本文關(guān)鍵詞：HCMV感染對(duì)HEL細(xì)胞MCM裝載因子Cdc6與Cdt1的影響　出處：《中南大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人巨細(xì)胞病毒 HEL細(xì)胞 Cdc6 Cdt1

【摘要】： 目的研究人類巨細(xì)胞病毒(human cytomegalovirus，HCMV)感染對(duì)人胚肺成纖維(human embryonic lung fibroblastic，HEL)細(xì)胞的MCM裝載因子Cdc6與Cdt1表達(dá)的影響，從分子水平上探討HCMV的發(fā)病機(jī)制。 方法用血清饑餓法同步化HEL細(xì)胞于G_0／G_1期，流式細(xì)胞術(shù)測(cè)細(xì)胞周期；用HCMV感染同步化的HEL細(xì)胞作為感染組，同時(shí)設(shè)模擬感染組為對(duì)照組。用倒置相差顯微鏡觀察兩組細(xì)胞形態(tài)改變至感染后7天。于感染后12h(hour)、24h、48h、72h和96h收獲細(xì)胞，提取總的RNA，采用半定量逆轉(zhuǎn)錄—聚合酶鏈反應(yīng)(RT—PCR)技術(shù)檢測(cè)Cdc6 mRNA的表達(dá)水平；用免疫細(xì)胞化學(xué)法檢測(cè)兩組細(xì)胞核內(nèi)HCMV Cdt1蛋白。 結(jié)果 1．模擬感染組未見(jiàn)細(xì)胞形態(tài)學(xué)改變，感染組感染后36h HEL細(xì)胞有形態(tài)學(xué)的改變即可見(jiàn)局部HEL細(xì)胞變圓、胞體變大、膨脹，7天時(shí)形態(tài)學(xué)的改變更加明顯，所有細(xì)胞均發(fā)生病變。 2．12h、24h、48h、72h和96h兩組HEL細(xì)胞Cdc6 mRNA表達(dá)水平均有顯著性差異(P＜0.05)，12h、24h和48h時(shí)感染組較模擬感染組明顯增加，72h和96h時(shí)模擬感染組較感染組明顯升高。感染組在感染后12h時(shí)Cdc6 mRNA表達(dá)水平最高，后逐漸下降，至72h時(shí)達(dá)最低水平，96h時(shí)又稍有回升；模擬感染組在感染12h時(shí)Cdc6mRNA表達(dá)水平最高，后逐漸下降，至48h時(shí)達(dá)最低水平，72h時(shí)后開始回升，96h時(shí)繼續(xù)回升。 3．12h、24h、和96h兩組HEL細(xì)胞Cdt1蛋白水平均有顯著性差異(P＜0.05)，而48h、72h兩組HEL細(xì)胞Cdt1蛋白水平無(wú)明顯差異(P＞0.05)。12h和96h時(shí)感染組較模擬感染組明顯降低，24h感染組較模擬感染組明顯升高。模擬感染組在感染后12h時(shí)Cdt1蛋白表達(dá)水平最高，后逐漸下降，至24h時(shí)達(dá)最低水平，96h又稍有回升；感染組在感染后12h Cdt1蛋白表達(dá)水平較低，24h達(dá)到最高峰，48h時(shí)急劇下降，一直維持較低的穩(wěn)定水平。 結(jié)論 1．HCMV誘導(dǎo)Cdc6 mRNA過(guò)度表達(dá)，導(dǎo)致Cdc6的積聚，最終將影響細(xì)胞周期的進(jìn)程。 2．HCMV誘導(dǎo)Cdt1蛋白表達(dá)延遲，從而影響了MCM復(fù)合物的裝載和pre-RC的形成，使細(xì)胞停滯在G_1期，阻止DNA的復(fù)制。
[Abstract]:Objective to study human cytomegalovirus cytomegalovirus. Human embryonic lung fibroblastic was infected with human fetal lung fibroblasts. The effects of MCM loading factor Cdc6 and Cdt1 on the expression of HCMV in Hell cells were studied in order to explore the pathogenesis of HCMV at molecular level. Methods the HEL cells were synchronized by serum starvation in the G _ 0 / G _ S _ 1 phase and the cell cycle was measured by flow cytometry. The synchronous HEL cells infected with HCMV were used as the infection group. At the same time, the simulated infection group was set up as the control group. The morphologic changes of the cells in the two groups were observed by inverted phase contrast microscope until 7 days after infection, and 24 hours and 48 hours after infection. The cells were harvested at 72 h and 96 h, the total RNAs were extracted and the expression level of Cdc6 mRNA was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The HCMV Cdt1 protein in the nuclei of the two groups was detected by immunocytochemistry. Results 1. There were no morphological changes in the simulated infection group, but there were morphological changes in the HEL cells 36 hours after infection. The local HEL cells became round, the cell bodies became larger and expanded. At 7 days, the morphological changes were more obvious, and all the cells had pathological changes. There was a significant difference in the expression of Cdc6 mRNA between the two groups (P < 0.05) and the expression of Cdc6 mRNA at 24 h (P < 0.05) and 96 h (P < 0.05). At 24h and 48h, the number of infected group was significantly higher than that of simulated infection group. At 72 h and 96 h, the expression of Cdc6 mRNA in the simulated infection group was significantly higher than that in the infected group. The expression of Cdc6 mRNA in the infected group reached the highest level at 12 h after infection, then decreased gradually, and reached the lowest level at 72 h after infection. At 96 h, there was a slight rise. In the simulated infection group, the expression of Cdc6mRNA reached its highest level at 12h, then decreased gradually, and continued to rise again at 48h after reaching the lowest level at 72h and after 96h. The levels of Cdt1 protein in HEL cells at 3.12h and 96h were significantly different (P < 0.05, P < 0.05), but at 48h, the levels of Cdt1 protein in HEL cells were significantly higher than those in control group (P < 0.05). There was no significant difference in Cdt1 protein level in HEL cells between the two groups at 72 h after infection (P > 0.05. 12 h and 96 h). The Cdt1 protein level in infected group was significantly lower than that in simulated infection group. The expression of Cdt1 protein in the simulated infection group was the highest at 12 h after infection, then decreased gradually, and then increased slightly at the lowest level at 24 h after infection. In the infected group, the expression of Cdt1 protein decreased sharply at 12 h after infection and reached the peak at 24 h, and maintained a low and stable level at 48 h after infection. Conclusion 1. HCMV-induced overexpression of Cdc6 mRNA leads to the accumulation of Cdc6, which will eventually affect the process of cell cycle. 2. HCMV-induced delayed expression of Cdt1 protein, which affected the loading of MCM complex and the formation of pre-RC. HCMV-induced cell arrest in G _ (1) phase and blocked the replication of DNA.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R373

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