【摘要】：膠質(zhì)瘤是臨床上最常見的原發(fā)性腦瘤,多形性膠質(zhì)母細(xì)胞瘤(Glioblastoma multiforme,GBM)是最具侵襲性的致命性腦腫瘤。轉(zhuǎn)錄激活因子5(activating transcription factor 5,ATF5)是抗凋亡蛋白的一種,在惡性膠質(zhì)瘤中高表達(dá)。人巨細(xì)胞病毒(Human Cytomegalovirus,HCMV)為皰疹病毒β亞科的病毒,目前的一些研究證實(shí)HCMV在膠質(zhì)瘤中存在,且?guī)追NHCMV病毒蛋白在膠質(zhì)瘤細(xì)胞中具有致癌性。本課題小組的前期實(shí)驗(yàn)已檢測了HCMV感染對(duì)膠質(zhì)瘤細(xì)胞侵襲和遷移的影響。為進(jìn)一步探討HCMV感染與膠質(zhì)瘤細(xì)胞干性轉(zhuǎn)化之間的關(guān)聯(lián),本實(shí)驗(yàn)構(gòu)建了體外培養(yǎng)的HCMV感染神經(jīng)膠質(zhì)瘤U251細(xì)胞模型和HCMV感染原代神經(jīng)神經(jīng)膠質(zhì)瘤細(xì)胞模型。以免疫熒光染色實(shí)驗(yàn)(Immunofluorescence,IF)檢測HCMV感染膠質(zhì)瘤細(xì)胞不同時(shí)間點(diǎn)HCMV即刻早期蛋白(HCMV IE2)、ATF5及腫瘤細(xì)胞干性標(biāo)記物CD133(Prominin 1)、Nestin(巢蛋白)的表達(dá)變化,結(jié)果顯示隨著IE2表達(dá)水平的增高,ATF5、CD133和Nestin的表達(dá)量逐漸增加。通過qPCR(Quantitative polymerase chain reaction)和Western-blot實(shí)驗(yàn)檢測HCMV IE2、ATF5、CD133和Notch通路受體Notch1基因水平和NICD(Notch intracellular domain)蛋白水平的表達(dá)變化,發(fā)現(xiàn)隨IE2表達(dá)量的增加,ATF5、CD133、Notch1(NICD)的表達(dá)均呈上升趨勢(shì),與IF實(shí)驗(yàn)結(jié)果吻合。慢病毒下調(diào)膠質(zhì)瘤細(xì)胞中ATF5的內(nèi)源性表達(dá),以HCMV感染,檢測HCMV IE2、ATF5、CD133和Notch1(NICD)在基因水平和蛋白水平的表達(dá)變化,實(shí)驗(yàn)結(jié)果顯示隨HCMV的感染CD133和Notch1(NICD)的表達(dá)并未發(fā)生明顯變化。以細(xì)胞毒實(shí)驗(yàn)分別檢測HCMV感染對(duì)膠質(zhì)瘤細(xì)胞和下調(diào)ATF5內(nèi)源性表達(dá)的膠質(zhì)瘤細(xì)胞增殖能力的影響,發(fā)現(xiàn)下調(diào)ATF5內(nèi)源性表達(dá)能明顯抑制HCMV感染的膠質(zhì)瘤細(xì)胞的增殖能力(p0.05)。細(xì)胞克隆形成實(shí)驗(yàn)表明,下調(diào)ATF5的內(nèi)源性表達(dá)可明顯抑制HCMV感染的膠質(zhì)瘤細(xì)胞在非貼壁培養(yǎng)條件下形成腫瘤球的能力(p0.05)。本實(shí)驗(yàn)研究分析了HCMV感染對(duì)U251細(xì)胞和原代神經(jīng)膠質(zhì)瘤細(xì)胞干性相關(guān)性狀的影響,干擾ATF5的表達(dá)能明顯的抑制HCMV感染促進(jìn)膠質(zhì)瘤細(xì)胞惡性轉(zhuǎn)化這一過程,這為進(jìn)一步闡釋HCMV感染與膠質(zhì)瘤細(xì)胞惡性轉(zhuǎn)化之間的分子機(jī)制奠定了理論基礎(chǔ)。
[Abstract]:Glioma is the most common primary brain tumor and glioblastoma pleomorphic (Glioblastoma multiforme,GBM) is the most aggressive and fatal brain tumor. Transcriptional activator 5 (activating transcription factor 5 (ATF5) is an anti-apoptotic protein, which is highly expressed in malignant gliomas. Human cytomegalovirus (Human Cytomegalovirus,HCMV) is a virus belonging to herpesvirus 尾 subfamily. Some studies have confirmed that HCMV exists in gliomas and several HCMV virus proteins are carcinogenic in glioma cells. Previous experiments of our team have examined the effects of HCMV infection on the invasion and migration of glioma cells. In order to further study the relationship between HCMV infection and dry transformation of glioma cells, the U251 cell model of HCMV infected glioma and the primary glioma cell model of HCMV infection in vitro were constructed. The expression of HCMV immediate early protein (HCMV IE2), ATF5 and Prominin 1), Nestin (nestin (CD133) in glioma cells infected with HCMV at different time points were detected by immunofluorescence staining (Immunofluorescence,IF). The results showed that the expression of ATF5,CD133 and Nestin increased with the increase of IE2 expression level. The expression of Notch1 gene and NICD (Notch intracellular domain) protein in HCMV IE2,ATF5,CD133 and Notch pathway receptors were detected by qPCR (Quantitative polymerase chain reaction) and Western-blot experiments. It was found that with the increase of IE2 expression, ATF5,CD133, expression increased. The expression of Notch1 (NICD) was on the rise, which coincided with the results of IF experiment. Lentivirus down-regulated the endogenous expression of ATF5 in glioma cells and detected the expression of HCMV IE2,ATF5,CD133 and Notch1 (NICD) at gene and protein levels by HCMV infection. The results showed that the expression of CD133 and Notch1 (NICD) did not change with HCMV infection. The effects of HCMV infection on the proliferation of glioma cells and glioma cells which down-regulated the endogenous expression of ATF5 were detected by cytotoxicity assay. It was found that down-regulation of ATF5 endogenous expression could significantly inhibit the proliferation of glioma cells infected with HCMV (p0.05). Cell clone formation assay showed that down-regulation of endogenous expression of ATF5 could significantly inhibit the ability of HCMV infected glioma cells to form tumor spheres in non-adherent culture (p0.05). In this study, we analyzed the effects of HCMV infection on the dry-related traits of U251 cells and primary glioma cells. Interfering with the expression of ATF5 could significantly inhibit the process of malignant transformation of glioma cells induced by HCMV infection. This provides a theoretical basis for further understanding the molecular mechanism between HCMV infection and malignant transformation of glioma cells.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.4

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本文編號(hào)：2300598