【摘要】： 目的研究人類巨細(xì)胞病毒(HCMV)能否感染體外培養(yǎng)的由人海馬神經(jīng)干細(xì)胞(NSCs)分化成的星形膠質(zhì)細(xì)胞(Astrocytes),并觀察病毒感染對(duì)細(xì)胞凋亡的影響,為探討先天性巨細(xì)胞病毒感染致腦發(fā)育異常的機(jī)制提供理論依據(jù)。 方法體外分離、培養(yǎng)人海馬NSCs,添加血清誘導(dǎo)其定向分化為星形膠質(zhì)細(xì)胞,倒置顯微鏡下觀察細(xì)胞的形態(tài)學(xué)特點(diǎn)并利用間接免疫熒光染色方法通過(guò)檢測(cè)其各自的標(biāo)志蛋白進(jìn)行鑒定。然后用HCMV AD169株感染人星形膠質(zhì)細(xì)胞(MOI=1),鏡下觀察感染后細(xì)胞的形態(tài)學(xué)變化,利用間接免疫熒光技術(shù)檢測(cè)細(xì)胞內(nèi)病毒基因產(chǎn)物-IE蛋白的表達(dá),采用流式細(xì)胞術(shù)PI染色和細(xì)胞涂片Annexin V-FITC/PI染色法檢測(cè)感染細(xì)胞的凋亡情況。同時(shí)收集感染細(xì)胞的培養(yǎng)液,接種到正常人胚肺二倍體細(xì)胞(MRC-5)中,觀察有無(wú)典型的HCMV致細(xì)胞病變出現(xiàn)。 結(jié)果體外原代培養(yǎng)的人海馬NSCs以神經(jīng)球的形式懸浮生長(zhǎng),絕大多數(shù)細(xì)胞都表達(dá)NSCs的標(biāo)記物-巢蛋白(nestin),誘導(dǎo)其分化后,細(xì)胞高表達(dá)星形膠質(zhì)細(xì)胞的標(biāo)志物—神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP)。HCMV AD169感染星形膠質(zhì)細(xì)胞7天后,細(xì)胞出現(xiàn)腫脹、變大、變圓等典型病變,胞核內(nèi)檢測(cè)到HCMV即刻早期基因的表達(dá)產(chǎn)物-IE蛋白。流式細(xì)胞術(shù)PI單染結(jié)果顯示:MOI=1時(shí),感染組細(xì)胞凋亡比例為14.72%±5.78%,明顯高于正常對(duì)照組7.59%±2.33%,兩組比較有統(tǒng)計(jì)學(xué)意義(t=3.18,P0.05)。細(xì)胞涂片Annexin V-FITC/PI染色結(jié)果也顯示,感染組細(xì)胞凋亡比例高于對(duì)照組。收集感染細(xì)胞的培養(yǎng)液并接種到HCMV的完全容許性細(xì)胞—MRC-5中,一周后細(xì)胞也出現(xiàn)上述典型病變,證明HCMV可在星形膠質(zhì)細(xì)胞內(nèi)增殖、復(fù)制并釋放到胞外。 結(jié)論(1)在體外培養(yǎng)的由人NSCs分化成的星形膠質(zhì)細(xì)胞中,HCMV AD169能夠建立增殖性感染,該細(xì)胞是HCMV的容許性細(xì)胞。 (2)HCMV感染誘導(dǎo)體外培養(yǎng)的由人NSCs分化成的星形膠質(zhì)細(xì)胞凋亡,這可能與先天性巨細(xì)胞病毒感染導(dǎo)致腦發(fā)育異常有關(guān)。
[Abstract]:Objective to study whether human cytomegalovirus (HCMV) can infect (Astrocytes), which is differentiated from human hippocampal neural stem cell (NSCs) in vitro, and to observe the effect of viral infection on apoptosis. It provides a theoretical basis for exploring the mechanism of abnormal brain development caused by congenital cytomegalovirus infection. Methods Human hippocampal NSCs, was isolated in vitro and cultured with serum to induce its directional differentiation into astrocytes. The morphological characteristics of the cells were observed under inverted microscope and identified by indirect immunofluorescence staining. Then the human astrocytes (MOI=1) were infected with HCMV AD169 strain. The morphological changes of the infected cells were observed under microscope, and the expression of IE protein, the intracellular viral gene product, was detected by indirect immunofluorescence technique. Flow cytometry PI staining and cell smear Annexin V-FITC/PI staining were used to detect the apoptosis of infected cells. At the same time, the culture medium of infected cells was collected and inoculated into normal embryonic lung diploid cells (MRC-5) to observe whether there were typical HCMV-induced cytopathic effects. Results Human hippocampal NSCs cultured in vitro was suspended in the form of neurosphere. Most of the cells expressed nestin (nestin), a marker of NSCs, to induce the differentiation of Hippocampal DNA. Seven days after the astrocytes were infected with glial fibrillar acidic protein (GFAP). HCMV AD169), a marker of high expression of astrocytes, the cells showed typical lesions such as swelling, enlargement and roundness. IE protein, an immediate early gene expression product of HCMV, was detected in the nucleus. The results of flow cytometry PI single staining showed that the proportion of apoptosis in the infected group was 14.72% 鹵5.78%, which was significantly higher than that in the normal control group (7.59% 鹵2.33%). There was significant difference between the two groups (t 鈮，

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