【作者】 李健宏；

【导师】 罗祖玉； Jean Rommelaere； Jean-No(?)l Freund；

【作者基本信息】 复旦大学 ， 生物物理学， 2005， 博士

【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),更多还原

【Abstract】 Autonomous parvoviruses are nonenveloped, linear-stranded, lytic DNA viruses which are cytotoxic to neoplastic cells while innocuous to their non-transformed counterparts, thus become a candidate of choice as vehicles in gene therapy. It is therefore of special importance to uncover the mechanism underlying parvovirus-host cell interaction. Previous studies have identified dozens of cellular factors which are necessary and helpful in viral life cycle and the some roles of viral proteins on the host cell have also been determined. However, the global impact of parvovirus infection on the host cell has not been investigated by far. Liver cancer is one of the most common cancer in China and southern Asia, the relationship between several hepatoma cell lines and parvovirus H-l have been investigated in the previous works, among them human hepatocellular carcinoma cell line QGY-7703 is the most well-studied one. In order to gain insight into the global cellular response at the transcriptional level and the possible cellular targets after parvovirus infection, QGY-7703-H-l was used as a model system and DNA microarray technology which simultaneously measure the transcript abundance of thousands of genes was employed to identify the target genes and pathways which underlie the mechanism of parvovirus-host interaction.The replication of parvovirus is strongly dependent on cellular factors associated with proliferation and differentiation, especially those related to the S-phase of the cell cycle. Conversely, the multiplication of virus can induce cell cycle arrest at S/G2 phase. With the aim of enhancing the fraction of cells expressing the viral genome as well as reducing the differences between samples under comparison due to virus interference with the cell cycle, synchronized cells were used to perform the experiments. Several approaches including serum starvation, isoleucine depletion, drug treatment and isoleucine plus drug treatment were tried to find the best method and optimal condition for synchronizing cells. Mimosine was found suitable to yield a highly synchronized QGY-7703 cell population. Cells were arrested at late Gl phase after drug treatment and proceed synchronously during at least one cycle. The cells were infected in the presence of this drug. The morality of cells and the replication of virus in synchronized cells after infection were monitored. The results show that after release from the cell cycle arrest cells NS1 protein and its mRNA increase over time,cell morality is low before 12h postrelease but increase over time after this time point. Two time points (6hrs and 12hrs postrelease) at which cells are synchronously in the same cell cycle stage, the NS1 expression is high and the morality is still low were selected for further study.The gene expression profiles at these time points were measured respectively using oligonucleotide microarray (Genechip Human Genome U133A, Affymetrix) that represent 22,000 human genes. In order to improve the reliability of the experiments, the quality of the samples, mainly the integrity of RNA at various steps in the experiment was monitored, the results demonstrated high quality of the samples. The experiment was performed in duplicates. The coefficient correlations of the two repeated but independent experiment are 0.994 (virus 1 vs virus 2) and 0.994 (control 1 vs control 2)at 6h postrelease; 0.980(virus 1 vs virus 2) and 0.975(control 1 vs control 2) at 12h postrelease. No significant difference at mRNA level were observed between H-l and mock-infected cells, although the NS1 positive cells was above 60%; 12h after release, the mRNA level of 38 genes changed in infected cells compared with uninfected cultures by the factor of 2.5, among which 29 genes are down-regulated and 9 up-regulated. Classification of these candidates into functional clusters identified mainly genes related to transcriptional regulation, signaling transduction and apoptosis, concerning many important pathways. Owing to the use of synchronized cell and the choice of earlier time points, the amount of genes whose expression are regulated is small, and the genes which are related to the traverse of cell cycle were not detected. Furthermore, the genes involved in immune reaction were also not found.10 of these genes were selected for confirmation by real-time RT-PCR (lightcycler). Using RNA sample either from microarray experiments or from independent experiments, the modulation of gene expression of all of them has been verified and the folds changes are rather close to each other, attesting the high reproducibility and reliability of microarray experiments. The results also showed that at 6h after release, the expression of these genes has been influenced, but to an extent that is below the standards set for selecting genes in microarray experiments, suggesting that NS1 is required to surpass a threshold in order to exert observable effect of virus. Changes of mRNA level of 7 of these verified genes after infection in a time course way were then followed by real-time RT-PCR and the results showed that MYC, DUSP2, DKK1, CEBPD, ARHB, and ID3 which are down-regulated at更多还原