HSV-1感染L-02细胞对核不均一性核糖核蛋白H2（hnRNP H2）表达影响

发布时间：2018-10-08 17:33

【摘要】： 疱疹病毒对于人类是一种危害严重的病原体,其中以单纯疱疹病毒(Herpessimplex virus,HSV)在人群中的感染最为普遍,HSV原发感染后,机体虽然能够产生特异性免疫反应,但并不能彻底消除病毒,病毒以潜伏感染的形式终生存在于宿主神经节细胞内,在一定状态下可以被重新激活进行裂解性增殖,从而损坏机体上皮组织。单纯疱疹病毒1型(Herpes simplex virus 1,HSV-1)感染能导致一系列的细胞效应,尤其是宿主细胞蛋白质合成途径在病毒侵入后出现明显变化,例如:宿主细胞mRNA被选择性或非特异性降解、mRNA转录被关闭、蛋白质被选择性地降解或保持稳定,甚至重新定义细胞蛋白功能。本课题借助比较蛋白质组学的技术平台,将HSV-1病毒感染的人正常肝细胞L-02与未感染细胞分别进行双向电泳(2-DE),作蛋白质组图,鉴定表达差异较为显著的蛋白点。在所发现的差异蛋白中,有一个为核不均一性核糖核蛋白H2(heterogeneousnuclear ribonucleoprotein H2,hnRNP H2),在细胞中具有正性调控mRNA剪切和加工和转运,当被HSV-1感染后表现为表达量显著下调,这一结果提示:病毒通过影响细胞转录、翻译调控及关闭细胞蛋白质等不同途径实现病毒蛋白的合成。其中对剪接及转运蛋白合成的下调,将直接导致pre-mRNA加工受阻。这对宿主细胞来说,意义显得十分重要。在此基础上,本课题通过Western blot,Northern blot等技术方法进一步验证其在病毒复制过程中不同时段对其表达影响。初步探索hnRNP H2这一重要的mRNA剪切和转运蛋白,在HSV-1感染过程中与病毒蛋白之间的相互作用。NorthernBlot实验则提示,在病毒感染的不同时期hnRNP H2 mRNA的表达量有比较显著的差异,随着病毒感染的进行,表达量显著下降。与此同时,Western blot与Northern Blot实验结果相吻合,即HSV-1感染L-02细胞4h、12h和24h后,检测到hnRNP蛋白随感染进行其表达逐步下调,在0-4h内下降最为明显,从而初步得出结论,可能是由于HSVⅠ的立早基因表达产物的参与,使hnRNP H2在病毒复制早期就开始显著下调。尽管上述细胞蛋白与病毒蛋白的具体作用机制还有待于进一步研究,但本课题为探讨病毒如何改变宿主蛋白合成途径提供了很多新的线索。
[Abstract]:Herpes simplex virus (Herpessimplex virus,HSV) is one of the most harmful pathogens in human. After the primary infection of herpes simplex virus (Herpessimplex virus,HSV), the body can produce specific immune response, but it can not completely eliminate the virus. The virus exists in the host ganglion cells in the form of latent infection for life, and can be reactivated in a certain state for lytic proliferation, thus damaging the skin tissue of the body. Herpes simplex virus type 1 (Herpes simplex virus 1) HSV-1) infection can cause a series of cellular effects, especially the host cell protein synthesis pathway changes obviously after the virus invades. For example, the mRNA of host cells is selectively or nonspecifically degraded, the transcription of mRNA is shut down, the proteins are selectively degraded or maintained stable, and the function of cellular proteins is even redefined. In this study, two dimensional electrophoresis (2-DE) was performed on L-02 and uninfected human hepatocytes infected with HSV-1 virus to identify the protein spots with significant difference. One of the differential proteins was nuclear heterogeneousribonucleoprotein H2 (heterogeneousnuclear ribonucleoprotein H2hnRNP H2), which positively regulated the shearing, processing and transport of mRNA in cells, and showed a marked down-regulation of the expression level when infected with HSV-1. These results suggest that viral proteins are synthesized through different pathways, such as affecting cell transcription, regulating translation and shutting down cell proteins. The down-regulation of splicing and transporter synthesis will directly block the processing of pre-mRNA. This is very important to the host cells. On this basis, Western blot,Northern blot and other technical methods were used to further verify the effect of virus replication on its expression at different times. HnRNP H2, an important mRNA splicing and transporter, was preliminarily explored. The interaction between hnRNP H2 and viral proteins in the process of HSV-1 infection. Northern blot showed that there were significant differences in the expression of hnRNP H2 mRNA at different stages of viral infection. With the infection of the virus, the expression level decreased significantly. At the same time, the results of Western blot and Northern Blot assay were consistent, that is, after HSV-1 was infected with L-02 cells for 12 h and 24 h, the expression of hnRNP protein was gradually down-regulated with the infection, and the decrease was most obvious within 0-4 h, so the preliminary conclusion was drawn. It is possible that hnRNP H2 is down-regulated at the early stage of viral replication due to the involvement of HSV I gene expression products. Although the specific mechanism of cellular protein and viral protein remains to be further studied, this study provides many new clues on how the virus changes the host protein synthesis pathway.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

【共引文献】