单纯疱疹病毒1型的分离与鉴定  

Isolation and identification of herpes simplex virus type 1

作　　者：袁蒙蒙 应瑞琼 李亚龙 李睿 张玉薇 刘广芝 张永辉 YUAN Mengmeng;YING Ruiqiong;LI Yalong;LI Rui;ZHANG Yuwei;LIU Guangzhi;ZHANG Yonghui(Department of Gynecology,Henan Provincial People's Hospital,Zhengzhou University People's Hospital,Zhengzhou,Henan 450003,China;Department of Gynecology,Henan University People's Hospital,Henan Provincial People's Hospital,Zhengzhou,Henan 450003,China;Henan International Joint Laboratory of Stem Cell Clinical Translation,Zhengzhou,Henan 450003,China;Henan Engineering Research Center of Immune Cell and Stem Cell Therapy,Zhengzhou,Henan 450003,China;Henan Key Laboratory of Stem Cell Differentiation and Modification,Zhengzhou,Henan 450003,China)

机构地区：[1]河南省人民医院郑州大学人民医院妇科,河南郑州450003 [2]河南大学人民医院河南省人民医院妇科,河南郑州450003 [3]河南省干细胞临床转化国际联合实验室,河南郑州450003 [4]河南省免疫细胞与干细胞治疗工程技术研究中心,河南郑州450003 [5]河南省干细胞分化与调控重点实验室,河南郑州450003

出　　处：《中华实用诊断与治疗杂志》2024年第4期348-353,共6页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省科技攻关计划项目(212102310462,222102310119,232102310039);河南省医学科技攻关计划联合共建项目(LHGJ20220046);2020年度留学人员科研择优项目([2020]70号)。

摘　　要：目的从口唇疱疹患者疱液中分离鉴定1株单纯疱疹病毒1型(HSV-1),并比较其与HSV-1(F)在卵巢癌细胞中的细胞毒性、病毒复制及诱导免疫活化能力。方法从口唇疱疹患者疱液样本中分离得到1株病毒,经基因测序、透射电镜形态学鉴定,将其命名为HSV-1(YMM)。分别用感染复数(MOI)=1、0.1的HSV-1(YMM)和HSV-1(F)感染SKOV3、ES2、ID8细胞,48h时采用结晶紫染色法、CCK-8法检测细胞活力以评价病毒的细胞毒性;分别用MOI=0.1的HSV-1(YMM)和HSV-1(F)感染Vero细胞,12、24、36、48、60、72h时测定病毒滴度以评价病毒复制能力;分别用MOI=1的HSV-1(YMM)和HSV-1(F)感染SKOV3细胞,24h时采用实时荧光定量PCR法检测干扰素(IFN)-α、IFN-βmRNA相对表达量,并进行比较。结果经病毒基因测序和形态学鉴定,成功分离出直径为100~125nm的球形包膜病毒HSV-1(YMM),基因序列与HSV-1(Sammple11-DOCK8)(MN401208.1)100%匹配。MOI=1的HSV-1(YMM)和HSV-1(F)对SKOV3、ES2、ID8细胞均有明显毒性作用;MOI=0.1的HSV-1(YMM)和HSV-1(F)对SKOV3、ES2、ID8细胞的毒性作用均明显小于MOI=1;MOI=1或0.1的HSV-1(YMM)与HSV-1(F)对SKOV3、ES2、ID8细胞的毒性作用均无明显差异。MOI=1的HSV-1(YMM)和HSV-1(F)感染SKOV3、ES2、ID8细胞48h时细胞活力均低于MOI=0.1(P<0.05);MOI=1或0.1的HSV-1(YMM)与HSV-1(F)感染SKOV3、ES2、ID8细胞48h时细胞活力比较差异均无统计学意义(P>0.05)。感染Vero细胞12、24、36、48、60h时HSV-1(YMM)病毒滴度与HSV-1(F)比较差异均无统计学意义(P>0.05),72h时HSV-1(YMM)病毒滴度[(7.4±4.6)×10^(7)PFUs/mL]高于HSV-1(F)[(2.3±0.5)×10^(7)PFUs/mL](t=2.857,P=0.046);HSV-1(YMM)和HSV-1(F)感染Vero细胞12~48h,随时间延长病毒滴度逐渐升高。HSV-1(YMM)感染SKOV3细胞后IFN-α、IFN-βmRNA相对表达量(11.9±7.8、24.1±3.8)均高于HSV-1(F)(4.1±2.5、12.2±2.2)(t=3.353,P=0.005;t=6.638,P=0.001)。结论从口唇疱疹患者疱液样本中分离的HSV-1(YMM)与HSV(F)的细胞毒性相近,HSV-1(YMObjective To isolate and identify a strain of herpes simplex virus type 1(HSV-1)from blister fluid of patients with herpes labialis,and to compare its cytotoxicity,viral replication,and immune activation ability with HSV-1(F)in ovarian cancer cells.Methods A virus was isolated from the blister fluid of a patient with orolabial herpes,and was named HSV-1(YMM)after identification by gene sequencing and transmission electron microscopy.SKOV3,ES2,and ID8 cells were infected with HSV-1(YMM)and HSV-1(F)at multiplicity of infection(MOI)of 1 and 0.1,respectively.Crystal violet staining and CCK-8 assay were used to detect the cell viability at 48 h to evaluate the cytotoxicity of the virus.Vero cells were infected with HSV-1(YMM)and HSV-1(F)at MOI of 0.1,respectively.At 12,24,36,48,60 and 72 h,the virus titer was measured to evaluate the virus replication ability.SKOV3 cells were infected with HSV-1(YMM)and HSV-1(F)at MOI of 1,respectively.The relative expressions of interferon(IFN)-αand IFN-βmRNAs were detected by real-time fluorescence quantitative PCR at 24 h,and were compared.Results After gene sequencing and morphological identification,a spherical enveloped virus with a diameter of 100 to 125 nm was isolated,and its gene sequence completely matched to that of HSV-1(Sammple11-DOCK8)(MN401208.1).HSV-1(YMM)and HSV-1(F)at MOI of 1 had obvious toxic effects on SKOV3,ES2 and ID8 cells.The cytotoxic effects of HSV-1(YMM)and HSV-1(F)on SKOV3,ES2 and ID8 cells at MOI of 0.1 were significantly less than those at MOI of 1.There were no significant differences in the cytotoxicities of HSV-1(YMM)and HSV-1(F)on SKOV3,ES2 and ID8 cells at MOI of 1 or 0.1.The cell viabilities of SKOV3,ES2 and ID8 cells infected with HSV-1(YMM)and HSV-1(F)at MOI of 1 were lower than those at MOI of 0.1(P<0.05).There were no significant differences in cell viabilities of SKOV3,ES2 and ID8 cells infected with HSV-1(YMM)and HSV-1(F)at MOI of 1 or 0.1(P>0.05).At 12,24,36,48 and 60 h after the Vero cells were infected,there were no significant differen

关 键 词：单纯疱疹病毒1型 细胞毒性 病毒复制 Ⅰ型干扰素 卵巢癌细胞 

分 类 号：R446.5[医药卫生—诊断学]