人巨细胞病毒感染恶性神经胶质瘤细胞对肿瘤干性的影响
发布时间：2018-10-30 16:54
【摘要】：胶质瘤是临床上最常见的原发性脑瘤,多形性胶质母细胞瘤(Glioblastoma multiforme,GBM)是最具侵袭性的致命性脑肿瘤。转录激活因子5(activating transcription factor 5,ATF5)是抗凋亡蛋白的一种,在恶性胶质瘤中高表达。人巨细胞病毒(Human Cytomegalovirus,HCMV)为疱疹病毒β亚科的病毒,目前的一些研究证实HCMV在胶质瘤中存在,且几种HCMV病毒蛋白在胶质瘤细胞中具有致癌性。本课题小组的前期实验已检测了HCMV感染对胶质瘤细胞侵袭和迁移的影响。为进一步探讨HCMV感染与胶质瘤细胞干性转化之间的关联,本实验构建了体外培养的HCMV感染神经胶质瘤U251细胞模型和HCMV感染原代神经神经胶质瘤细胞模型。以免疫荧光染色实验(Immunofluorescence,IF)检测HCMV感染胶质瘤细胞不同时间点HCMV即刻早期蛋白(HCMV IE2)、ATF5及肿瘤细胞干性标记物CD133(Prominin 1)、Nestin(巢蛋白)的表达变化,结果显示随着IE2表达水平的增高,ATF5、CD133和Nestin的表达量逐渐增加。通过qPCR(Quantitative polymerase chain reaction)和Western-blot实验检测HCMV IE2、ATF5、CD133和Notch通路受体Notch1基因水平和NICD(Notch intracellular domain)蛋白水平的表达变化,发现随IE2表达量的增加,ATF5、CD133、Notch1(NICD)的表达均呈上升趋势,与IF实验结果吻合。慢病毒下调胶质瘤细胞中ATF5的内源性表达,以HCMV感染,检测HCMV IE2、ATF5、CD133和Notch1(NICD)在基因水平和蛋白水平的表达变化,实验结果显示随HCMV的感染CD133和Notch1(NICD)的表达并未发生明显变化。以细胞毒实验分别检测HCMV感染对胶质瘤细胞和下调ATF5内源性表达的胶质瘤细胞增殖能力的影响,发现下调ATF5内源性表达能明显抑制HCMV感染的胶质瘤细胞的增殖能力(p0.05)。细胞克隆形成实验表明,下调ATF5的内源性表达可明显抑制HCMV感染的胶质瘤细胞在非贴壁培养条件下形成肿瘤球的能力(p0.05)。本实验研究分析了HCMV感染对U251细胞和原代神经胶质瘤细胞干性相关性状的影响,干扰ATF5的表达能明显的抑制HCMV感染促进胶质瘤细胞恶性转化这一过程,这为进一步阐释HCMV感染与胶质瘤细胞恶性转化之间的分子机制奠定了理论基础。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.4
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相关期刊论文 前4条
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