本文選題：人巨細(xì)胞病毒 + 小鼠大腦皮質(zhì)細(xì)胞�。� 參考：《安徽醫(yī)科大學(xué)》2006年碩士論文

【摘要】：目的：研究人巨細(xì)胞病毒(human cytomegalovirus，HCMV)在體外可以增殖性感染新生小鼠大腦皮質(zhì)細(xì)胞；在體內(nèi)可以先天性潛伏感染小鼠大腦皮質(zhì)細(xì)胞，并且可以被再激活。 方法：體外試驗(yàn)：選用HCMV AD169株和3株HCMV臨床分離株作為本次實(shí)驗(yàn)的毒種，并設(shè)立經(jīng)高溫滅活后HCMV AD169株對(duì)照組。對(duì)所有毒株進(jìn)行50％組織細(xì)胞感染量(TCID_(50))測(cè)定，然后在長(zhǎng)成單層的Balb／c新生小鼠的大腦皮質(zhì)細(xì)胞和HF細(xì)胞6孔板上分別接種相同滴度的病毒懸液0.1ml／孔。逐日鏡下觀察HCMV特征性細(xì)胞致病變效應(yīng)(CPE)，同時(shí)采用PCR、RT-PCR、間接免疫熒光檢測(cè)HCMV特異性基因IE和UL83的復(fù)制和蛋白表達(dá)，確定HCMV感染狀態(tài)。分別在感染后第1、2、3、4、5天收集細(xì)胞及其培養(yǎng)上清液，采用TCID_(50)法檢測(cè)被感染的新生小鼠大腦皮質(zhì)細(xì)胞培養(yǎng)物上清的病毒滴度，每種病毒標(biāo)本分兩組，每組重復(fù)三次；并且在不同的時(shí)間點(diǎn)固定細(xì)胞片進(jìn)行HCMV特異性早期蛋白和pp65蛋白免疫熒光檢測(cè)；透射電鏡觀察大腦皮質(zhì)細(xì)胞內(nèi)HCMV病毒顆粒。體內(nèi)試驗(yàn)：利用已建立的HCMV先天性潛伏感染的小鼠模型。試驗(yàn)分為三組：HCMV先天潛伏感染組、HCMV先天性潛伏感染再激活組(小鼠腹腔注射環(huán)磷酰胺150mg／kg，每6天1次，共3次)、HF細(xì)胞對(duì)照組小鼠。在各組小鼠達(dá)18～20月齡時(shí)，，分別在各組隨機(jī)選取小鼠5只。取HCMV先天性潛伏感染組、HCMV先天性潛伏感染再激活組小鼠和HF細(xì)胞對(duì)照組小鼠大腦皮質(zhì)細(xì)胞與HF細(xì)胞建立共培養(yǎng)體系。利用PCR、RT-PCR、間接免疫熒光檢測(cè)HCMV特異性基因IE和UL83的復(fù)制和蛋白表達(dá)。 結(jié)果：體外試驗(yàn)研究：將TCID_(50)為1.7×10~3／ml HCMV AD169株病毒懸液接種在新生小鼠大腦皮質(zhì)細(xì)胞培養(yǎng)物上，在感染后第2天可見(jiàn)神經(jīng)細(xì)胞形態(tài)明顯改變：鏡下
[Abstract]:Aim: to investigate whether human cytomegalovirus (HCMV) can proliferate and infect neonatal mouse cerebral cortex cells in vitro and can be reactivated in vivo by congenital latent infection. Methods: in vitro test: HCMV AD169 strain and 3 clinical isolates of HCMV were selected as the virus strains in this experiment, and the control group of HCMV AD169 strain after high temperature inactivation was set up. All the strains were detected by 50% histocyte infection and then inoculated with the same titer of virus suspension 0.1ml/ holes on the 6-well plates of the cerebral cortex cells and HF cells of the monolayer Balb/c newborn mice. The characteristic cytopathic effect of HCMV was observed daily. Meanwhile, the replication and protein expression of HCMV specific genes IE and UL83 were detected by indirect immunofluorescence, and the status of HCMV infection was determined. The cells and their supernatants were collected on the first day after infection and the supernatant of culture was collected. The virus titers of the supernatant of cerebral cortex cell culture of infected newborn mice were detected by TCID-50 method. Each kind of virus specimen was divided into two groups and repeated three times in each group. The HCMV specific early protein and pp65 protein were detected by immunofluorescence at different time points, and the HCMV virus granules in cerebral cortex cells were observed by transmission electron microscope. In vivo test: a mouse model of congenital latent infection of HCMV was established. The experiment was divided into three groups: HCMV congenital latent infection group and HCMV congenital latent infection reactivation group (mice were injected cyclophosphamide 150 mg / kg, once every 6 days, 3 times). At the age of 18 to 20 months, 5 mice were randomly selected in each group. The coculture system of cerebral cortex cells and HF cells was established in mice of HCMV congenital latent infection group and HF cell control group. The replication and protein expression of HCMV specific genes IE and UL83 were detected by indirect immunofluorescence. Results: in vitro, the virus suspension of 1. 7 脳 10~3/ml HCMV AD169 strain was inoculated on the culture of cerebral cortex cells of newborn mice. The morphology of neurons was obviously changed on the second day after infection.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R373

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