【摘要】：目的： 1、探討人巨細(xì)胞病毒(HCMV)感染對(duì)惡性神經(jīng)膠質(zhì)瘤U87細(xì)胞增殖的影響,檢測(cè)HCMV感染后U87細(xì)胞中轉(zhuǎn)錄激活因子5(Activating transcription factor5,ATF5)及相關(guān)基因的表達(dá)； 2、探討HCMV感染促進(jìn)膠質(zhì)瘤細(xì)胞惡性轉(zhuǎn)化是否通過(guò)調(diào)控ATF5通路來(lái)完成,為治療膠質(zhì)瘤提供一個(gè)新思路。 方法： 1、HCMV感染惡性膠質(zhì)瘤細(xì)胞U87不同時(shí)間,MTT法檢測(cè)HCMV感染對(duì)細(xì)胞增殖的影響； 2、熒光定量PCR及Western-blot檢測(cè)HCMV感染U87細(xì)胞0、12、24、48h時(shí)ATF5、Bcl-2及BAX的表達(dá)情況； 3、以慢病毒為載體的靶向ATF5小干擾RNA構(gòu)建載體,感染U87細(xì)胞后,熒光顯微鏡觀察其感染效率,并檢測(cè)ATF5的表達(dá)水平；選擇干擾效率最高的一組(siATF5U87)用于下一步實(shí)驗(yàn)； 4、MTT法檢測(cè)HCMV感染對(duì)siATF5U87細(xì)胞增殖的影響,TUNEL法檢測(cè)HCMV感染對(duì)siATF5U87細(xì)胞凋亡的影響； 5、Western-blot檢測(cè)干擾ATF5功能后HCMV感染對(duì)U87細(xì)胞內(nèi)Bcl-2及BAX的表達(dá)情況,研究HCMV感染增加細(xì)胞惡性性狀可能的分子機(jī)制。 結(jié)果： 1、MTT結(jié)果顯示HCMV感染U87細(xì)胞0、12、24、48h,細(xì)胞增殖水平高于對(duì)照組； 2、Real-time PCR及Western-blot結(jié)果顯示HCMV感染U87細(xì)胞后,ATF5表達(dá)水平增加,Bcl-2/BAX的比值也隨之升高,說(shuō)明HCMV感染促進(jìn)了U87細(xì)胞抗凋亡能力的增強(qiáng)； 3、對(duì)照慢病毒LiCON055及含靶向干擾ATF5的小干擾RNA質(zhì)粒的慢病毒LV-ATF5-RNAi(8842)、LV-ATF5-RNAi(8843)和LV-ATF5-RNAi(8844)分別感染U87細(xì)胞,熒光顯微鏡下觀察其感染效率都在95%以上Wetern-blot檢測(cè)各組ATF5表達(dá)水平,分別為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,選取8842組進(jìn)行實(shí)驗(yàn)。 4、干擾ATF5功能后,與空載體組相比,HCMV感染對(duì)干擾ATF5U87細(xì)胞的增殖有明顯的抑制作用(p0.05),細(xì)胞凋亡率明顯增加； 5.Western-blot結(jié)果顯示,與空載體組比較,HCMV感染對(duì)干擾ATF5U87細(xì)胞抗凋亡因子Bcl-2和促凋亡因子BAX的表達(dá)變化無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。 結(jié)論： 1、HCMV感染可能通過(guò)調(diào)控ATF5通路增加細(xì)胞惡性性狀； 2、預(yù)防HCMV感染,對(duì)抑制惡性神經(jīng)膠質(zhì)瘤的惡性轉(zhuǎn)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 鹵0.09 and 0.61 鹵0.06 鹵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.
【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】：2014
【分類(lèi)號(hào)】：R739.4

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本文編號(hào)：2185863