摘要
目的探究索拉非尼对汉滩病毒(HTNV)的体外抗病毒活性。方法使用不同浓度索拉非尼处理HTNV感染的A549细胞。采用CCK-8法检测索拉非尼对A549细胞的毒性作用,确定药物的安全工作浓度;采用斑点形成单位实验、实时荧光定量PCR、Western blot方法和免疫荧光实验检测索拉非尼对HTNV复制的抑制作用。结果索拉非尼对A549细胞的CC_(50)为26.88μmol/L,确定其安全工作浓度在20μmol/L以下。与HTNV感染组比较,斑点形成实验表明索拉非尼(10μmol/L)可降低A549细胞上清中HTNV滴度(t=30.33,P<0.05);RT-qPCR实验表明索拉非尼(10μmol/L)处理可使HTNV S基因相对表达量呈现下调(t=7.75,P<0.05);Western blot实验表明不同浓度索拉非尼(20、10、1μmol/L)处理均能使HTNV NP核蛋白表达降低,与HTNV感染组比较差异有统计学意义(t=39.00、25.86、24.39,P均<0.05);免疫荧光实验显示20、10、1μmol/L索拉非尼处理组感染率分别为(32.70±6.76)%、(50.26±7.18)%、(63.98±5.98)%,均低于HTNV感染组的感染率(100.00±11.80)%,差异有统计学意义(t=12.12、8.82、6.67,P均<0.05)。结论索拉非尼在体外能够抑制HTNV在宿主细胞中的复制,在安全工作浓度20μmol/L以下,索拉非尼对HTNV复制的抑制作用依赖于药物浓度,随药物浓度增加而增强。
Objective To explore the antiviral activity of sorafenib against Hantaan virus(HTNV)in vitro.Methods HTNV infected A549 cells were treated with sorafenib at different concentrations.The toxicity of sorafenib on A549 cells wasdetected by CCK-8 method to determine the safe working concentration.Focus-forming unit assay,real-time quantitativePCR assay,Western blot assay and immunofluorescence test,were used to detect the inhibitory effect of sorafenib on HTNVreplication.Results The CC_(50)of sorafenib on A549 cells was 26.88μmol/L,and its safe working concentration was below20μmol/L.Compared with the HTNV infection group,the focus forming unit assay showed that sorafenib(10μmol/L)could reduce the HTNV titer in the supernatant of A549 cells(t=30.33,P<0.05);RT-q PCR showed that sorafenib(10μmol/L)treatment could down-regulate the relative expression of HTNV S gene(t=7.75,P<0.05);Western blot showedthat different concentrations of sorafenib(20,10,1μmol/L)could reduce HTNV NP nuclear protein level,and thecorrelation between them was negative(t=39.00,25.86,24.39,P<0.05);Immunofluorescence experiment showed that theinfection rates of the sorafenib treatment groups(20,10,1μmol/L)were(32.70±6.76)%,(50.26±7.18)%,and(63.98±5.98)%respectively,which were all lower than the infection rates of the HTNV infection group(100.00±11.80)%,thedifferences were statistically significant(t=12.12,8.82,6.67,P<0.05).Conclusions Sorafenib could effectively inhibit thereplication of HTNV in host cells in vitro.Below a safe working concentration of 20μmol/L,the inhibitory effect of sorafenibon the replication of HTNV depends on the concentration,and increases with the increasing of drug concentration.
作者
冯禹楠
张晓晓
应旗康
董宇航
王芳
吴兴安
FENG Yu⁃nan;ZHANG Xiao⁃xiao;YING Qi⁃kang;DONG Yu⁃hang;WANG Fang;WU Xing⁃an(Department of Microbiology and Pathogenic Biology,School of Basic Medicine,Air Force Medical University,Xi'an,Shaanxi 710032,China)
出处
《热带医学杂志》
CAS
2022年第8期1037-1042,1173,共7页
Journal of Tropical Medicine
基金
国家自然科学基金(81971563)
陕西省自然科学基础研究计划(2021JM-219)
陕西省重点研发计划(2019ZDLSF02-03)
关键词
索拉非尼
汉滩病毒
复制
抑制
Sorafenib
Hantaan virus
Replication
Inhibitory
作者简介
冯禹楠(1995-),男,硕士研究生,从事分子病毒学研究;通信作者:吴兴安,E⁃mail:wuxingan@fmmu.edu.cn;通信作者:王芳,E⁃mail:wangf07@fmmu.edu.cn
