HCMV感染对恶性神经胶质瘤细胞增殖、凋亡的影响及作用机制的研究
发布时间：2018-08-16 11:34
【摘要】：目的： 1、探讨人巨细胞病毒(HCMV)感染对恶性神经胶质瘤U87细胞增殖的影响,检测HCMV感染后U87细胞中转录激活因子5(Activating transcription factor5,ATF5)及相关基因的表达； 2、探讨HCMV感染促进胶质瘤细胞恶性转化是否通过调控ATF5通路来完成,为治疗胶质瘤提供一个新思路。 方法： 1、HCMV感染恶性胶质瘤细胞U87不同时间,MTT法检测HCMV感染对细胞增殖的影响； 2、荧光定量PCR及Western-blot检测HCMV感染U87细胞0、12、24、48h时ATF5、Bcl-2及BAX的表达情况； 3、以慢病毒为载体的靶向ATF5小干扰RNA构建载体,感染U87细胞后,荧光显微镜观察其感染效率,并检测ATF5的表达水平；选择干扰效率最高的一组(siATF5U87)用于下一步实验； 4、MTT法检测HCMV感染对siATF5U87细胞增殖的影响,TUNEL法检测HCMV感染对siATF5U87细胞凋亡的影响； 5、Western-blot检测干扰ATF5功能后HCMV感染对U87细胞内Bcl-2及BAX的表达情况,研究HCMV感染增加细胞恶性性状可能的分子机制。 结果： 1、MTT结果显示HCMV感染U87细胞0、12、24、48h,细胞增殖水平高于对照组； 2、Real-time PCR及Western-blot结果显示HCMV感染U87细胞后,ATF5表达水平增加,Bcl-2/BAX的比值也随之升高,说明HCMV感染促进了U87细胞抗凋亡能力的增强； 3、对照慢病毒LiCON055及含靶向干扰ATF5的小干扰RNA质粒的慢病毒LV-ATF5-RNAi(8842)、LV-ATF5-RNAi(8843)和LV-ATF5-RNAi(8844)分别感染U87细胞,荧光显微镜下观察其感染效率都在95%以上Wetern-blot检测各组ATF5表达水平,分别为0.67±0.03、0.38±0.09、0.61±0.06、0.68±0.06,以LV-ATF5-RNAi(8842)抑制效率最高,选取8842组进行实验。 4、干扰ATF5功能后,与空载体组相比,HCMV感染对干扰ATF5U87细胞的增殖有明显的抑制作用(p0.05),细胞凋亡率明显增加； 5.Western-blot结果显示,与空载体组比较,HCMV感染对干扰ATF5U87细胞抗凋亡因子Bcl-2和促凋亡因子BAX的表达变化无统计学意义(p0.05)。 结论： 1、HCMV感染可能通过调控ATF5通路增加细胞恶性性状； 2、预防HCMV感染,对抑制恶性神经胶质瘤的恶性转化有一定作用。
[Abstract]:Objective: 1 to investigate the effect of human cytomegalovirus (HCMV) infection on the proliferation of malignant glioma U87 cells, and to detect the expression of transcription activator 5 (Activating transcription factor5ATF5 and related genes in U87 cells after HCMV infection. 2. To investigate whether HCMV infection can promote malignant transformation of glioma cells by regulating ATF5 pathway, and to provide a new idea for the treatment of glioma. Methods: 1the effect of HCMV infection on the proliferation of malignant glioma cell U87 was detected by MTT assay at different time points, and the expression of ATF5Bcl 2 and BAX in U87 cells infected with HCMV for 48 h was detected by fluorescence quantitative PCR and Western-blot. 3. The vector was constructed by targeting ATF5 small interfering RNA with lentivirus as vector. After U87 cells were infected, the infection efficiency was observed by fluorescence microscope, and the expression level of ATF5 was detected by fluorescence microscope, and the group with the highest interference efficiency (siATF5U87) was selected for the next experiment. (4) the effect of HCMV infection on the proliferation of siATF5U87 cells was detected by MTT assay. The apoptosis of siATF5U87 cells was detected by Tunel method, and the expression of Bcl-2 and BAX in U87 cells by HCMV infection after interfering with ATF5 function was detected by Western-blot. To study the molecular mechanism of HCMV infection increasing malignant character of cell. Results: 1MTT results showed that the proliferation level of U87 cells infected with HCMV for 48 h was higher than that of the control group, and the expression level of ATF5 in U87 cells was increased after HCMV infection with real-time PCR and Western-blot, and the ratio of Bcl-2 / Bax was also increased after HCMV infection. The results showed that the anti-apoptosis ability of U87 cells was enhanced by HCMV infection. (3) LV-ATF5-RNAi (8843) and LV-ATF5-RNAi (8844) were infected with LV-ATF5-RNAi (8843) and LV-ATF5-RNAi (8844), respectively. The positive rate of LV-ATF5-RNAi (8842) was the highest in 8842 groups, and the inhibitory efficiency of LV-ATF5-RNAi (8842) was the highest. 4. After interfering with ATF5 function, the expression of ATF5 was detected by fluorescence microscope in more than 95% of the groups, 0.38 卤0.09 and 0.61 卤0.06 卤0.06, respectively. Compared with the empty vector group, HCMV infection inhibited the proliferation of ATF5U87 cells significantly (p0.05), and the apoptosis rate increased significantly. There was no significant difference in the expression of anti-apoptotic factor (Bcl-2) and pro-apoptotic factor (BAX) between HCMV infection and empty vector group (p0.05). Conclusion: 1HCMV infection may increase the malignant character of the cells by regulating the ATF5 pathway, and (2) preventing HCMV infection may inhibit the malignant transformation of malignant glioma.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4
本文编号：2185863
[Abstract]:Objective: 1 to investigate the effect of human cytomegalovirus (HCMV) infection on the proliferation of malignant glioma U87 cells, and to detect the expression of transcription activator 5 (Activating transcription factor5ATF5 and related genes in U87 cells after HCMV infection. 2. To investigate whether HCMV infection can promote malignant transformation of glioma cells by regulating ATF5 pathway, and to provide a new idea for the treatment of glioma. Methods: 1the effect of HCMV infection on the proliferation of malignant glioma cell U87 was detected by MTT assay at different time points, and the expression of ATF5Bcl 2 and BAX in U87 cells infected with HCMV for 48 h was detected by fluorescence quantitative PCR and Western-blot. 3. The vector was constructed by targeting ATF5 small interfering RNA with lentivirus as vector. After U87 cells were infected, the infection efficiency was observed by fluorescence microscope, and the expression level of ATF5 was detected by fluorescence microscope, and the group with the highest interference efficiency (siATF5U87) was selected for the next experiment. (4) the effect of HCMV infection on the proliferation of siATF5U87 cells was detected by MTT assay. The apoptosis of siATF5U87 cells was detected by Tunel method, and the expression of Bcl-2 and BAX in U87 cells by HCMV infection after interfering with ATF5 function was detected by Western-blot. To study the molecular mechanism of HCMV infection increasing malignant character of cell. Results: 1MTT results showed that the proliferation level of U87 cells infected with HCMV for 48 h was higher than that of the control group, and the expression level of ATF5 in U87 cells was increased after HCMV infection with real-time PCR and Western-blot, and the ratio of Bcl-2 / Bax was also increased after HCMV infection. The results showed that the anti-apoptosis ability of U87 cells was enhanced by HCMV infection. (3) LV-ATF5-RNAi (8843) and LV-ATF5-RNAi (8844) were infected with LV-ATF5-RNAi (8843) and LV-ATF5-RNAi (8844), respectively. The positive rate of LV-ATF5-RNAi (8842) was the highest in 8842 groups, and the inhibitory efficiency of LV-ATF5-RNAi (8842) was the highest. 4. After interfering with ATF5 function, the expression of ATF5 was detected by fluorescence microscope in more than 95% of the groups, 0.38 卤0.09 and 0.61 卤0.06 卤0.06, respectively. Compared with the empty vector group, HCMV infection inhibited the proliferation of ATF5U87 cells significantly (p0.05), and the apoptosis rate increased significantly. There was no significant difference in the expression of anti-apoptotic factor (Bcl-2) and pro-apoptotic factor (BAX) between HCMV infection and empty vector group (p0.05). Conclusion: 1HCMV infection may increase the malignant character of the cells by regulating the ATF5 pathway, and (2) preventing HCMV infection may inhibit the malignant transformation of malignant glioma.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4
【参考文献】
相关期刊论文 前4条
1 曾而明;罗达亚;王红梅;李东海;洪涛;;胶质瘤组织中人类巨细胞病毒的感染研究[J];南昌大学学报(医学版);2010年06期
2 谭泽明,李艺,罗敏华;HCMV增殖机制研究进展[J];生命科学;2005年01期
3 崔寰;王明丽;;人巨细胞病毒感染与肿瘤相关性研究进展[J];微生物与感染;2008年03期
4 汪晓婷;王明丽;;人巨细胞病毒相关肿瘤调节作用的研究新进展[J];微生物与感染;2011年03期
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