细小病毒H-1感染对人肝癌细胞系QGY-7703基因表达影响的研究
发布时间：2019-03-12 14:43
【摘要】：细小病毒是一组无囊膜,线状,裂解性的DNA病毒,它们对转化细胞具有杀伤效应而对相应的非转化细胞则无害,是基因治疗的候选载体。因此揭示细小病毒-宿主相互作用的机制尤为重要。前人的研究已识别了不少在细小病毒生活史中必需的或有助于病毒增殖的细胞因子,也认识了病毒蛋白在宿主细胞中的部分作用。但细小病毒对宿主细胞分子的全面影响至今尚未被认识。 肝癌是中国和南亚地区最常见的疾病之一。几株肝癌细胞系和细小病毒H-1的相互关系已在以往的工作中进行过研究,其中肝癌细胞系QGY-7703是研究最为深入的一个。为了了解细小病毒感染后细胞在转录水平上的全面反应以及病毒可能的细胞靶因子,以QGY-7703-H-1作为一个模型系统,采用了能同时监测上千基因表达变化的基因芯片技术,以期识别和细小病毒-宿主相互作用机制有关的细胞靶因子基因及其可能参与的途径。 细小病毒的复制紧紧依赖于和增殖及分化有关的细胞因子,尤其是和细胞周期S-期有关的因子。反过来,病毒的增殖又使细胞周期停滞在S/G2期。为了提高细胞群中表达病毒基因组细胞的比例同时减少病毒对细胞周期的干扰使样品产生的差异,采用同步化的细胞进行实验。试验了血清饥饿法,异亮氨酸饥饿法,药物阻断法,以及异亮氨酸饥饿结合药物阻断等多种方法,发现草氨酸处理法既简便又能获得所需的高度同步化的细胞群。草氨酸处理使细胞周期被阻滞在G1末期,药物释放后细胞能同步化地经过一个周期。 细胞在该药物存在下被病毒感染。对感染后不同时间的同步化细胞的死亡率,病毒的复制情况进行了监测,结果表明在细胞周期阻滞释放后,NS1蛋白及其mRNA的量随时间的增加而增加,细胞死亡率在释放12小时之前很低但之后也随时间而增加。细胞仍同步地处于同一细胞周期阶段,NS1表达较高,而细胞死亡率仍较低的两个时间点(释放后6小时和12小时)被选择进行芯片实验。 用代表22,000个人类基因的寡聚核苷酸芯片(Genechip Human Genome U133A,Aflymetrix)对这两个时间点的基因表达谱进行了研究。为了保证实验结果的可靠性,对实验样品的质量主要是实验不同阶段RNA的完整性进行了多步骤的监测,表明各个步骤样品均具有很高的质量。芯片实验的重复次数为两次,两次独立而重复芯片实验在6小时时间点的相关系数(coefficient correlation)分别为0.994(病毒感染1vs病毒感染2)和0.994(对照1vs对照2),在12小时时间点的相关系数分别为0.980(病毒感染1vs病毒感染2)和0.975(对照1vs对照2),
[Abstract]:Parvovirus is a group of non-enveloped, linear, lytic DNA viruses, which have killing effect on transformed cells and harmless to corresponding non-transformed cells. Parvoviruses are candidate vectors for gene therapy. Therefore, it is very important to reveal the mechanism of parvovirus-host interaction. Previous studies have identified many cytokines that are necessary in the life cycle of parvovirus or contribute to virus proliferation, as well as the role of viral proteins in host cells. However, the overall effect of parvovirus on host cell molecules has not yet been recognized. Liver cancer is one of the most common diseases in China and South Asia. The relationship between several hepatoma cell lines and parvovirus H _ (1) has been studied in the past. Among them, the hepatoma cell line (QGY-7703) is the most in-depth study. In order to understand the overall response of parvovirus infected cells at the transcription level and the possible cell target factors of the virus, QGY-7703-H-1 was used as a model system. In order to identify cell target factor genes related to parvovirus-host interaction mechanism and their possible pathways, a gene chip technique that can simultaneously monitor the expression of thousands of genes has been adopted in order to identify cell target factor genes related to the mechanism of parvovirus-host interaction. Parvovirus replication closely depends on cytokines related to proliferation and differentiation, especially those related to S-phase of cell cycle. In turn, the proliferation of the virus stalls the cell cycle in the S/G2 phase. In order to increase the proportion of virus genome cells expressed in the cell population and reduce the difference of cell cycle caused by virus interference, synchronous cells were used to experiment. Several methods, such as serum starvation, isoleucine starvation, drug blocking, isoleucine starvation combined with drug blocking, were tested. It was found that the oxalic acid treatment was simple and able to obtain the highly synchronized cell populations needed. Oxalic acid treatment blocks the cell cycle at the end of G1, and cells can pass through a cycle synchronously after drug release. The cells were infected with the virus in the presence of the drug. The death rate of synchronized cells and replication of virus were monitored at different time after infection. The results showed that the amount of NS1 protein and its mRNA increased with the increase of time after cell cycle arrest release. Cell mortality is low 12 hours before release but increases over time. The cells were still in the same cell cycle phase, the expression of NS1 was high, and the two time points with low cell mortality (6 hours and 12 hours after release) were selected for microarray experiments. The gene expression profiles of 22000 human genes were studied by using oligonucleotide chip (Genechip Human Genome U133A (Aflymetrix). In order to ensure the reliability of the experimental results, the quality of the experimental samples is mainly the integrity of the RNA in different stages of the experiment. The results show that the samples of each step have very high quality. The correlation coefficients (coefficient correlation) were 0.994 (1 vs 2) and 0.994 (control 1vs control 2), respectively, at the 6-hour time point in which the microarray experiment was repeated twice, and the correlation coefficients of the two independent repeats were 0.994 (1 vs 2) and 0.994 (control group 2) at the 6-hour time point respectively. The correlation coefficients were 0.980 (viral infection 1 vs 2) and 0.975 (control 1vs control 2) at the 12-hour time point, respectively.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R735.7;R373
本文编号：2438875
[Abstract]:Parvovirus is a group of non-enveloped, linear, lytic DNA viruses, which have killing effect on transformed cells and harmless to corresponding non-transformed cells. Parvoviruses are candidate vectors for gene therapy. Therefore, it is very important to reveal the mechanism of parvovirus-host interaction. Previous studies have identified many cytokines that are necessary in the life cycle of parvovirus or contribute to virus proliferation, as well as the role of viral proteins in host cells. However, the overall effect of parvovirus on host cell molecules has not yet been recognized. Liver cancer is one of the most common diseases in China and South Asia. The relationship between several hepatoma cell lines and parvovirus H _ (1) has been studied in the past. Among them, the hepatoma cell line (QGY-7703) is the most in-depth study. In order to understand the overall response of parvovirus infected cells at the transcription level and the possible cell target factors of the virus, QGY-7703-H-1 was used as a model system. In order to identify cell target factor genes related to parvovirus-host interaction mechanism and their possible pathways, a gene chip technique that can simultaneously monitor the expression of thousands of genes has been adopted in order to identify cell target factor genes related to the mechanism of parvovirus-host interaction. Parvovirus replication closely depends on cytokines related to proliferation and differentiation, especially those related to S-phase of cell cycle. In turn, the proliferation of the virus stalls the cell cycle in the S/G2 phase. In order to increase the proportion of virus genome cells expressed in the cell population and reduce the difference of cell cycle caused by virus interference, synchronous cells were used to experiment. Several methods, such as serum starvation, isoleucine starvation, drug blocking, isoleucine starvation combined with drug blocking, were tested. It was found that the oxalic acid treatment was simple and able to obtain the highly synchronized cell populations needed. Oxalic acid treatment blocks the cell cycle at the end of G1, and cells can pass through a cycle synchronously after drug release. The cells were infected with the virus in the presence of the drug. The death rate of synchronized cells and replication of virus were monitored at different time after infection. The results showed that the amount of NS1 protein and its mRNA increased with the increase of time after cell cycle arrest release. Cell mortality is low 12 hours before release but increases over time. The cells were still in the same cell cycle phase, the expression of NS1 was high, and the two time points with low cell mortality (6 hours and 12 hours after release) were selected for microarray experiments. The gene expression profiles of 22000 human genes were studied by using oligonucleotide chip (Genechip Human Genome U133A (Aflymetrix). In order to ensure the reliability of the experimental results, the quality of the experimental samples is mainly the integrity of the RNA in different stages of the experiment. The results show that the samples of each step have very high quality. The correlation coefficients (coefficient correlation) were 0.994 (1 vs 2) and 0.994 (control 1vs control 2), respectively, at the 6-hour time point in which the microarray experiment was repeated twice, and the correlation coefficients of the two independent repeats were 0.994 (1 vs 2) and 0.994 (control group 2) at the 6-hour time point respectively. The correlation coefficients were 0.980 (viral infection 1 vs 2) and 0.975 (control 1vs control 2) at the 12-hour time point, respectively.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R735.7;R373
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