本文關(guān)鍵詞：人巨細(xì)胞病毒感染對人胚肺成纖維細(xì)胞周期及復(fù)制前復(fù)合物的影響　出處：《中南大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人巨細(xì)胞病毒 人胚肺成纖維細(xì)胞 人巨細(xì)胞病毒IE_172蛋白 細(xì)胞周期 復(fù)制前復(fù)合物 Cdc10依賴性轉(zhuǎn)錄因子1 細(xì)胞分裂周期基因產(chǎn)物6 微小染色質(zhì)維持蛋白2 微小染色質(zhì)維持蛋白8 復(fù)制抑制因子Geminin

【摘要】： 目的 研究人巨細(xì)胞病毒(human cytomegalovirus,HCMV)感染人胚肺成纖維細(xì)胞(human embryonic lung fibroblast,HELF)后對其細(xì)胞周期及復(fù)制前復(fù)合物(pre- replication complex,pre-RC)的影響。探討HCMV感染的發(fā)病機(jī)制。 方法 1、血清饑餓法同步化HELF于G_0/G_1期,用HCMV感染同步化的HELF作為感染組(細(xì)胞數(shù)=1×10~6),同時(shí)設(shè)模擬感染組為對照組(細(xì)胞數(shù)=1×10~6)。用倒置相差顯微鏡觀察兩組細(xì)胞形態(tài)學(xué)變化至感染后7天,于感染后12、24、48、72和96小時(shí)收獲細(xì)胞。 2、流式細(xì)胞儀檢測HELF細(xì)胞周期。 3、免疫細(xì)胞化學(xué)法檢測兩組HELF細(xì)胞核內(nèi)HCMV IE_172蛋白,Cdt1蛋白水平。 4、提取總RNA,用半定量逆轉(zhuǎn)錄—聚合酶鏈反應(yīng)(RT-PCR)檢測兩組HELF復(fù)制前復(fù)合物Cdt1 mRNA、Cdc6 mRNA、Mcm2 mRNA、Mcm8 mRNA的表達(dá)水平。 5、RT-PCR法檢測兩組HELF復(fù)制抑制因子Geminin的表達(dá)水平。 結(jié)果 1、兩組HELF細(xì)胞形態(tài)學(xué)變化: 感染組HELF感染后72h時(shí)即可見局部細(xì)胞變圓,膨脹,細(xì)胞體及細(xì)胞核巨大化,7天時(shí)可見所有細(xì)胞均發(fā)生病變;模擬感染組HELF各時(shí)間點(diǎn)均未見細(xì)胞形態(tài)學(xué)改變。 2、兩組HELF HCMV IE_172蛋白陽性產(chǎn)物表達(dá): 感染組HELF于感染后12h時(shí)細(xì)胞核內(nèi)即出現(xiàn)呈棕黃色顆粒的HCMV IE_172蛋白產(chǎn)物的表達(dá);模擬感染組HELF各時(shí)間點(diǎn)均未出現(xiàn)HCMV IE_172蛋白陽性產(chǎn)物。 3、兩組HELF細(xì)胞周期檢測結(jié)果: 感染組在感染后12h時(shí)有70.4%的細(xì)胞處于G_1期,24h時(shí)有69.9%的細(xì)胞處于G_1期;模擬感染組在感染后12h時(shí)有65.7%的細(xì)胞處于G_1期,24h時(shí)有43.8%的細(xì)胞處于G_1期。感染組HELF停滯G_1期于較模擬感染組明顯增多,兩組比較,差異均有統(tǒng)計(jì)學(xué)意義(P＜0.01)。 4、兩組HELF Cdt1 mRNA表達(dá)水平: 模擬感染組在感染后12h時(shí)Cdt1 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。感染組在感染后12h時(shí)Cdt1 mRNA表達(dá)水平較低,24h時(shí)稍有升高,48h達(dá)最高峰,72h時(shí)開始下降,96h時(shí)大致維持72h水平。兩組比較,12h、24h和48h時(shí)Cdt1 mRNA表達(dá)水平差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05),12h和24h時(shí)感染組較模擬感染組明顯降低,48h時(shí)感染組較模擬感染組明顯升高。模擬感染組Cdt1 mRNA表達(dá)水平的高峰出現(xiàn)在感染后12h,而感染組的高峰則出現(xiàn)在感染后48h,較模擬感染組明顯滯后。 5、兩組HELF Cdt1蛋白水平: 12h、24h、和96h時(shí)兩組HELF Cdt1蛋白水平差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05),48h、72h時(shí)兩組HELF Cdt1蛋白水平差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。12h和96h時(shí)感染組較模擬感染組明顯降低,24h時(shí)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時(shí)又稍有回升;感染組在感染后12h時(shí)Cdt1蛋白表達(dá)水平較低,24h時(shí)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。 6、兩組HELF Cdc6 mRNA表達(dá)水平: 12h、24h、48h、72h和96h時(shí)兩組HELF Cdc6 mRNA表達(dá)水平差異均有統(tǒng)計(jì)學(xué)意義(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)Cdc6 mRNA表達(dá)水平最高,后逐漸下降,至48h時(shí)達(dá)最低水平,72h后開始回升,96h時(shí)繼續(xù)回升。 7、兩組HELF Mcm2 mRNA、Mcm8 mRNA表達(dá)水平: 12h、24h、48h、72h、96h時(shí)兩組HELF Mcm2 mRNA、Mcm8mRNA表達(dá)水平差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05),12h和24h時(shí)感染組表達(dá)水平低于模擬感染組,48h、72h、96h時(shí)感染組表達(dá)水平明顯高于模擬感染組。模擬感染組Mcm2 mRNA、Mcm8 mRNA表達(dá)水平的高峰出現(xiàn)在感染后24h,而感染組表達(dá)水平的高峰則出現(xiàn)在感染后48h,較模擬感染組有明顯滯后。 8、兩組HELF Geminin mRNA表達(dá)水平: 12h、24h、48h和72h時(shí)兩組HELF Geminin mRNA表達(dá)水平差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05),12h和24h時(shí)感染組較模擬感染組明顯增加,48h和72h時(shí)感染組較模擬感染組明顯降低。感染組在感染后12h時(shí)Geminin mRNA表達(dá)水平最高,模擬感染組在感染后48h達(dá)最高峰。 結(jié)論 1、人巨細(xì)胞病毒感染人胚肺成纖維細(xì)胞后在其細(xì)胞核內(nèi)進(jìn)行復(fù)制、增殖。 2、人巨細(xì)胞病毒感染阻滯了人胚肺成纖維細(xì)胞周期,使大部分細(xì)胞停滯在G_1期。 3、人巨細(xì)胞病毒IE_172蛋白的表達(dá)提示人巨細(xì)胞病毒的早期感染。 4、人巨細(xì)胞病毒感染使人胚肺成纖維細(xì)胞復(fù)制前復(fù)合物主要成份Cdt1 mRNA、Mcm2 mRNA、Mcm8 mRNA的表達(dá)水平遲滯、Cdt1蛋白表達(dá)延遲。Cdc6 mRNA高水平表達(dá)。 5、人巨細(xì)胞病毒感染能誘導(dǎo)人胚肺成纖維細(xì)胞復(fù)制抑制因子Geminin異常表達(dá),使Geminin/pre-RC平衡失調(diào),阻止復(fù)制前復(fù)合物的形成,DNA合成障礙。這可能是人巨細(xì)胞病毒感染導(dǎo)致人胚肺成纖維細(xì)胞周期阻滯于G_1期的機(jī)制。
[Abstract]:objective
Objective to study the effects of human cytomegalovirus (HCMV) infection on the cell cycle and the replication complex (pre- replication complex, pre-) of human embryonic lung fibroblast (human embryonic lung fibroblast), and to explore the pathogenesis of infection.
Method
1, serum starvation synchronization HELF in G_0/G_1 phase, with the synchronization of HCMV infection as HELF infection group (cell number =1 * 10~6), and simulated infection group (cell number =1 * 10~6). Two groups were observed by the inverted microscope cell morphological changes to 7 days after infection, infection after 96 hours the cells were harvested and 12,24,48,72.
2, HELF cell cycle was detected by flow cytometry.
3, the level of HCMV IE_172 protein and Cdt1 protein in the nucleus of HELF was detected by immunocytochemical method.
4, the total RNA was extracted and semi quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression level of HELF complex, Cdt1 mRNA, Cdc6 mRNA, Mcm2 mRNA and Mcm8 mRNA in the two groups.
5, RT-PCR was used to detect the expression level of the two groups of HELF replicating inhibitor Geminin.
Result
1, two groups of HELF cell morphological changes:
Infection group HELF infection 72h after that, local cells became round and expanded, cell body and nucleus were huge. 7 days, all cells were found to be lesions. There was no morphological change in HELF at each time point in the simulated infection group.
2, two groups of HELF HCMV IE_172 protein positive products were expressed as follows:
The expression of HCMV IE_172 protein appeared in the nuclei of infected group HELF at 12h after infection, and there was no HCMV IE_172 protein positive product at all time points at HELF in the simulated infection group.
3, two groups of HELF cell cycle detection results:
In the G_1 infection group in 12h after infection of 70.4% cells, 24h 69.9% cells in G_1 phase; the mock infection group in 65.7% 12h after infection of the cells in the G_1 phase, 24h 43.8% cells in the G_1 phase. HELF in G_1 infection group stagnation infection group increased significantly compared with that of, the two groups, the differences were statistically significant (P < 0.01).
4, two groups of HELF Cdt1 mRNA expression levels:
The mock infection group after infection of 12h Cdt1 mRNA expression level is the highest, then gradually decreased to the lowest level at 72h, 96h rebounded slightly. The infection group after infection 12h Cdt1 expression level of mRNA is low, 24h increased slightly, 48h reached the peak and began to decline at 72h, 96h roughly maintain 72h level. 12h comparison, two groups had significantly different Cdt1 mRNA expression of 24h and 48h (P < 0.05), 12h and 24h infection group compared with the mock infection group decreased significantly, 48h infection group compared with mock infection group increased significantly. Simulated infection group Cdt1 mRNA level of the peak at 12h after infection, and the infection group reached a peak at 48h after infection, compared with the mock infection group was significantly delayed.
5, two groups of HELF Cdt1 protein levels:
12h, 24h, 96h and HELF Cdt1 protein levels in the two groups differences were statistically significant (P < 0.05), 48h, the difference was not statistically significant 72h HELF Cdt1 protein levels in the two groups (P > 0.05).12h and 96h infection group compared with the mock infection group decreased significantly, 24h infection group compared with the mock infection group obviously increased. The mock infection group after infection of 12h Cdt1 protein expression level is the highest, then gradually decreased to the lowest level at 24h, 96h rebounded slightly after infection of 12h infection group; the expression of Cdt1 protein level is low, reached a peak of 24h, 48h declined sharply, maintained a stable level low.
6, two groups of HELF Cdc6 mRNA expression levels:
12h, 24h, 48h level showed significant differences in two groups of HELF Cdc6 mRNA expression of 72h and 96h (P < 0.05), 12h, 24h and 48h infection group compared with the mock infection group was significantly increased, 72h and 96h simulated infection group than in infected group increased significantly. The infection group after infection of 12h Cdc6 mRNA the highest expression level, then decreased gradually, to the lowest level at 72h, 96h rebounded slightly; in the simulation when infected with 12h, the expression level of mRNA Cdc6 decreased gradually after the highest infection group, 48h, to the lowest level at 72h, and began to rise, 96h continues to rise.
7, two groups of HELF Mcm2 mRNA, Mcm8 mRNA expression level:
12h, 24h, 48h, 72h, 96h of the two groups of HELF Mcm2 mRNA, Mcm8mRNA expression levels were statistically significant difference (P < 0.05), 12h and 24h infection group expression level is lower than the mock infection group, 48h, 72h, 96h infection group was significantly higher than that simulated infection group. Simulated infection group Mcm2 mRNA. The expression of Mcm8 mRNA peaked at 24h after infection, and the infection group, the expression levels reached a peak at 48h after infection, compared with mock infection group had a significant lag.
8, two groups of HELF Geminin mRNA expression levels:
12h, 24h levels were statistically significant difference of two groups of HELF Geminin mRNA expression of 48h and 72h (P < 0.05), 12h and 24h infection group compared with the mock infection group was significantly increased, 48h and 72h infection group compared with mock infection group decreased significantly. The infection group after infection of 12h Geminin mRNA the highest expression level. The mock infection group at 48h after infection and reached the peak.
conclusion
1, human cytomegalovirus (HCMV) is infected with human embryonic lung fibroblasts and replicates and proliferate in its nucleus.
2, human cytomegalovirus infection blocks the cycle of human embryonic lung fibroblasts, which makes most of the cells stagnate in the G_1 phase.
3, the expression of human cytomegalovirus IE_172 protein suggests early infection of human cytomegalovirus (CCMV).
4, human cytomegalovirus infection causes the expression level of Cdt1 mRNA, Mcm2 mRNA, Mcm8 mRNA in human embryonic lung fibroblast. The expression level of Cdt1 is delayed, and the expression level of Cdt1 is delayed by.Cdc6 mRNA.
5, human cytomegalovirus infection to human embryonic lung fibroblast replication inhibitor induced abnormal expression of Geminin, Geminin/pre-RC balance, prevent the formation of pre replication complex, disorder of DNA synthesis. This may be the mechanism of human cytomegalovirus infection in human embryonic lung fibroblast cell cycle arrest in G_1 phase.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R373

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