HCMV感染对神经胶质瘤细胞缝隙连接基因及蛋白的影响

发布时间：2018-03-28 19:28

本文选题：神经胶质瘤细胞　切入点：人巨细胞病毒　出处：《青岛大学》2017年硕士论文

【摘要】：人巨细胞病毒(Human cytomegalovirus,HCMV)属于β亚科疱疹病毒,在人群中感染非常普遍,一系列的研究显示HCMV与神经胶质瘤细胞的发病机制关系密切。在脑组织中,细胞之间传递信息主要依靠于细胞缝隙连接通讯(gap juncion intercelluar communication,GJIC),细胞缝隙连接通讯是机体中传导信号最直接的通讯方式,另外,神经胶质瘤细胞之间的GJIC被预测在胶质瘤侵袭中起到关键性的作用,并且,脑胶质瘤的侵润性是临床中治疗的难点。缝隙连接基因及蛋白的表达和功能异常与多种神经系统疾病相关,此方面的研究在未来将是新的方向和重点。本研究拟探讨HCMV感染对神经胶质瘤细胞缝隙连接基因及蛋白的影响,并深入了解缝隙连接蛋白在肿瘤病理机制中的作用。分别培养并使用HCMV ADl69株(MOI=1)感染原代神经胶质瘤细胞和U251细胞,使用相差显微镜观察细胞的形态学变化,观察结果显示原代神经胶质瘤细胞及U251细胞经HCMV感染后逐渐变大变圆。检测HCMV感染原代神经胶质瘤细胞及U251细胞后缝隙连接基因GJA8和GJB1的表达,结果显示缝隙连接基因GJA8和GJB1在原代神经胶质瘤细胞及U251细胞中均可表达。Real-time PCR检测原代神经胶质瘤细胞及U251细胞经HCMV感染后缝隙连接基因GJA8和GJB1的表达,结果表明原代神经胶质瘤细胞经HCMV感染0h、12h、24h、48h后,GJB1的相对表达水平下降(p㩳0.05),GJA8在24h后相对表达水平下降(p㩳0.05),在24h之前无明显变化(p㧐0.05);U251细胞经HCMV感染0h、12h、24h、48h后,GJA8和GJB1的相对表达量均是下降趋势(p㩳0.05)。再使用Western-Blot检测原代神经胶质瘤细胞及U251细胞经HCMV感染后缝隙连接蛋白Cx50及Cx32的表达,结果表明,原代神经胶质瘤细胞经HCMV感染0h、12h、24h、48h后,Cx32的相对表达量减少(p㩳0.05),Cx50的相对表达量在24h之前无明显变化(p㧐0.05),24h后相对表达量出现减少趋势(p㩳0.05);U251细胞经HCMV感染0h、12h、24h、48h后,Cx50和Cx32的相对表达量均是下降趋势(p㩳0.05)。使用细胞划痕法检测HCMV感染对U251细胞的迁移能力的影响,结果显示经HCMV感染的U251细胞的侵袭转移能力增强。以上这些实验结果表明原代神经胶质瘤细胞和U251细胞经HCMV感染后,缝隙连接基因及蛋白的表达量有所差异,此研究可为缝隙连接基因及蛋白的表达与肿瘤的恶性程度的相关性提供一定的理论基础。
[Abstract]:Human cytomegalovirus (human cytomegalovirus) belongs to the 尾 subfamily herpes virus and is very common in the population. A series of studies have shown that HCMV is closely related to the pathogenesis of glioma cells. The transmission of information between cells mainly depends on cell gap junction communication gap juncion intercelluar communication, gap junction communication is the most direct mode of communication in the body, in addition, GJIC between glioma cells is predicted to play a key role in glioma invasion, and, Invasion of glioma is a difficult point in clinical treatment. The expression and function of gap junction gene and protein are related to many nervous system diseases. This study aims to explore the effects of HCMV infection on gap junction genes and proteins in glioma cells. In addition, the role of gap junction protein in the pathological mechanism of tumor was studied. The primary glioma cells and U251 cells were infected with HCMV ADl69 strain, and the morphologic changes of the cells were observed by phase contrast microscope. The results showed that the primary glioma cells and U251 cells became large and round after HCMV infection. The expression of gap junction gene GJA8 and GJB1 in primary glioma cells and U251 cells infected with HCMV was detected. The results showed that gap junction gene GJA8 and GJB1 could be expressed in primary glioma cells and U251 cells. Real-time PCR was used to detect the expression of gap junction genes GJA8 and GJB1 in primary glioma cells and U251 cells after HCMV infection. The results showed that the relative expression level of GJB1 in primary glioma cells was decreased after infection with HCMV for 12 h or 24 h and 48 h later. The relative expression level of GJA8 decreased after 24 hours. 0. 05%, no significant change before 24 hours. The relative expression of GJA8 and GJB1 in the cells infected with HCMV for 48 h were decreased. Western-Blot was used to detect the expression of gap junction protein Cx50 and Cx32 in primary glioma cells and U251 cells infected by HCMV. The results showed that the relative expression of Cx32 in primary glioma cells was decreased after HCMV infection for 24 h or 48 h after HCMV infection. The relative expression of Cx50 was not changed significantly before 24 hours. The relative expression level decreased after 24 hours. The relative expression of Cx50 and Cx32 in the cells infected with HCMV for 24 h and 48 h after HCMV infection showed a decreasing trend. The effect of HCMV infection on the migration ability of U251 cells was detected by cell scratch method. The results showed that the invasion and metastasis ability of U251 cells infected with HCMV was enhanced. These results indicated that the primary glioma cells and U251 cells were infected by HCMV. The expression of gap junction genes and proteins is different. This study may provide a theoretical basis for the correlation between the expression of gap junction genes and proteins and the malignancy of tumors.
【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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