本文選題：致腎盂腎炎大腸桿菌　切入點(diǎn)：正常人腎盂移行上皮原代細(xì)胞　出處：《天津醫(yī)科大學(xué)》2006年碩士論文

【摘要】：目的:建立正常人腎盂移行上皮原代細(xì)胞的培養(yǎng)方法,進(jìn)行致腎盂腎炎大腸桿菌(pyelonephritic E.coli or uropathogenic E.coli,UPEC)粘附特性的研究,并探討UPEC132毒力因子對(duì)宿主細(xì)胞的毒性作用。方法:采用PCR及復(fù)合PCR方法分別擴(kuò)增并檢測(cè)UPEC132的papC,hly及cnf1毒力基因。進(jìn)行溶血試驗(yàn)檢測(cè)UPEC132的溶血素。選擇正常人腎臟的腎盂組織進(jìn)行原代細(xì)胞培養(yǎng),分別比較兩種常用培養(yǎng)方法及含血清培養(yǎng)基(RPMI1640等)與添加成分的無血清培養(yǎng)基(KSFM等)的不同培養(yǎng)基體系的培養(yǎng)結(jié)果,進(jìn)行顯微鏡形態(tài)學(xué)觀察、計(jì)數(shù)、HE染色、鑒定及成功率和污染率的統(tǒng)計(jì),確定原代培養(yǎng)體系。進(jìn)行UPEC132的細(xì)菌粘附試驗(yàn),經(jīng)Giemsa染色運(yùn)用顯微鏡攝影技術(shù)觀察UPEC132與正常人腎盂移行上皮原代細(xì)胞、二倍體細(xì)胞系(Vero)及人移行上皮癌細(xì)胞系(EJ)粘附后的形態(tài)學(xué)改變,計(jì)數(shù)并統(tǒng)計(jì)15至180分鐘不同時(shí)間段的粘附率及粘附指數(shù),比較UPEC對(duì)三種細(xì)胞的粘附特性及與宿主細(xì)胞的相互作用。試驗(yàn)中以標(biāo)準(zhǔn)株UPECJ96為陽性對(duì)照,無菌毛代表株E.coli K-12p678-54為陰性對(duì)照。試驗(yàn)數(shù)據(jù)運(yùn)用SPSS軟件進(jìn)行t檢驗(yàn),x~2檢驗(yàn)等統(tǒng)計(jì)學(xué)處理。結(jié)果:凝膠電泳結(jié)果顯示UPEC132的papC,hly和cnf1基因片斷擴(kuò)增均為陽性。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R378.21

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