基因沉默ATF5联合更昔洛韦对HCMV阳性胶质瘤U87细胞增殖及病毒复制的影响

发布时间：2018-06-17 02:29

本文选题：人巨细胞病毒 + 转录激活因子5　；参考：《青岛大学》2017年硕士论文

【摘要】：神经胶质瘤是最常见的原发性中枢神经系统肿瘤,尽管采取积极的治疗措施,预后依然很差。在神经胶质瘤环境中,人巨细胞病毒(human cytomegalovirus,HCMV)基因及转录产物的表达随着肿瘤恶性程度的增高而增高,其感染与神经胶质瘤关系密切。转录激活因子5(Activating transcription factor 5,ATF5)是肿瘤标志性转录因子,在神经胶质瘤中特异性高表达。本文在神经胶质瘤环境中,探讨基因沉默ATF5联合抗病毒药物更昔洛韦(ganciclovir,GCV)对HCMV阳性胶质瘤U87细胞增殖及病毒复制的影响。研究首先筛选合适的GCV作用浓度;利用慢病毒介导的小干扰RNA靶向干扰ATF5基因的表达,构建ATF5下调稳定表达的LV-ATF5-RNAi U87细胞系;低感染复数(multiplicity of infection,MOI)的HCMV感染LV-ATF5-RNAi U87细胞,并用含GCV的培养基培养。在该条件下,CCK-8法检测细胞的增殖活性;Real-time PCR、western-blot分别检测HCMV即刻早期(IE)、早期(UL44)、晚期(UL99)基因及蛋白表达水平;空斑形成试验检测HCMV子代病毒复制水平。结果表明:成功构建ATF5下调稳定表达的LV-ATF5-RNAi U87细胞。基因沉默ATF5联合GCV共同作用,可以较好的抑制HCMV阳性神经胶质瘤细胞的增殖活性;基因沉默ATF5联合GCV共同作用对HCMV在mRNA、蛋白水平、子代病毒复制水平上的抑制结果一致,都明显大于GCV或者沉默ATF5单独作用的抑制效率。以上结果提示,基因沉默ATF5联合GCV可以较好的遏制HCMV病毒的复制以及神经胶质瘤的恶性增殖作用。因此,基因沉默ATF5联合GCV共同作用可能为临床上治疗神经胶质瘤提供一个新的方法。
[Abstract]:Glioma is the most common primary central nervous system tumor. In glioma environment, the expression of human cytomegalovirus (HCMV) gene and transcription products of human cytomegalovirus (HCMV) increases with the increase of tumor malignancy, and its infection is closely related to glioma. Transcriptional activator 5(Activating transcription factor 5 (ATF 5) is a tumor marker transcription factor, which is specifically overexpressed in gliomas. To investigate the effects of gene silencing ATF5 combined with ganciclovirr GCVV on the proliferation and replication of HCMV positive glioma U87 cells in glioma environment. In this study, we first selected suitable GCV concentration, constructed LV-ATF5-RNAi U87 cell line, which down-regulated stable expression of LV-ATF5-RNAi U87 cell line, and HCMV infected LV-ATF5-RNAi U87 cell line with low infection complex multiplicity of infective moi, using lentivirus-mediated small interfering RNA targeted interference ATF5 gene expression, and constructed LV-ATF5-RNAi U87 cell line with down-regulated expression of ATF5-RNAi U87 cells. The culture medium containing GCV was used. Under this condition, the cell proliferation activity was detected by CCK-8 method and the expression of HCMV gene and protein were detected by real-time PCRRX western-blot, respectively, and the replication level of HCMV progeny virus was detected by plaque formation test. The results showed that ATF5 down-regulated stable expression of LV-ATF5-RNAi U87 cells. Gene silencing ATF5 combined with GCV could inhibit the proliferation of HCMV positive glioma cells. Both of them were significantly more effective than those of GCV or silencing ATF 5 alone. These results suggest that gene silencing ATF5 combined with GCV can inhibit the replication of HCMV virus and the malignant proliferation of glioma. Therefore, the combined action of gene silencing ATF 5 and GCV may provide a new method for the treatment of glioma.
【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】：R739.41

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