837细胞活力抑制(NDV3-NK效靶比=1、2:t=﹣10.17、﹣25.94,均P<0.001)、乳酸脱氢酶(NDV3-NK效靶比=1、2:t=13.70、9.75,均P<0.001)释放、N端GSDMB(NDV2-NK效靶比=1、2:t=6.80、3.07,均P<0.05)均增加,且差异有统计学意义。SW837细胞出现质膜气泡化及肿胀等焦亡特征,而SW480细胞虽出现活力抑制和细胞破坏,但焦亡表现不明显。结论NDV激活的NK细胞能通过分泌GZMA诱导结直肠癌细胞焦亡效应,尤其在GSDMB高表达的细胞中效果显著。Objective To investigate the mechanism of pyroptosis production in colorectal cancer cells by Newcastle disease virus(NDV)via natural killer(NK)cell activation through granzyme A(GZMA)secretion,with specific focus on the role of gasdermin B(GSDMB)expression level in this process.Methods NK cells were co-cultured with NDV at different concentrations[NDV1,2,3:0.001,0.010,0.100 hemagglutinin/mL]for 16 h,and blank control(culture medium only)as well as non-stimulated NK cell control were set.All groups were subsequently incubated with high(SW837)or low(SW480)GSDMB expression colorectal cancer cells.Quantitative PCR and ELISA were used to detect the gene expression and protein secretion levels,respectively,of effector molecules in NK cells.Cell proliferation,cytotoxicity and lactate dehydrogenase(LDH)release were measured to assess the cell viability and pyroptosis in 2 tumor cell lines.Cell morphology observation and Western blot of GSDMB cleavage were applied to verify NK cell-mediated pyroptosis effect.Results Quantitative PCR and ELISA results indicated that NDV stimulation upregulated the gene expression and protein secretion levels of GZMA,IFN-γ,and perforin in NK cells.The highest protein secretion levels of GZMA(t=6.70,P=0.003)and IFN-γ(t=13.66,P<0.001)were reached in NDV3 stimulation,and were statistically significantly higher than nonstimulated NK control.Comparing with non-stimulated NK cell control treated,cell viability inhibition[NDV3-NK at effector-to-target ratio(E/T)=1,2:t=﹣10.17,﹣25.94,both P<0.001],LDH release(NDV3-NK at E/T=1,2:t=13.70,9.75,both P<0.001),and N-terminal GSDMB(NDV2-NK at E/T=1,2:t=6.80,3.07,both P<0.05)of with stimulated NK cell treated SW837 all increased statistically significantly.Pyroptosis-related features such as plasma membrane blebbing and swelling were observed in SW837,but not in SW480 cells,even though viability inhibition and cell damage were seen in SW480.Conclusion NDV-activated NK cells induce pyroptosis in colorectal cancer cells via GZMA secretion,particularly

关 键 词：新城疫病毒 焦亡 结直肠肿瘤 自然杀伤细胞 颗粒酶A 

分 类 号：R735.3[医药卫生—肿瘤]