本文選題：博爾納病病毒 + U87細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景：博爾納病病毒(borna disease virus, BDV)是一種非節(jié)段負(fù)股單鏈RNA病毒(NNS),具有高度嗜神經(jīng)性并以非細(xì)胞溶解的方式在感染細(xì)胞中復(fù)制BDV能實驗性感染許多像兔、猴直到雞等范圍內(nèi)脊椎動物。流行病學(xué)調(diào)查發(fā)現(xiàn)BDV的自然感染宿主范圍也很廣泛。文獻(xiàn)報道了在馬、羊、兔、猴、貓、狗和駱駝中BDV感染造成的致死性腦炎。無癥狀感染在世界各地多種動物物種中也有報道。因此BDV很可能感染所有溫血動物。大量的流行病學(xué)調(diào)查發(fā)現(xiàn),人類的神經(jīng)精神疾病和BDV感染有一定聯(lián)系,因此BDV又被稱為“情感病毒”。感染新生大鼠導(dǎo)致學(xué)習(xí)記憶能力下降,其神經(jīng)病理學(xué)變化基礎(chǔ)皮質(zhì)萎縮、小腦發(fā)育不良、海馬齒狀腦回顆粒細(xì)胞神經(jīng)元的減少、小腦浦肯野細(xì)胞的減少。BDV誘導(dǎo)神經(jīng)元的凋亡明確的分子機(jī)制目前還需進(jìn)一步的研究和確認(rèn)。目的：本文通過BDV病毒Hu-H1株感染人腦膠質(zhì)母細(xì)胞瘤U87細(xì)胞,對病毒感染后U87細(xì)胞增殖所率和凋亡情況作一個初步的研究,對BDV與細(xì)胞的相互作用規(guī)律有一個初步的認(rèn)識。方法：1通過反復(fù)凍融持續(xù)感染BDV的OL細(xì)胞來獲取病毒液。2.病毒液的濃度采用免疫細(xì)胞化學(xué)的方法檢測p40蛋白陽性細(xì)胞集落,計算病毒液的滴度。3.采用CCK8試劑盒檢測感染后U87細(xì)胞和未感染細(xì)胞在1-7天的增殖情況。4.采用Annexin V/PI雙染流式細(xì)胞儀檢測感染和正常U87細(xì)胞在1、3、5天時細(xì)胞凋亡比例的變化。5.PI單染流式細(xì)胞儀檢測U87細(xì)胞感染后24h內(nèi)周期有無阻滯情況。6.免疫印跡法WB檢測U87細(xì)胞中凋亡相關(guān)蛋白Bax和Bcl-2在感染后的變化。結(jié)果：1.經(jīng)免疫細(xì)胞化學(xué)顯色方法測得Hu-H1滴度為6×10^4FFU/mL。2.BDV感染U87后3天左右即可觀察到p40蛋白的表達(dá)。說明BDV可以持續(xù)感染U87細(xì)胞。3.經(jīng)CCK8法檢測,BDV感染的U87細(xì)胞增殖速率在第1天時與對照組無差異；在第2-4天時,其增殖速率低于對照組細(xì)胞,BDV抑制了細(xì)胞的增殖；5-6天時感染組細(xì)胞增殖速率開始加快,超過了對照組,BDV促進(jìn)了細(xì)胞的增殖。4.BDV對凋亡的影響：感染組在第1天時和對照組無差異；感染后第3天,感染組細(xì)胞凋亡高于對照組,此時BDV促進(jìn)了U87細(xì)胞的凋亡；第5天時,感染組細(xì)胞凋亡又低于對照組,此時BDV抑制了U87的凋亡。5.BDV感染U87細(xì)胞后,細(xì)胞周期在血清饑餓24h,此時感染組和對照組無差異,說明血清饑餓有效阻滯了周期。在6h、15h和24h,感染組的增殖指數(shù)都比對照組低,說明BDV在這段時間對細(xì)胞周期有干擾作用,妨礙了細(xì)胞的生長增殖。6.Bcl-2蛋白在第1天時感染組和對照組無差異；第3和5天,感染組表達(dá)水平低于對照組。Bax蛋白表達(dá)在第1天時也無差異,第3和5天時,感染組明顯高于對照組。結(jié)論：本實驗中,BDV Hu-H1病毒株能順利感染U87細(xì)胞,并能夠?qū)87細(xì)胞的增殖和凋亡產(chǎn)生影響。在前階段,病毒抑制了細(xì)胞的增殖,促進(jìn)了細(xì)胞的凋亡；在后一階段,病毒有可能轉(zhuǎn)而促進(jìn)細(xì)胞增殖,抑制細(xì)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R96

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