坦布苏病毒诱导昆明鼠脾细胞凋亡  

Apoptosis of Kunming mice spleen cells induced by Tembusu virus

作　　者：提金凤 李志杰[1] 刁有祥 李舫[1] TI Jinfeng;LI Zhijie;DIAO Youxiang;LI Fang(Shandong Vocational Animal Science and Veterinary College,Weifang,Shandong 261061,China;College of Animal Science and Technology,Shandong Agricultural University,Tai′an,Shandong 271018,China)

机构地区：[1]山东畜牧兽医职业学院,山东潍坊261061 [2]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2021年第4期640-645,共6页Chinese Journal of Veterinary Science

基　　金：山东省高等学校科技发展计划资助项目(J16LF58,J17KB104);山东省家禽产业体系岗位专家资助项目(SDAIT-11-05);潍坊市科技发展计划资助项目(2018GX051)。

摘　　要：为探讨坦布苏病毒(TMUV)能否诱导昆明鼠脾细胞凋亡,采用体内和体外2种途径、不同检测方法进行验证。体内试验:TMUV脑内接种3周龄昆明鼠,分别取病毒感染3,5,7d后昆明鼠的脾脏,采用琼脂糖凝胶法观察其DNA Ladder。结果显示,攻毒3d后,脾脏没有出现明显的梯状DNA Ladder,攻毒5,7d后,脾脏出现了明显的特异性梯状DNA Ladder图谱。通过无菌研磨脾脏获得游离脾细胞,AnnexinV-FITC/PI双染色后采用流式细胞仪检测脾细胞的细胞凋亡情况。结果显示,攻毒后3d,脾细胞中凋亡细胞比例与对照组相比差异显著(P<0.05);攻毒后5,7d,脾细胞中凋亡细胞比例与对照组细胞相比差异极显著(P<0.01)。体外试验:无菌取3周龄昆明鼠脾细胞,接种不同剂量TMUV,将分别感染病毒24,48h的脾细胞AnnexinV-FITC/PI双染色,通过流式细胞仪和荧光显微镜检测脾细胞的细胞凋亡情况。结果显示,病毒感染后24,48h的脾细胞中,凋亡细胞比例与对照组相比差异显著(P<0.05)。病毒感染后24,48h脾细胞在荧光显微镜下均出现了数量较多呈现绿色荧光的细胞,且感染后48h更多。由此可见,TMUV可诱导昆明鼠发生显著的脾细胞凋亡,感染病毒时间较长,脾细胞凋亡越明显。本研究通过体内和体外2种途径、采用不同检测方法明确了TMUV能诱导昆明鼠脾细胞发生显著细胞凋亡,为进一步了解TMUV的致病机制提供理论支撑。The apoptosis of Kunming mice spleen cells induced by Tembusu virus(TMUV)was investigated in vivo and vitro.In vivo,3-week-old Kunming mice were inoculated with TMUV by the intracerebral(i.c.)route and the spleens were collected at 3days post infection(dpi),5dpi and 7dpi.DNA was examined by electrophoresis in 1%agarose gel.The result showed that no DNA Ladder was observed at 3dpi but specific DNA Ladder was observed at 5dpi and 7dpi.Spleen cells were collected and stained doubly by Annexin V-FITC and PI,and apoptosis was detected by flow cytometry.The result showed that there was a significant difference in the proportion of apoptotic spleen cells between the experimental group and the control group at 3dpi(P<0.05).There was an extremely significant difference in the proportion of apoptotic spleen cells between the experimental group and the control group at 5dpi and 7dpi(P<0.01).In vitro,spleen cells of 3-weekold Kunming mice were inoculated with TMUV and collected at 24hours post infection(hpi)and 48hpi.The cells were stained doubly by Annexin V-FITC and PI.Apoptosis of splenic cells was detected by flow cytometry and fluorescence microscope.The result showed that there was a significant difference in the proportion of apoptotic spleen cells between the experimental group and the control group at 24hpi and 48hpi(P<0.05).Many green fluorescent cells were observed at 24hpi and 48hpi under the fluorescence microscope and there were more green fluorescent cells at 48hpi,indicating that apoptosis of spleen cells could be induced by TMUV in Kunming mice.With the increase of TMUV infection time,the apoptosis of splenocytes was more obvious.This study confirmed that TMUV can induce apoptosis of spleen cells of Kunming mice in vivo and in vitro,which lays the foundation for the study of TMUV pathogenic mechanism.

关 键 词：坦布苏病毒 昆明鼠脾细胞 细胞凋亡 DNA Ladder图谱 

分 类 号：S852.65[农业科学—基础兽医学]