博尔纳病病毒Hu-H1感染对人胶质母细胞瘤细胞的增殖及凋亡影响的初步研究

发布时间：2018-04-24 18:09

本文选题：博尔纳病病毒 + U87细胞　；参考：《重庆医科大学》2015年硕士论文

【摘要】：背景：博尔纳病病毒(borna disease virus, BDV)是一种非节段负股单链RNA病毒(NNS),具有高度嗜神经性并以非细胞溶解的方式在感染细胞中复制BDV能实验性感染许多像兔、猴直到鸡等范围内脊椎动物。流行病学调查发现BDV的自然感染宿主范围也很广泛。文献报道了在马、羊、兔、猴、猫、狗和骆驼中BDV感染造成的致死性脑炎。无症状感染在世界各地多种动物物种中也有报道。因此BDV很可能感染所有温血动物。大量的流行病学调查发现,人类的神经精神疾病和BDV感染有一定联系,因此BDV又被称为“情感病毒”。感染新生大鼠导致学习记忆能力下降,其神经病理学变化基础皮质萎缩、小脑发育不良、海马齿状脑回颗粒细胞神经元的减少、小脑浦肯野细胞的减少。BDV诱导神经元的凋亡明确的分子机制目前还需进一步的研究和确认。目的：本文通过BDV病毒Hu-H1株感染人脑胶质母细胞瘤U87细胞,对病毒感染后U87细胞增殖所率和凋亡情况作一个初步的研究,对BDV与细胞的相互作用规律有一个初步的认识。方法：1通过反复冻融持续感染BDV的OL细胞来获取病毒液。2.病毒液的浓度采用免疫细胞化学的方法检测p40蛋白阳性细胞集落,计算病毒液的滴度。3.采用CCK8试剂盒检测感染后U87细胞和未感染细胞在1-7天的增殖情况。4.采用Annexin V/PI双染流式细胞仪检测感染和正常U87细胞在1、3、5天时细胞凋亡比例的变化。5.PI单染流式细胞仪检测U87细胞感染后24h内周期有无阻滞情况。6.免疫印迹法WB检测U87细胞中凋亡相关蛋白Bax和Bcl-2在感染后的变化。结果：1.经免疫细胞化学显色方法测得Hu-H1滴度为6×10^4FFU/mL。2.BDV感染U87后3天左右即可观察到p40蛋白的表达。说明BDV可以持续感染U87细胞。3.经CCK8法检测,BDV感染的U87细胞增殖速率在第1天时与对照组无差异；在第2-4天时,其增殖速率低于对照组细胞,BDV抑制了细胞的增殖；5-6天时感染组细胞增殖速率开始加快,超过了对照组,BDV促进了细胞的增殖。4.BDV对凋亡的影响：感染组在第1天时和对照组无差异；感染后第3天,感染组细胞凋亡高于对照组,此时BDV促进了U87细胞的凋亡；第5天时,感染组细胞凋亡又低于对照组,此时BDV抑制了U87的凋亡。5.BDV感染U87细胞后,细胞周期在血清饥饿24h,此时感染组和对照组无差异,说明血清饥饿有效阻滞了周期。在6h、15h和24h,感染组的增殖指数都比对照组低,说明BDV在这段时间对细胞周期有干扰作用,妨碍了细胞的生长增殖。6.Bcl-2蛋白在第1天时感染组和对照组无差异；第3和5天,感染组表达水平低于对照组。Bax蛋白表达在第1天时也无差异,第3和5天时,感染组明显高于对照组。结论：本实验中,BDV Hu-H1病毒株能顺利感染U87细胞,并能够对U87细胞的增殖和凋亡产生影响。在前阶段,病毒抑制了细胞的增殖,促进了细胞的凋亡；在后一阶段,病毒有可能转而促进细胞增殖,抑制细胞凋亡。
[Abstract]:Background: Borna disease virus (DVV) is a non-segmental negative-stranded RNA virus, which has high neurophilic properties and replicates BDV in infected cells in a non-cellular manner. Monkey to chicken and so on within the range of vertebrates. Epidemiological investigation found that the host range of natural infection of BDV is also very extensive. Fatal encephalitis caused by BDV infection has been reported in horses, sheep, rabbits, monkeys, cats, dogs and camels. Asymptomatic infections are also reported in many animal species around the world. So BDV is likely to infect all warm-blooded animals. A large number of epidemiological investigations have found that human neuropsychiatric diseases are associated with BDV infection, so BDV is also called "affective virus". Infection of neonatal rats resulted in decreased learning and memory ability, atrophy of basic cortex, hypoplasia of cerebellum, and decrease of granular cell neurons in dentate gyrus of sea horse. The clear molecular mechanism of apoptosis induced by BDV in cerebellar Purkinje cells needs to be further studied and confirmed. Objective: to study the proliferation rate and apoptosis of human glioblastoma U87 cells infected with BDV virus Hu-H1 strain, and to understand the interaction between BDV and cells. Methods the virus solution. 2. 2 was obtained by repeated freezing and thawing of OL cells infected with BDV. The concentration of the virus was detected by immunocytochemical method, and the titer of the virus was calculated. CCK8 kit was used to detect the proliferation of U87 and uninfected U87 cells in 1 to 7 days after infection. Annexin V/PI double staining flow cytometry was used to detect the change of apoptosis ratio of U87 cells after infection and normal U87 cells at the 5th day after infection. 5. Pi single staining flow cytometry was used to detect the cell cycle arrest within 24 hours after infection. The changes of apoptosis-related proteins Bax and Bcl-2 in U87 cells were detected by Western blot. The result is 1: 1. The expression of p40 protein was observed about 3 days after U87 infection with Hu-H1 titer of 6 脳 10 ^ 4FFU/mL.2.BDV by immunocytochemical staining. These results indicate that BDV can continuously infect U87 cells. 3. The proliferation rate of U87 cells infected with CCK8 was not different from that of the control group on the first day, but the proliferation rate of U87 cells infected with CCK8 was lower than that of the control group on the 2nd and 4th day, and the proliferation rate of the infected U87 cells began to accelerate at 5-6 days after infection. The effect of BDV on cell proliferation was higher than that of control group. 4. The effect of BDV on apoptosis: there was no difference between the infected group and the control group on the first day; on the third day after infection, the apoptosis of the infected group was higher than that of the control group, and at this time, BDV promoted the apoptosis of U87 cells. The apoptosis of the infected group was lower than that of the control group. At this time, BDV inhibited the apoptosis of U87 cells. 5. After U87 cells were infected with BDV, the cell cycle was hungry in serum for 24 hours, but there was no difference between the infected group and the control group at this time, which indicated that serum starvation effectively blocked the cell cycle. The proliferation index of the infected group was lower than that of the control group at 6 h and 24 h, indicating that BDV interfered with the cell cycle during this period, and inhibited the proliferation of the cells. 6. There was no difference between the infected group and the control group on the 1st day, but on the 3rd and 5th day, the proliferation index of the infected group was not different from that of the control group. The expression level of Bax protein in the infected group was lower than that in the control group, and the expression of Bax protein in the infected group was significantly higher than that in the control group on the 3rd and 5th day. Conclusion: the Hu-H1 virus strain can infect U87 cells successfully, and it can affect the proliferation and apoptosis of U87 cells. In the former stage, the virus inhibited cell proliferation and promoted cell apoptosis, while in the latter stage, the virus may turn to promote cell proliferation and inhibit cell apoptosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R96

【相似文献】