【摘要】：細(xì)小病毒是一組無囊膜,線狀,裂解性的DNA病毒,它們對轉(zhuǎn)化細(xì)胞具有殺傷效應(yīng)而對相應(yīng)的非轉(zhuǎn)化細(xì)胞則無害,是基因治療的候選載體。因此揭示細(xì)小病毒-宿主相互作用的機(jī)制尤為重要。前人的研究已識別了不少在細(xì)小病毒生活史中必需的或有助于病毒增殖的細(xì)胞因子,也認(rèn)識了病毒蛋白在宿主細(xì)胞中的部分作用。但細(xì)小病毒對宿主細(xì)胞分子的全面影響至今尚未被認(rèn)識。 肝癌是中國和南亞地區(qū)最常見的疾病之一。幾株肝癌細(xì)胞系和細(xì)小病毒H-1的相互關(guān)系已在以往的工作中進(jìn)行過研究,其中肝癌細(xì)胞系QGY-7703是研究最為深入的一個。為了了解細(xì)小病毒感染后細(xì)胞在轉(zhuǎn)錄水平上的全面反應(yīng)以及病毒可能的細(xì)胞靶因子,以QGY-7703-H-1作為一個模型系統(tǒng),采用了能同時監(jiān)測上千基因表達(dá)變化的基因芯片技術(shù),以期識別和細(xì)小病毒-宿主相互作用機(jī)制有關(guān)的細(xì)胞靶因子基因及其可能參與的途徑。 細(xì)小病毒的復(fù)制緊緊依賴于和增殖及分化有關(guān)的細(xì)胞因子,尤其是和細(xì)胞周期S-期有關(guān)的因子。反過來,病毒的增殖又使細(xì)胞周期停滯在S/G2期。為了提高細(xì)胞群中表達(dá)病毒基因組細(xì)胞的比例同時減少病毒對細(xì)胞周期的干擾使樣品產(chǎn)生的差異,采用同步化的細(xì)胞進(jìn)行實驗。試驗了血清饑餓法,異亮氨酸饑餓法,藥物阻斷法,以及異亮氨酸饑餓結(jié)合藥物阻斷等多種方法,發(fā)現(xiàn)草氨酸處理法既簡便又能獲得所需的高度同步化的細(xì)胞群。草氨酸處理使細(xì)胞周期被阻滯在G1末期,藥物釋放后細(xì)胞能同步化地經(jīng)過一個周期。 細(xì)胞在該藥物存在下被病毒感染。對感染后不同時間的同步化細(xì)胞的死亡率,病毒的復(fù)制情況進(jìn)行了監(jiān)測,結(jié)果表明在細(xì)胞周期阻滯釋放后,NS1蛋白及其mRNA的量隨時間的增加而增加,細(xì)胞死亡率在釋放12小時之前很低但之后也隨時間而增加。細(xì)胞仍同步地處于同一細(xì)胞周期階段,NS1表達(dá)較高,而細(xì)胞死亡率仍較低的兩個時間點(釋放后6小時和12小時)被選擇進(jìn)行芯片實驗。 用代表22,000個人類基因的寡聚核苷酸芯片(Genechip Human Genome U133A,Aflymetrix)對這兩個時間點的基因表達(dá)譜進(jìn)行了研究。為了保證實驗結(jié)果的可靠性,對實驗樣品的質(zhì)量主要是實驗不同階段RNA的完整性進(jìn)行了多步驟的監(jiān)測,表明各個步驟樣品均具有很高的質(zhì)量。芯片實驗的重復(fù)次數(shù)為兩次,兩次獨立而重復(fù)芯片實驗在6小時時間點的相關(guān)系數(shù)(coefficient correlation)分別為0.994(病毒感染1vs病毒感染2)和0.994(對照1vs對照2),在12小時時間點的相關(guān)系數(shù)分別為0.980(病毒感染1vs病毒感染2)和0.975(對照1vs對照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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R735.7;R373

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