鼠巨细胞病毒感染RAW264.7细胞的体外实验研究

目的 体外观察鼠巨细胞病毒(murine cytomegalovirus,MCMV)对鼠巨噬细胞株RAW264.7的感染特点及其对细胞增殖和凋亡的影响.方法 分别以感染复数(multiplicity of infection,MOI)为1、0.1、0.01的MCMV Smith株感染RAW细胞,于感染后6、12、24、36、48、72、96和120 h收集细胞和培养上清,光镜下观察细胞病变,透射电镜观察感染细胞内病毒颗粒和细胞超微结构变化;免疫细胞化学检测MCMV早期抗原(early antigen,EA)表达以及蚀斑形成实验监测病毒增殖情况;MTT法和流式细胞术分别评估感染细胞增殖与凋亡的改变,以MCMV敏感的鼠胚胎成纤维细胞为阳性对照.结果 MCMV感染后24～48 h可见RAW细胞肿胀和脱落,电镜显示RAW胞浆中存在完整的病毒颗粒且细胞器肿胀明显;病毒感染RAW细胞后6 h(MOI:1和0.1)～12 h(MOI=0.01)可见病毒EA的阳性表达;感染后24 h培养上清中病毒滴度即明显上升,至96～120 h达高峰;RAW细胞增殖在感染后72～120 h受到明显抑制;当MOI=0.1时,感染后72～120 h细胞死亡率明显增高,但凋亡率改变不显著.结论 巨噬细胞株RAW264.7在体外对MCMV易感,较鼠胚胎成纤维细胞更快形成溶细胞性的产毒性感染;病毒可抑制细胞增殖但不影响细胞凋亡,为深入探讨CMV相关免疫学致病机制提供了一个良好的体外细胞模型.

Study on the infection of macrophage strain RAW264.7 by murine cytomegalovirus in vitro

Objective To study the feature that murine cytomegalovirus(MCMV)infect macrophage strain RAW264.7 and the influence of virus infection on proliferation and apoptosis of RAW264.7 in vitro.Methods RAW was infected by MCMV Smith with multiplicity of infection(MOI)1,0.1 and 0.01,respectivelv.The cells and culture supernatant were collected at 6,12,24,36,48,72,96 and 120 h post-infection(P.i.).Cytopathic effect(CPE)was found with microscope.Virus particles and uhrastructural changes of RAW were observed by transmission electron microscope(TEM). Early antigen(EA)expression was assaved bv immunohistochemical method.The proliferation of MCMV was studied by plaque formation assay.The influence of virus infection on proliferation and apoptosis of RAW were measured by MTT method and flow cytometry.The mouse embryo fibroblast(MEF)susceptible to MCMV infection was positive contro1.Results RAW was swollen and desquamated on 24-48 h P.i..The full-grown virus particles and swollen organelles in RAW were displayed with TEM.Preliminary positive expression of EA was demonstra ted from 6 h(MOI=1 and 0.1)to 12 h(MOI=0.01)P.i..Virus titer in RAw supernatant increased obviouslv on 24 h p.i.and reached the peak on 96-120 h P.i..The proliferation of RAW could be obviously inhibited by MCMV on 72-120 h p.i..When infected by virus with MOI=0.1,necrotic cells of RAW increased on 72-120 h D.i.and the influence of MCMV infection on apoptosis of RAW was not obvious.Conclusion Macrophage strain RAW264.7 is susceptible to MCMV,and it emerges faster cytolytic and productive infection than MEF.MCMV can inhibit the proliferation of RAW but not influence the apoptosis of it.These results can provide a practical experimental model for studying immunological pathogenic mechanism of cytomegalovirus in vitro.