致肾盂肾炎大肠杆菌体外细胞粘附的初步研究

发布时间：2018-04-07 16:31

本文选题：致肾盂肾炎大肠杆菌　切入点：正常人肾盂移行上皮原代细胞　出处：《天津医科大学》2006年硕士论文

【摘要】：目的:建立正常人肾盂移行上皮原代细胞的培养方法,进行致肾盂肾炎大肠杆菌(pyelonephritic E.coli or uropathogenic E.coli,UPEC)粘附特性的研究,并探讨UPEC132毒力因子对宿主细胞的毒性作用。方法:采用PCR及复合PCR方法分别扩增并检测UPEC132的papC,hly及cnf1毒力基因。进行溶血试验检测UPEC132的溶血素。选择正常人肾脏的肾盂组织进行原代细胞培养,分别比较两种常用培养方法及含血清培养基(RPMI1640等)与添加成分的无血清培养基(KSFM等)的不同培养基体系的培养结果,进行显微镜形态学观察、计数、HE染色、鉴定及成功率和污染率的统计,确定原代培养体系。进行UPEC132的细菌粘附试验,经Giemsa染色运用显微镜摄影技术观察UPEC132与正常人肾盂移行上皮原代细胞、二倍体细胞系(Vero)及人移行上皮癌细胞系(EJ)粘附后的形态学改变,计数并统计15至180分钟不同时间段的粘附率及粘附指数,比较UPEC对三种细胞的粘附特性及与宿主细胞的相互作用。试验中以标准株UPECJ96为阳性对照,无菌毛代表株E.coli K-12p678-54为阴性对照。试验数据运用SPSS软件进行t检验,x~2检验等统计学处理。结果:凝胶电泳结果显示UPEC132的papC,hly和cnf1基因片断扩增均为阳性。
[Abstract]:Aim: to establish a culture method of normal human transitional epithelial cells (TECs), to study the adhesion characteristics of pyelonephritic E.coli or uropathogenic E.coli UPECs, and to investigate the toxic effects of UPEC132 virulence factors on host cells.Methods: PCR and compound PCR were used to amplify and detect the UPEC132 PAPCICHly and cnf1 virulence genes respectively.The hemolysin of UPEC132 was detected by hemolysis test.The primary cell culture of normal human renal pelvis was carried out, and the results of two common culture methods and different culture systems containing serum medium RPMI1640 and serum-free medium KSFM were compared, respectively.The primary culture system was determined by microscopic morphological observation, HE staining, identification and statistics of success rate and contamination rate.The bacterial adhesion test of UPEC132 was carried out. The morphological changes of UPEC132 were observed by Giemsa staining after adhesion with primary cells of normal human renal pelvis transitional epithelium, diploid cell lines and human transitional cell carcinoma cell lines.The adhesion rate and adhesion index of UPEC in different time periods from 15 to 180 minutes were counted, and the adhesion characteristics of UPEC to three kinds of cells and their interaction with host cells were compared.The standard strain UPECJ96 was used as positive control and the representative sterile pili strain E.coli K-12p678-54 as negative control.The test data were processed by SPSS software.Results: the results of gel electrophoresis showed that UPEC132 was positive for the amplification of PAPCCAHly and cnf1 gene fragments.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R378.21

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