本文選題：FMDV + 牛鼻咽粘膜上皮細胞��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學》2017年碩士論文

【摘要】：口蹄疫(FMD)是由口蹄疫病毒(FMDV)引起的感染偶蹄類動物的一種高度接觸性、急性熱性傳染病,該病傳播速度快、途徑廣,嚴重威脅畜牧業(yè)的經(jīng)濟發(fā)展。因此,對FMDV感染宿主細胞的致病機制研究對防治口蹄疫具有重要意義。鼻咽粘膜上皮細胞和肺泡上皮細胞是FMDV侵染宿主的主要靶點細胞,對其體外分離培養(yǎng)并建立FMDV宿主細胞感染模型的研究可以為下一步深入開展口蹄疫致病機制的研究提供良好的素材和基礎(chǔ)。本研究以偶蹄類動物牛為實驗材料,體外成功分離培養(yǎng)牛鼻咽粘膜上皮細胞和牛肺泡上皮細胞,經(jīng)純化后對其進行免疫熒光等技術(shù)鑒定與FMDV感染實驗,初步建立FMDV感染宿主細胞的基礎(chǔ)研究模型。具體研究結(jié)果如下:(1)細胞分離體系的建立:取牛鼻咽部粘膜及肺部組織,分別用組織塊貼壁法和酶消化法分離原代細胞,選取機械刮除法與分時間消化法純化牛鼻咽粘膜上皮細胞,用機械刮除法純化牛肺泡上皮細胞;(2)細胞的培養(yǎng)及鑒定:對成功分離后的牛鼻咽粘膜及肺泡上皮細胞進行傳代培養(yǎng)操作,分別對P5代細胞進行細胞增殖曲線、染色體數(shù)目分析,利用免疫熒光技術(shù)經(jīng)廣譜角蛋白染色鑒定為上皮細胞;(3)FMDV感染細胞模型的建立:將牛鼻咽粘膜上皮細胞和牛肺泡上皮細胞感染O型FMDV,細胞均回縮變圓發(fā)生病變,提取其總RNA并反轉(zhuǎn)錄得到cDNA,用FMDV特異性引物進行PCR擴增后得到420 bp大小的目的片段,對其進行測序分析后確定FMDV已成功感染兩種上皮細胞。
[Abstract]:Foot-and-mouth disease (FMD) is a highly contact and acute thermal infectious disease caused by foot-and-mouth disease virus (FMDV). The disease spreads rapidly and has a wide range of routes, which seriously threatens the economic development of animal husbandry.Therefore, it is of great significance to study the pathogenesis of FMDV infection in host cells for the prevention and treatment of foot-and-mouth disease.Nasopharyngeal mucosal epithelial cells and alveolar epithelial cells are the main target cells of FMDV infection host.The study of FMDV host cell infection model in vitro can provide a good material and foundation for further research on the pathogenesis of foot-and-mouth disease.In this study, bovine nasopharyngeal mucosal epithelial cells and bovine alveolar epithelial cells were isolated and cultured successfully from cloven-hoofed cattle in vitro. The bovine nasopharyngeal mucosal epithelial cells and bovine alveolar epithelial cells were purified and identified by immunofluorescence and FMDV infection.The basic research model of FMDV infected host cells was established.The specific results are as follows: (1) Establishment of cell isolation system: primary cells were isolated from bovine nasopharyngeal mucosa and lung tissue by tissue mass adherence method and enzyme digestion method, respectively.The bovine nasopharyngeal mucosal epithelial cells were purified by mechanical scraping and time-divided digestion.The culture and identification of bovine alveolar epithelial cells (BALECs) by mechanical curettage: the successfully isolated bovine nasopharyngeal mucosa and alveolar epithelial cells were subcultured, and the proliferation curve and chromosome number of P5 passage cells were analyzed respectively.The model of FMDV infection in epithelial cells was established by using immunofluorescence technique and identified by broad-spectrum keratin staining. When bovine nasopharyngeal mucosal epithelial cells and bovine alveolar epithelial cells were infected with type O FMDV, the cells retracted and changed to round lesions.The total RNA was extracted and reverse transcribed to obtain the cDNA. the target fragment of 420bp was obtained by PCR amplification with FMDV specific primers. After sequencing, it was confirmed that FMDV had been successfully infected with two kinds of epithelial cells.
【學位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S855.3

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