新城疫病毒诱导细胞周期停滞的研究

发布时间：2018-03-20 10:38

本文选题：新城疫病毒　切入点：细胞周期　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：新城疫(ND)是由新城疫病毒(NDV)引起的严重的家禽传染病,给家禽业产生重大的经济损失。NDV也可以在人的一些肿瘤细胞内复制,引起肿瘤细胞的凋亡,目前已经被用作人类癌症治疗的工具。我们在之前的研究中注意到,NDV感染正处于对数生长期的细胞后,细胞生长会马上停止,并于24小时后大量凋亡。因此,研究NDV感染与细胞周期的关系,一方面可以加深了解病毒入侵后促进复制的机制,另一方面还能够更多了解NDV特异性嗜肿瘤细胞的机制,为NDV作为杀肿瘤药物提供更为深入的理论基础。本研究使用NDV感染HeLa细胞,感染24h后,固定细胞,使用流式细胞术检测细胞周期各时相的分布,结果发现NDV感染诱导细胞周期停滞在G0/G1期。在判定NDV感染对细胞周期影响的基础上,检测UV处理及未处理NDV感染细胞后,细胞周期的变化情况,结果显示NDV感染所诱导的细胞周期停滞依赖于病毒在细胞中的复制能力。为了进一步了解病毒感染进程与细胞周期的关系,我们在NDV感染细胞后不同时间点处理细胞,进行流式细胞术分析,检测对细胞周期影响的时间点。结果发现在NDV感染细胞16h时,细胞周期发生改变。最后使用NDV感染禽源细胞(DF-1细胞或CEF细胞),结果显示NDV感染同样可以使禽源细胞停滞在G0/G1期。本研究分析了 NDV感染诱导细胞周期改变的分子机制。将感染后的细胞样品进行Western Blot检测,从细胞周期调控分子Rb蛋白的磷酸化,细胞周期蛋白(cyclin)的表达等多个层面,分析细胞周期改变的分子机制。实验证明cyclinD1和cyclinE1下调,从而在分子水平上吻合了病毒诱导细胞周期阻滞于G0/G1期的结果。随后的机制显示,NDV感染通过调控cyclin D1相关信号通路的重要分子β-catenin,从而直接调控了 cyclin D1的转录水平,最终影响了细胞周期的进程。本研究进一步探索了细胞周期的改变对病毒复制所带来的影响。通过饥饿以及药物处理等方法,将细胞分别同步化在G0、G1、G2、M期,平行建立非同步化细胞对照,各组分别接毒,感染24小时后收取细胞上清,检测上清中病毒滴度,进行数据分析。将收取上清后的细胞裂解,提取蛋白,Western Blot分析NP蛋白的表达,将Western Blot结果量化处理后数据分析,检测非同步化细胞以及各细胞周期时相中感染病毒的复制情况。病毒滴度与NP蛋白表达水平的结果均显示当细胞周期停滞于G0/G1期时,最有利于病毒复制。综上所述,本研究通过对NDV感染后与宿主细胞细胞周期调控之间的相互关系,阐明NDV感染影响细胞周期的机制,以及病毒通过调控细胞周期,对病毒复制的影响及原理。并通过NDV感染影响细胞周期的细胞模型,分析病毒调控细胞周期的分子机制。为深入和拓宽病毒学的基础研究以及肿瘤治疗的应用研究提供理论和实验基础。
[Abstract]:Newcastle disease (NDV) is a serious infectious disease of poultry caused by Newcastle disease virus (NDV). NDV can also replicate in some human tumor cells and cause apoptosis of tumor cells. It's been used as a tool for cancer treatment in humans. In previous studies, we've noticed that after NDV infection is in the logarithmic growth phase of cells, cell growth stops immediately, and a large number of apoptosis occurs 24 hours later. By studying the relationship between NDV infection and cell cycle, we can understand the mechanism of replication after virus invasion, on the other hand, we can understand more about the mechanism of NDV specific tumor cells. In this study, NDV was used to infect HeLa cells, 24 hours after infection, fixed cells, and flow cytometry was used to detect the distribution of cell cycle. The results showed that NDV infection induced cell cycle arrest in G _ 0 / G _ 1 phase. On the basis of judging the effect of NDV infection on cell cycle, the changes of cell cycle after UV treatment and untreated NDV infection were detected. The results showed that cell cycle arrest induced by NDV infection depended on the ability of virus replication in cells. In order to further understand the relationship between viral infection process and cell cycle, we treated cells at different time points after NDV infection. Flow cytometry was used to detect the time point of cell cycle. The results showed that the cells were infected with NDV for 16 h. Cell cycle changes occurred. Finally, NDV was used to infect fowl-derived cells of DF-1 cells or CEF cells. The results showed that NDV infection could also cause avian origin cells to stagnate in G _ 0 / G _ 1 phase. In this study, the cell cycle changes induced by NDV infection were analyzed. Submechanisms. Western Blot detection was performed on infected cell samples. The molecular mechanism of cell cycle change was analyzed from several aspects, such as the phosphorylation of RB protein and the expression of cyclin. The results showed that cyclinD1 and cyclinE1 were down-regulated. Therefore, the results of virus induced cell cycle arrest in G _ 0 / G _ 1 phase were consistent at molecular level. The subsequent mechanism showed that cyclin D1 infection directly regulated the transcription level of cyclin D1 by regulating the important molecule 尾 -catenin, which is an important molecule in the signal pathway associated with cyclin D1. In this study, we further explored the effect of cell cycle changes on viral replication. By starvation and drug treatment, cells were synchronized in G0 / G1 / G2 / M phase, respectively. The cell supernatant was collected 24 hours after infection, the virus titer in the supernatant was detected, and the data was analyzed. The protein was extracted from the supernatant to analyze the expression of NP protein by Western Blot. After quantifying the Western Blot results, the replication of the infected virus was detected in the asynchronous cells and in each cell cycle. The results of viral titer and NP protein expression level showed that when the cell cycle stopped at the G _ 0 / G _ 1 phase, both the viral titer and the NP protein expression level showed that the cell cycle was stagnant in the G _ 0 / G _ 1 phase. To sum up, the relationship between NDV infection and host cell cycle regulation is used to elucidate the mechanism of NDV infection affecting cell cycle, and the virus regulates cell cycle. The effect and principle of virus replication, and the cell model that affects cell cycle through NDV infection, The molecular mechanism of viral regulation of cell cycle is analyzed, which provides a theoretical and experimental basis for further and broadening the basic research of virology and the application of tumor therapy.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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