本文選題：新城疫病毒　切入點：細胞周期　出處：《揚州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：新城疫(ND)是由新城疫病毒(NDV)引起的嚴重的家禽傳染病,給家禽業(yè)產(chǎn)生重大的經(jīng)濟損失。NDV也可以在人的一些腫瘤細胞內(nèi)復(fù)制,引起腫瘤細胞的凋亡,目前已經(jīng)被用作人類癌癥治療的工具。我們在之前的研究中注意到,NDV感染正處于對數(shù)生長期的細胞后,細胞生長會馬上停止,并于24小時后大量凋亡。因此,研究NDV感染與細胞周期的關(guān)系,一方面可以加深了解病毒入侵后促進復(fù)制的機制,另一方面還能夠更多了解NDV特異性嗜腫瘤細胞的機制,為NDV作為殺腫瘤藥物提供更為深入的理論基礎(chǔ)。本研究使用NDV感染HeLa細胞,感染24h后,固定細胞,使用流式細胞術(shù)檢測細胞周期各時相的分布,結(jié)果發(fā)現(xiàn)NDV感染誘導(dǎo)細胞周期停滯在G0/G1期。在判定NDV感染對細胞周期影響的基礎(chǔ)上,檢測UV處理及未處理NDV感染細胞后,細胞周期的變化情況,結(jié)果顯示NDV感染所誘導(dǎo)的細胞周期停滯依賴于病毒在細胞中的復(fù)制能力。為了進一步了解病毒感染進程與細胞周期的關(guān)系,我們在NDV感染細胞后不同時間點處理細胞,進行流式細胞術(shù)分析,檢測對細胞周期影響的時間點。結(jié)果發(fā)現(xiàn)在NDV感染細胞16h時,細胞周期發(fā)生改變。最后使用NDV感染禽源細胞(DF-1細胞或CEF細胞),結(jié)果顯示NDV感染同樣可以使禽源細胞停滯在G0/G1期。本研究分析了 NDV感染誘導(dǎo)細胞周期改變的分子機制。將感染后的細胞樣品進行Western Blot檢測,從細胞周期調(diào)控分子Rb蛋白的磷酸化,細胞周期蛋白(cyclin)的表達等多個層面,分析細胞周期改變的分子機制。實驗證明cyclinD1和cyclinE1下調(diào),從而在分子水平上吻合了病毒誘導(dǎo)細胞周期阻滯于G0/G1期的結(jié)果。隨后的機制顯示,NDV感染通過調(diào)控cyclin D1相關(guān)信號通路的重要分子β-catenin,從而直接調(diào)控了 cyclin D1的轉(zhuǎn)錄水平,最終影響了細胞周期的進程。本研究進一步探索了細胞周期的改變對病毒復(fù)制所帶來的影響。通過饑餓以及藥物處理等方法,將細胞分別同步化在G0、G1、G2、M期,平行建立非同步化細胞對照,各組分別接毒,感染24小時后收取細胞上清,檢測上清中病毒滴度,進行數(shù)據(jù)分析。將收取上清后的細胞裂解,提取蛋白,Western Blot分析NP蛋白的表達,將Western Blot結(jié)果量化處理后數(shù)據(jù)分析,檢測非同步化細胞以及各細胞周期時相中感染病毒的復(fù)制情況。病毒滴度與NP蛋白表達水平的結(jié)果均顯示當(dāng)細胞周期停滯于G0/G1期時,最有利于病毒復(fù)制。綜上所述,本研究通過對NDV感染后與宿主細胞細胞周期調(diào)控之間的相互關(guān)系,闡明NDV感染影響細胞周期的機制,以及病毒通過調(diào)控細胞周期,對病毒復(fù)制的影響及原理。并通過NDV感染影響細胞周期的細胞模型,分析病毒調(diào)控細胞周期的分子機制。為深入和拓寬病毒學(xué)的基礎(chǔ)研究以及腫瘤治療的應(yīng)用研究提供理論和實驗基礎(chǔ)。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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