发热伴血小板减少综合征病毒致细胞程序性死亡的研究  

Study on programmed cell death caused by severe fever with thrombocytopenia syndrome virus

作　　者：杨柱 刘畅[1] 秦泽明 温红玲[1] 黄涛[2] 赵丽[1] Yang Zhu;Liu Chang;Qin Zeming;Wen Hongling;Huang Tao;Zhao Li(Department of Laboratory Science of Sanitary Microbiology,School of Public Health,Shandong University,Key Laboratory of Shandong Province During the 13th Five-year Plan,Jinan 250012,China;handong Center for Disease Control and Prevention,Jinan 250014,China)

机构地区：[1]山东大学公共卫生学院卫生微生物检验学系、山东省"十三五"高校重点实验室,济南250012 [2]山东省疾病预防控制中心,济南250014

出　　处：《中华实验和临床病毒学杂志》2023年第1期1-6,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：山东省自然科学基金(ZR2020MH301)。

摘　　要：目的研究发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus, SFTSV)感染致细胞程序性死亡(programmed cell death, PCD)形式, 并进一步于蛋白水平探讨细胞焦亡的存在, 为研究SFTSV致病机制提供新的思路。方法用不同感染复数(multiplicity of infection, MOI)SFTSV感染Vero细胞, 逐日观察细胞病变效应(cytopathic effect, CPE), CCK-8法检测细胞活力、LDH释放试验检测细胞膜受损情况, 以判定病毒的最佳感染量与细胞死亡时间。使用不同PCD抑制剂分别对Vero细胞进行预处理后感染SFTSV, CCK-8法检测细胞活力, 判断SFTSV致PCD死亡形式。采用Western blot检测细胞焦亡相关蛋白的含量, 进一步探讨焦亡的存在。结果 SFTSV以MOI=10感染Vero细胞后48 h、72 h为后续实验最佳感染量与时间, 此时细胞CPE明显, 细胞活力降至对照组的51%、41%(P<0.001、P<0.001), LDH释放量达最大酶活性释放孔的24%、37%, 为对照组LDH释放量的3.8、3.4倍(P<0.001、P<0.001)。不同PCD抑制剂对SFTSV致细胞死亡抑制结果显示, 泛caspase抑制剂和受体相互作用丝氨酸苏氨酸激酶3(receptor-interacting serine/threonine-protein kinase 3, RIP3)抑制剂在48 h、72 h均有抑制作用, 48 h其细胞活力为病毒对照组的2.1、1.6倍, 72 h为2.3、1.7倍, 且泛caspase抑制剂的作用显著高于其他抑制剂组, caspase-1、caspase-3抑制剂仅在48 h有抑制作用, 细胞活力为病毒对照组的1.5、1.3倍。SFTSV感染Vero细胞后, 随着时间延长, caspase-1、IL-1β的表达量逐渐增加, 至48 h分别达对照组的3.4、9.5倍(P<0.001、P<0.001)。结论 SFTSV感染Vero细胞可导致多种形式PCD, 包括凋亡、焦亡、程序性坏死, 且PCD过程中可检测到焦亡相关蛋白活化, 进一步探讨了焦亡的存在。Objective To investigate the form of programmed cell death(PCD)induced by severe fever with thrombocytopenia syndrome virus(SFTSV)infection in Vero cells and further explore the existence of pyroptosis,so as to provide new ideas for studying the pathogenic mechanism of SFTSV.Methods Vero cells were infected with SFTSV at different multiplicity of infection(MOI),cytopathic effect(CPE)was observed daily,cell viability was detected by CCK-8 method,and cell membrane damage was detected by LDH release test to determine the optimal amount of virus infection and cell death time.Vero cells were pretreated with different PCD inhibitors and infected with SFTSV.CCK-8 kit was used to detect the cell viability and determine the death form of PCD caused by SFTSV.The expression of pyroptosis related proteins was detected by Western blotting to further explore the existence of pyroptosis.Results At 48 h and 72 h after SFTSV infected Vero cells with MOI=10,the optimal infection amount and time of subsequent experiments were observed.At this time,the CPE of cells was obvious,the cell viability decreased to 51%and 41%of the control group(P<0.001,P<0.001),and the LDH release amount reached 24%and 37%of the maximum enzyme activity release wells,were 3.8,3.4 times of LDH release in the control group(P<0.001,P<0.001).The inhibition of SFTSV-induced cell death by different PCD inhibitors showed that pan-caspase inhibitor and receptor-interacting serine/threonine-protein kinase 3(RIP3)inhibitor had inhibitory effects at 48 h and 72 h.The cell viability was 2.1 and 1.6 times of the viral control group at 48 h,2.3 and 1.7 times of the viral control group at 72 h,and the effect of pan-caspase inhibitor was significantly higher than that of the other inhibitor groups.Caspase-1 and caspase-3 inhibitors only had inhibitory effect at 48 h,and the cell viability was 1.5 and 1.3 times of the viral control group.After SFTSV infection of Vero cells,the expressions of caspase-1 and IL-1βincreased gradually with the prolongation of time,and reached

关 键 词：发热伴血小板减少综合征病毒 细胞程序性死亡 细胞焦亡 

分 类 号：R512.8[医药卫生—内科学]