HCMV感染对胶质瘤细胞U87自噬的影响及意义

发布时间：2018-01-01 08:39

本文关键词：HCMV感染对胶质瘤细胞U87自噬的影响及意义　出处：《青岛大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:1.人巨细胞病毒(human cytomegalovirus,HCMV)感染神经胶质瘤细胞U87,通过观察自噬体形态及数量的变化、微管相关蛋白1轻链3(LC3)表达水平的变化,从而研究HCMV感染对U87细胞自噬水平所产生的影响。2.HCMV感染U87后,检测自噬相关基因Beclin1及其蛋白的表达量,并检测细胞活性,从而探讨HCMV感染在神经胶质瘤在发生、发展过程中起到的作用及意义。方法与材料:1.HCMV AD169(MOI=5)感染神经胶质瘤细胞U87后,通过相差显微镜观察病毒感染6h、12h、24h、48h后的形态学改变。2.在HCMV AD169(MOI=5)病毒感染U87细胞6h、48h后,用透射电镜观察自噬小体的数目及形态。用HCMV AD169(MOI=5)感染神经胶质瘤U87细胞6h、12h、24h、48h;并在各对应时间点用未感染的细胞作对照组。收集细胞并用Western-blot和免疫荧光对LC3编码蛋白表达水平进行检测。3.用HCMV AD169(MOI=5)感染神经胶质瘤U87细胞,分别在6h、12h、24h、48h后收集细胞;同时将未感染的细胞作为对照。用RT-PCR检测基因Beclin1的表达;并用Western-blot和免疫荧光对Beclin1蛋白进行检测。4.CCK-8法对神经胶质瘤细胞U87的增殖活性的情况进行检测。结果:1.HCMV感染U87细胞6h、12h、24h、48h后,细胞形态明显发生变化。2.在电镜下观察HCMV感染U87细胞6h、48h后的自噬小体数目及形态。结果显示,相比较6h的自噬体数目,在48h时自噬体数量明显减少;并于6h、8h时在镜下分别观察到初始自噬小体(Initial autophagic vacuole,AVi)和晚期自噬小体(Late/Degradative autophagic vacuole,AVd),自噬小体均具膜结构,且在胞浆内可观察到未完全降解或者已降解的细胞器等。Western-blot结果显示,HCMV感染U87细胞6 h、12h、24 h、48 h后,LC3-II相对表达水平逐渐下降(P0.05);LC3-II/I相对表达量也呈下调趋势(P0.01)。而未感染组LC3-II、LC3-II/I蛋白相对表达量未有明显变化(P0.05)。其结果与免疫荧光结果一致,均证实LC3蛋白表达水平明显减少。3.PCR检测结果显示,与对照组相比,感染组Beclin1m RNA相对表达量逐渐减少(P㩳0.01)。Western-blot结果显示,与未感染组相比,HCMV感染U87细胞6 h、12h、24 h、48 h,Beclin1相对表达量逐渐下降(P0.05)。其结果与免疫荧光结果一致,证实Beclin1表达水平明显减少。4.CCK-8结果显示,HCMV感染U87细胞6 h、12h、24 h、48 h后,细胞活性逐渐增高(P㩳0.01)。结论:1.HCMV感染胶质瘤U87细胞后,自噬受到抑制。2.HCMV感染神经胶质瘤U87细胞导致Beclin1基因及蛋白表达量下降,且细胞活性逐渐增强,即HCMV诱导胶质瘤细胞U87的增殖可能受Beclin1蛋白的调控。
[Abstract]:Objective to observe the morphology and quantity of autophagy in human cytomegalovirus (HCMV) infected glioma cell line U87. The change of microtubule-associated protein 1 light chain 3LC3) expression, so as to study the effect of HCMV infection on the autophagy level of U87 cells. 2. HCMV infection with U87. To detect the expression of autophagy related gene Beclin1 and its protein, and to detect the cell activity, HCMV infection was detected in glioma. Methods: 1. HCMV AD169 and MOI5) were infected with U87 glioma cells, and the virus infection was observed by phase contrast microscope for 6 h. The morphological changes of U87 cells were observed at 12h, 24h and 48h, respectively. 2. After HCMV AD169 moi 5) virus was infected with U87 cells for 6 h and 48 h later. The number and morphology of autophagy bodies were observed by transmission electron microscope. U87 cells were infected with HCMV AD169 moi 5 for 24 h or 48 h. The uninfected cells were used as control group at each corresponding time point. Western-blot and immunofluorescence were used to detect the expression level of LC3 coding protein. 3. HCMV was used to detect the expression of LC3 protein. AD169 (. The glioma U87 cells were infected with MOI5. The cells were collected at 6h ~ 12h ~ 24h ~ (th) after 48 h. At the same time, uninfected cells were used as control. RT-PCR was used to detect the expression of Beclin1. Beclin1 protein was detected by Western-blot and immunofluorescence. 4. The proliferative activity of glioma cell U87 was detected by CCK-8 method. Results:. 1. HCMV was infected with U87 cells for 6 h. The morphology of U87 cells was observed under electron microscope. The number and morphology of autophagy were observed under electron microscope after HCMV infection for 6 h or 48 h. Compared with the number of autophagy at 6 h, the number of autophagy decreased significantly at 48 h. Initial autophagic vacuole was observed under microscope at 6 h or 8 h. Aviv) and late stage autophagic autophagic Vacuoleus. The autophagy has membrane structure. Western-blot results showed that HCMV infected U87 cells for 6 h and 12 h for 24 h. 48 h later, the relative expression level of LC3-II decreased gradually. The relative expression of LC3-II/I was also down-regulated (P 0.01), while LC3-II was not infected. There was no significant change in the relative expression of LC3-II/I protein. The results were consistent with those of immunofluorescence. All of them confirmed that the expression of LC3 protein was significantly decreased. 3. The results showed that the relative expression of Beclin1m RNA in infected group was gradually decreased compared with the control group. 3. Compared with the control group, HCMV was infected with U87 cells for 6 h, 12 h and 24 h and 48 h, respectively. The relative expression of Beclin1 decreased gradually, and the results were consistent with the results of immunofluorescence, which confirmed that the level of Beclin1 expression was significantly decreased. 4. The results of CCK-8 showed that. The cell activity of U87 cells increased gradually after HCMV infection for 24 h or 48 h after infection with U87 cells for 6 h or 12 h. Conclusion 1. HCMV infected U87 glioma cells. Inhibition of autophagy. 2. HCMV-infected glioma U87 cells decreased the expression of Beclin1 gene and protein, and the cell activity increased gradually. That is, the proliferation of U87 cells induced by HCMV may be regulated by Beclin1 protein.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.41

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