汉坦病毒感染体外培养乳大鼠大脑皮层星形胶质细胞的初步研究

发布时间：2018-04-12 14:19

本文选题：汉坦病毒 + 大脑皮层　；参考：《第四军医大学》2008年硕士论文

【摘要】： 汉坦病毒(Hantavirus, HTVs)属布尼亚病毒科(Bunyaviridae)汉坦病毒属(Genus hantavirus),是引起肾综合征出血热(HFRS)和汉坦病毒肺综合征(HPS)的致病病毒。汉滩病毒(HTNV)、汉城病毒(SEOV)、多布拉戈一贝尔格莱德病毒(DOBV)和普马拉病毒(PUUV)分别引起重、中、轻程度不同的HFRS。已证实HTVs在许多实验动物体内呈泛嗜性感染,且乳鼠脑对HTVs高度易感,易致致死性脑炎,这也是HTVs致死性的主要原因之一。HTVs侵入机体首先在血管内皮细胞增殖,后释放入血,研究证实病毒可经血脑屏障进入脑组织。星形胶质细胞终足包绕着毛细血管,是血脑屏障的重要组成成分。但目前对于HTVs感染星形胶质细胞尚鲜见报道,为此,我们首先建立了乳大鼠大脑皮层星形胶质细胞的体外培养体系,分别用HTNV 76-118和SEOV L99毒株感染细胞,为进一步研究HTVs的致病机制提供实验依据。我们通过应用出生24 h内的Sprague Dawley乳鼠,建立乳大鼠大脑皮层星形胶质细胞的体外培养体系;分别用HTNV 76-118株和SEOV L99株病毒感染乳大鼠大脑皮层星形胶质细胞,于感染后1 d , 2 d , 3 d , 4 d , 5 d , 6 d和7 d收集细胞,免疫荧光染色、Western印迹和RT-PCR检测病毒感染细胞病毒NP和S基因片段的变化;用Hoechst33342染色、Annexin-V、Western印迹和RT-PCR检测病毒感染和星形胶质细胞间相互作用。结果发现:汉坦病毒感染星形胶质细胞1 d即可在细胞中检测到病毒抗原和少量病毒S片段RNA扩增产物,在病毒感染5 d、6 d和7 d,病毒NP表达量和病毒S片段RNA扩增产物量较病毒感染后1 d显著增多,随着感染时间的推移细胞阳性率逐渐增加(P㩳0.01)。且汉坦病毒感染后,未见凋亡细胞,Hsp70轻微增加趋势,GFAP呈增加趋势。本实验成功地建立了乳大鼠大脑皮层星形胶质细胞体外培养体系,并证实其可作为HTNV和SEOV感染的靶细胞。随着感染时间的推移病毒感染细胞数和病毒量显著增加,但未见明显的细胞损伤效应。进一步证实HTVs感染后,Hsp70和GFAP反应性增高,且GFAP和病毒NP存在共定位,可能Hsp70和GFAP共同参与了HTVs发病过程。本研究中星形胶质细胞体外培养的建立,成功地病毒感染以及病毒感染后Hsp70和GFAP的变化为进一步研究病毒与靶细胞间相互作用及揭示HFRS发病机制提供实验基础。
[Abstract]:Hantavirus (HTVs) belongs to Bunyaviridaevirus (Genus hantavirusus), which causes hemorrhagic fever with renal syndrome (HFRSs) and Hantavirus lung syndrome (HPSs).Hantaan virus (HTNVV), Seoul virus (SEOV), Doblago-Belgrade virus (DOBV) and Pumala virus (PUUV) caused severe, moderate and light HFRSs, respectively.It has been proved that HTVs is a panotropic infection in many experimental animals, and the brain of the newborn is highly susceptible to HTVs and is prone to fatal encephalitis. This is also one of the main causes of the fatal HTVs. HTVs invades the body first in the vascular endothelial cells and then releases into the blood.Studies have shown that viruses can enter the brain through the blood-brain barrier.Astrocytes, an important component of the blood-brain barrier, surround the capillaries.However, there are few reports on astrocytes infected with HTVs. For this reason, we first established an in vitro culture system of astrocytes in the cerebral cortex of neonatal rats, and infected the cells with HTNV 76-118 and SEOV L99, respectively.To provide experimental basis for further study of the pathogenesis of HTVs.We established an in vitro culture system of cerebral cortex astrocytes from Sprague Dawley mice within 24 hours of birth, and infected them with HTNV 76-118 strain and SEOV L99 strain respectively.Cells were collected on day 1, day 2, day 3, day 4, day 5, day 6 and day 7 after infection. The changes of NP and S gene fragments were detected by Western blot and RT-PCR.Hoechst33342 staining was used to detect viral infection and astrocyte interaction.The results showed that the virus antigen and a small amount of RNA amplification products of virus S fragment could be detected in the astrocytes infected with Hantavirus for 1 day.On the 6th and 7th day of virus infection, the expression of NP and the amount of RNA amplification products of virus S fragment were significantly higher than those of 1 day after infection, and the positive rate of the cells increased gradually with the passage of infection time.After Hantavirus infection, there was no slight increase of Hsp70 in apoptotic cells.In this experiment, we successfully established the culture system of cerebral cortex astrocytes in milk rats, and confirmed that it can be used as target cells for HTNV and SEOV infection.With the passage of time, the number of infected cells and the amount of virus increased significantly, but there was no obvious cell damage effect.It was further confirmed that the reactivity of Hsp70 and GFAP was increased after HTVs infection, and that there was co-localization between GFAP and viral NP, which suggested that Hsp70 and GFAP might be involved in the pathogenesis of HTVs.In this study, the establishment of astrocytes in vitro, the successful infection of virus and the changes of Hsp70 and GFAP after virus infection provide experimental basis for further studying the interaction between virus and target cells and revealing the pathogenesis of HFRS.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

【相似文献】