本文選題：小反芻獸疫病毒 + 山羊子宮內(nèi)膜上皮細(xì)胞　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：小反芻獸疫(Peste des petits ruminants,PPR)是由小反芻獸疫病毒(Peste des petits ruminants virus,PPRV)引起的感染小反芻獸的一類動物傳染病。近年來,PPRV的研究大多集中在蛋白功能以及疫苗制備等方面,有關(guān)PPRV入侵宿主細(xì)胞的途徑研究少有報道。本試驗?zāi)康脑谟谔骄縋PRV對山羊子宮內(nèi)膜上皮細(xì)胞(Goat endometrial epithelial cells,EEC)的感染特性,以及病毒進入宿主細(xì)胞的內(nèi)吞途徑,從而為研究該病毒對宿主細(xì)胞的感染機制提供相關(guān)的理論基礎(chǔ)及科學(xué)依據(jù)。研究結(jié)果如下:1.獲得了PPRV在山羊子宮內(nèi)膜上皮細(xì)胞上的增殖規(guī)律。PPRV接種于EEC細(xì)胞后24 h到48 h后細(xì)胞無明顯的形態(tài)學(xué)變化,72h發(fā)現(xiàn)細(xì)胞發(fā)生典型的細(xì)胞病變(cytopathic effect,CPE),細(xì)胞聚集在一起,形成合胞體,折光性變強等,96 h后細(xì)胞出現(xiàn)裂解、死亡現(xiàn)象。EEC接種PPRV N75-1株后,激光共聚焦觀察確定該病毒進入細(xì)胞的時間在1-2 h內(nèi)。收取1 h、3 h、6 h、9 h、12 h、24 h的細(xì)胞上清,TCID50檢測發(fā)現(xiàn)病毒滴度在感染后24 h達(dá)到最高;收取0 h、3 h、6 h、9 h、12 h、24 h的細(xì)胞樣品,Western Blot檢測表明N蛋白的表達(dá)在感染后12 h表達(dá)量最大,24h有所下降,獲得了PPRV在EEC細(xì)胞上的增殖規(guī)律。2.PPRV入侵EEC不通過網(wǎng)格蛋白介導(dǎo)的內(nèi)吞途徑。用內(nèi)吞途徑的網(wǎng)格蛋白抑制劑氯丙嗪(CPZ)處理EEC,接毒后激光共聚焦顯微鏡觀察進入細(xì)胞的PPRV與對照組相比無明顯差異;Western Blot以及TCID50檢測發(fā)現(xiàn)N蛋白的表達(dá)和毒價亦無明顯變化。將網(wǎng)格蛋白的的siRNA轉(zhuǎn)染EEC中,接毒后Western Blot以及TCID50檢測N蛋白的表達(dá)以及PPRV毒價亦無明顯變化。實驗結(jié)果表明,PPRV入侵EEC不通過網(wǎng)格蛋白介導(dǎo)的內(nèi)吞途徑。3.PPRV入侵EEC是通過Caveola介導(dǎo)的Qg吞途徑。用膜穴樣凹陷內(nèi)吞途徑抑制劑甲基-β-環(huán)糊精(MβCD)和制霉素(Nystatin)兩種藥物處理EEC,接種PPRV后激光共聚焦顯微鏡觀察到進入細(xì)胞的病毒相對于對照組明顯減少。Western Blot檢測發(fā)現(xiàn),藥物處理組PPRV-N蛋白表達(dá)與對照相比均顯著減少。將Caveolin-1的siRNA轉(zhuǎn)染EEC后接種PPRV,Western Blot以及TCID50檢測發(fā)現(xiàn)N蛋白表達(dá)和病毒滴度明顯降低。實驗結(jié)果表明,PPRV入侵EEC是通過Caveola介導(dǎo)的Qg吞途徑。綜上所述,本研究通過特異性化學(xué)抑制劑阻斷、siRNA干擾方法確定,PPRV入侵EEC是依賴膜穴樣凹陷介導(dǎo)的內(nèi)吞途徑。研究結(jié)果為了解PPRV的致病機制和尋找新的藥物治療靶點提供理論依據(jù)。
[Abstract]:Peste des petits ruminants (PPRV) is a kind of infectious disease of small ruminants caused by Peste des petits ruminants virus (PPRV).In recent years, the research of PPRV is mainly focused on protein function and vaccine preparation. There are few reports on the pathway of PPRV invading host cells.The purpose of this study was to investigate the characteristics of PPRV infection on goat endometrial epithelial cells (Goat endometrial epithelial cells) and the endocytosis pathway of the virus into host cells.Therefore, it provides a theoretical and scientific basis for the study of the infection mechanism of the virus on host cells.The results are as follows: 1.The rule of proliferation of PPRV in goat endometrial epithelial cells was obtained. There were no obvious morphological changes in EEC cells after inoculation with PPRV for 24 h to 48 h. The typical cytopathic effect PPRV cells were found to have a typical cytopathic effect PPRV, and the cells gathered together to form syncytosomes.After 96 h of refractive change, the cells were lysed. The death phenomenon. After inoculating PPRV N75-1 strain, the laser confocal observation confirmed that the time of the virus entering the cells was within 1-2 hours.TCID50 titer of cell supernatant was detected at 24 h after infection, and the highest titer of TCID50 was found at 24 h after infection, and the expression of N protein was decreased at 12 h after infection by Western Blot analysis of cell samples collected for 0 h ~ 3 h ~ 6 h ~ 9 h ~ 9 h ~ 12 h ~ 24 h.The proliferation of PPRV on EEC cells was obtained. 2. PPRV invades EEC through endocytosis without grid protein.There was no significant difference in the expression of N protein and the toxic value between the PPRV cells and the control group by laser confocal microscopy after treatment with chlorpromazine (CPZ), a grid protein inhibitor of endocytosis pathway. The expression of N protein and the toxic value of N protein were also found to be unchanged by TCID50.The siRNA of griddle protein was transfected into EEC. The expression of N protein and PPRV level were not significantly changed by Western Blot and TCID50.The results showed that the invasion of EEC by PPRV was not mediated by the endocytosis pathway mediated by grid protein. 3. The invasion of EEC by PPRV was mediated by Caveola.EECs were treated with two drugs, methyl- 尾 -cyclodextrin (M 尾 CDD) and Nystatin (2). After inoculation with PPRV, the number of viruses entering the cells was significantly decreased compared with the control group. Western Blot analysis showed that the number of viruses entering the cells was significantly lower than that in the control group.The expression of PPRV-N protein in the drug treated group was significantly lower than that in the control group.The expression of N protein and the titer of the virus were significantly decreased after the siRNA of Caveolin-1 was transfected into EEC, and the expression of N protein and the titer of the virus were detected by TCID50 assay.The results showed that PPRV invasion of EEC was mediated by Caveola-mediated QG-swallowing pathway.To sum up, the method of blocking siRNA interference by specific chemical inhibitors confirmed that PPRV invasion of EEC was a membrane-like hole-mediated endocytosis pathway.The results provide theoretical basis for understanding the pathogenesis of PPRV and finding new therapeutic targets.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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