牛鼻咽粘膜及肺泡上皮细胞分离培养、鉴定与FMDV感染研究

发布时间：2018-04-18 14:48

本文选题：FMDV + 牛鼻咽粘膜上皮细胞　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：口蹄疫(FMD)是由口蹄疫病毒(FMDV)引起的感染偶蹄类动物的一种高度接触性、急性热性传染病,该病传播速度快、途径广,严重威胁畜牧业的经济发展。因此,对FMDV感染宿主细胞的致病机制研究对防治口蹄疫具有重要意义。鼻咽粘膜上皮细胞和肺泡上皮细胞是FMDV侵染宿主的主要靶点细胞,对其体外分离培养并建立FMDV宿主细胞感染模型的研究可以为下一步深入开展口蹄疫致病机制的研究提供良好的素材和基础。本研究以偶蹄类动物牛为实验材料,体外成功分离培养牛鼻咽粘膜上皮细胞和牛肺泡上皮细胞,经纯化后对其进行免疫荧光等技术鉴定与FMDV感染实验,初步建立FMDV感染宿主细胞的基础研究模型。具体研究结果如下:(1)细胞分离体系的建立:取牛鼻咽部粘膜及肺部组织,分别用组织块贴壁法和酶消化法分离原代细胞,选取机械刮除法与分时间消化法纯化牛鼻咽粘膜上皮细胞,用机械刮除法纯化牛肺泡上皮细胞;(2)细胞的培养及鉴定:对成功分离后的牛鼻咽粘膜及肺泡上皮细胞进行传代培养操作,分别对P5代细胞进行细胞增殖曲线、染色体数目分析,利用免疫荧光技术经广谱角蛋白染色鉴定为上皮细胞;(3)FMDV感染细胞模型的建立:将牛鼻咽粘膜上皮细胞和牛肺泡上皮细胞感染O型FMDV,细胞均回缩变圆发生病变,提取其总RNA并反转录得到cDNA,用FMDV特异性引物进行PCR扩增后得到420 bp大小的目的片段,对其进行测序分析后确定FMDV已成功感染两种上皮细胞。
[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.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S855.3

【参考文献】