TNF-α增强膀胱上皮细胞清除细胞内肺炎克雷伯杆菌

研究TNF-α对膀胱上皮细胞内肺炎克雷伯杆菌生存的影响。方法：以肺炎克雷伯杆菌侵入体外培养的人膀胱上皮细胞（T24细胞）为模型,观察在TNF-α等细胞因子处理条件下,不同时间点细胞内细菌数量变化。结果：单独使用TNF-α使T24细胞内的肺炎克雷伯杆菌K5株细菌数量明显减少,联合使用TNF-α与IFN-γ使胞内活菌数量更显著的减少。而IL-1β对细胞内活菌数量无明显影响。过氧化氢酶可以有效抑制TNF-α与IFN-γ刺激的＂1＂24细胞抗菌作用,而一氧化氮合酶抑制剂L-NAME无抑制作用。结论：TNF-α能够增强膀胱上皮细胞对抗细胞内肺炎克雷伯杆菌,抗菌机制与细胞产生活性氧（ROS）有关。     

膀胱上皮细胞清除胞内大肠杆菌

目的:了解大肠杆菌与膀胱上皮细胞的相互关系,观察大肠杆菌在人膀胱上皮细胞株T24中的动态变化。方法:采用大肠杆菌标准株K12和大肠杆菌临床分离株299侵袭T24细胞,并用庆大霉素杀死细胞外的细菌,分别于细菌侵袭细胞后的4、24及48 h用TritonX-100裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌数。结果:两株大肠杆菌在T24细胞内都不能生长,随着时间的推移,细胞内大肠杆菌活菌数量呈不断减少趋势。24 h细胞内活菌数下降为4 h活菌数的15%。48h细胞内活菌数进一步减少。结论:膀胱上皮细胞清除进入细胞内的大肠杆菌,这可能是泌尿道天然免疫防御的一个重要防御机     

腺病毒介导突变Rip2基因对膀胱上皮细胞清除克雷伯杆菌的影响

目的探讨受体相互作用蛋白2（Rip2）在克雷伯杆菌所致的尿路感染中的作用。方法使用含有突变Rip2基因（mRip2）、绿色荧光蛋白（GFP）基因的复制缺陷腺病毒（Ad-mRip2-GFP）感染人膀胱上皮癌细胞T24细胞，表达GFP为转染成功标志，观察转染率，同时感染含有GFP基因腺病毒做对照，利用克雷伯杆菌临床分离株Kp5与细胞37℃孵育4、24、48小时，用平板培养菌落计数活菌数。结臬刺激后，在感染Ad-mRip2-GFP的T24细胞清除胞内Kp5能力下降，与Ad-GFP组比较P〈0．01。结论Rip2可以阻断膀胱上皮细胞的清除克雷伯杆菌作用，提示Rip2在膀胱上皮细胞的天然免疫中可能起重要作用。     

角质形成细胞对胞内金黄色葡萄球菌抗菌作用研究

目的:观察金黄色葡萄球菌在人角质形成细胞株HaCaT中生存的动态变化,了解金黄色葡萄球菌与皮肤角质形成细胞之间的相互关系。方法:用金黄色葡萄球菌标准株ATCC25923侵袭HaCaT细胞,分别于细菌进入细胞后的4、24、48、72h裂解细胞,释放出细胞内的活细菌,用平板菌落计数法计数胞内活菌。结果:金黄色葡萄球菌ATCC25923株在进入HaCaT细胞的24h有一定生长,但实验48h细胞内活菌数量明显减少。蛋白激酶C激活剂PMA和腺苷酸环化酶激活剂FSK可以促进HaCaT细胞清除胞内细菌。结论:皮肤角质形成细胞清除进入细胞内的金黄色葡萄球菌,可能是皮肤天然免疫的一种防御机制,而PMA和FSK增强细胞的抗菌作用,提示角质形成细胞抗菌活性与NADPH氧化酶相关。     

呼吸道上皮细胞与致病菌相互作用--上皮细胞内吞细菌实验研究

目的：呼吸道上皮细胞在防御这类机会致病菌感染时发挥了重要的作用。本研究旨在探讨上皮细胞能否清除胞内的绿脓杆菌，胞内模式识别受体Nods蛋白家族是否参与胞内杀菌，防御素是否能通过促经克雷伯杆菌粘附到上皮细胞而被上皮细胞内在化而清除。方法：首先用绿脓杆菌活菌刺激支气管原代上皮细胞及A549细胞，活菌细胞共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续孵育4小时，24小时后，用TritonX-100溶解细胞，采用平板菌落计数法计数胞内活菌数。绿脓杆菌与细胞按不同比例共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续培养24小时后收集细胞培养上清，酶联免疫吸附法（ELISA）检测IL-8的表达量。为进一步研究Nods蛋白是否在胞内杀菌及活菌在胞内引起IL-8分泌中发挥作用，我们用超声处理的绿脓杆菌菌体成分刺激使细胞膜通透性增加的温和去污剂digitonin处理和未使用digitonin处理A549细胞，ELISA检测细胞IL-8表达水平。胞内模式识别受体Nod1，Nod2可以识别菌体成分，用RT-PCR检测肺上皮细胞Nodl，Nod2的表达。其次，     

HNP对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响

目的：探讨呼吸道抗感染防御机制,观察人中性粒细胞α-防御素（HNP）对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响。方法：从人中性粒细胞分离纯化HNP。A549细胞与HNP（20μg·ml^-1）及两株肺炎克雷伯杆菌临床分离株共同孵育4小时,用平板培养茵落计数法测定与细胞粘附的活茵数。结果：在HNP存在的情况下,Kp03.33株细菌对肺上皮细胞的粘附提高了12倍（P〈0.01）,Kp03116株细菌对上皮细胞的粘附提高了5倍（p〈0.01）结论：HNP显著增强肺炎克雷伯杆菌粘附于呼吸道上皮细胞,可能有利于呼吸道清除细菌。     

膀胱上皮细胞对大肠杆菌抗菌作用观察

目的：了解膀胱上皮细胞和大肠杆菌的相互作用,观察其对大肠杆菌杀菌作用的动态变化。方法：采用人膀胱上皮细胞株T24和大肠杆菌标准株K12共同孵育,分别与作用后的15分钟、30分钟、60分钟、90分钟、120分钟,用Triton X-100释放粘附在细胞上及细胞内的活菌,用平板菌落计数法计数活菌数。结果：T24对大肠杆菌K12有杀菌作用,在相互作用15分钟时即表现出来,且在30分钟时杀菌作用最强,与对照组相比细菌减少53％。结论：膀胱上皮细胞对大肠杆菌有杀菌作用,这可能是泌尿道抵抗外来细菌感染的一种天然防御机制。     

人气管上皮细胞对绿脓杆菌抗菌作用观察

目的：探讨呼吸道上皮细胞与呼吸道致病菌之间的相互关系,观察呼吸道上皮细胞对绿脓杆菌的抗菌作用。方法：（1）贴壁生长的人呼吸道上皮细胞株HBE-16与绿脓杆菌标准株ATCC27853共孵育,庆大霉素杀死胞外菌,动态观察上皮细胞内活细菌数;（2）HBE-16细胞与绿脓杆菌共同悬浮于细胞培养基,在不同孵育时间点用平板菌落计数法计数活菌数。结果：绿脓杆菌不能在HBE-16细胞内生长,并被细胞逐渐清楚。悬浮状态下的HBE-16细胞对绿脓杆菌有一定杀菌作用。结论：呼吸道上皮细胞对胞内外绿脓杆菌的抗菌作用,可能是呼吸道抵抗细菌感染的一种天然免疫防御机制。     

肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL-8的研究

目的 :探讨肺炎克雷伯杆菌 (Klebsiellapneumoniae ,Kp)分泌因子及活菌诱导肺上皮细胞株表达和分泌IL 8的状况。方法 :用临床分离株Kp0 3 1 1 6、Kp0 3 1 83的细菌培养上清或活菌刺激肺上皮细胞株A5 49和SPC A 1 ,酶联免疫吸附实验 (ELISA)检测细胞IL 8表达量。结果 :①两株Kp培养上清分别刺激A5 49或SPC A 1后 ,IL 8表达量均有显著增高 (P <0 .0 1 ) ,且随上清刺激浓度增加而上升。②两株Kp及DH5 (活菌分别与SPC A 1共孵育 2h ,用庆大霉素杀死胞外菌 ,继续孵育 2 4h后两株Kp诱导细胞IL 8表达量均显著高于DH5α(P <0 .0 1 ) ,且随细菌数 /细胞数比例增大而上升。结论 :Kp分泌因子及活菌都能够诱导肺上皮细胞IL 8表达 ,而活菌上调IL 8的效应较培育上清分泌因子更显著 ,提示肺上皮细胞在Kp感染刺激的肺部炎症反应中起重要作用。     

α-防御素保护小鼠抵抗肺炎克雷伯杆菌肺部感染的实验观察

目的:探讨人中性粒细胞α-防御素对小鼠感染肺炎克雷伯杆菌肺炎模型动物的保护作用。方法:①用克雷伯肺炎杆菌临床分离株K4与不同浓度α-防御素在37℃孵育两小时,平板培养菌落计数法计数活细菌数。②用K4和α-防御素混合经肺部感染昆明种小鼠,观察小鼠生存率。结果:①K4与α-防御素共孵育的菌落数和PBS对照组菌落数比较没有显著性差异。②肺部注入肺炎克雷伯杆菌混合α-防御素组小鼠的死亡率低于单纯注入肺炎克雷伯杆菌的对照组,且生存时间延长(P<0.05)。结论:α-防御素可以保护小鼠抵抗细菌性肺炎,保护机制可能不是α-防御素的直接杀菌,而可能是通过增强上皮细胞天然免疫功能。     

Invasion of cultured human epithelial cells by Klebsiella pneumoniae isolated from the urinary tract.

The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091, isolated from a urine sample of a patient with a urinary tract infection, invaded human epithelial cells from the bladder and ileocecum and persisted for days in vitro. Electron microscopic studies demonstrated that K. pneumoniae was always contained in endosomes. The internalization mechanism(s) triggered by K. pneumoniae was studied by invasion assays conducted with different inhibitors that act on prokaryotic and eukaryotic cell structures and processes. Chloramphenicol inhibition of bacterial uptake revealed that bacterial de novo protein synthesis was essential for efficient invasion by K. pneumoniae and S. typhi. Interference with receptor-mediated endocytosis by g-strophanthin or monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular K. pneumoniae cells, but not S. typhi cells. The depolymerization of microfilaments by cytochalasin D inhibited the uptake of both bacteria. Microtubule depolymerization caused by colchicine, demecolcine, or nocodazole and the stabilization of microtubules with taxol reduced only the invasion ability of K. pneumoniae. S. typhi invasion was unaffected by microtubule depolymerization or stabilization. These data suggest that the internalization mechanism triggered by K. pneumoniae 3091 is strikingly different from the solely microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria, such as enteroinvasive Escherichia coli, Salmonella, Shigella, and Yersinia strains.     

Evidence for pili-mediated adherence of Klebsiella pneumoniae to rat bladder epithelial cells in vitro.

The possible role of pili in the pathogenesis of urinary tract infections caused by Klebsiella pneumoniae was studied in an in vitro mixture of a phosphate-buffered saline suspension of rat bladder epithelial cells and phosphate-buffered saline-washed K. pneumoniae. Nonpiliated and piliated populations derived from a single K. pneumoniae strain were obtained by controlling the total time of growth in broth medium. The piliated phase demonstrated a significant increase in adherence when compared to the nonpiliated phase. Incubation of the bacteria and epithelial cell mixture at 4 and 37 degrees C resulted in no differences in adherence; optimal adherence occurred at pH 5. Pretreatment of the bacteria with enzymes to destroy the pili resulted in a decrease in adherence, as did killing the bacteria by various means before adherence testing. Pretreatment of the epithelial cells with certain saccharides inhibited bacterial adherence. Finally, a 96% decrease in adherence was observed after coincubation of bacteria and epithelial cells with papain-treated antipili antibodies. Thus, it appears that pili on the surface of K. pneumoniae mediate attachment of the bacteria to rat bladder epithelial cells.     

In vitro studies of epithelium-associated crystallization caused by uropathogens during urinary calculi development

Infectious urinary stones account for about 10% of all urinary stones. In 50% of cases urolithiasis is a recurrent illness, which can lead to the loss of a kidney if not properly treated. One of the reasons for recurrence of the disease may be the ability of bacteria to invade urothelial cells, persist in the host cells and serve as potential reservoirs for infection. Various uropathogens are associated with the formation of bacteria-induced urinary stones but Proteus mirabilis is the most commonly isolated (70%). An in vitro model was used in this study to analyze intracellular growth and crystallization in the presence of P. mirabilis, Klebsiella pneumoniae and Escherichia coli. Human ureter (Hu 609) and bladder (HCV 29) epithelial cell lines were infected with bacteria and incubated (3–72 h) in the presence of synthetic urine and amikacin to prevent extracellular bacterial growth. During the incubation the number of bacteria (CFU/ml) inside epithelial cells and the intensity of crystallization were established. Crystallization was determined as an amount of a calcium radioisotope. The chosen strains of uropathogens were able to invade both types of epithelial cells but the Hu 609 cells were invaded to a higher extent. However, crystallization occurred only in the presence of P. mirabilis strains which were invasive and urease-positive. The highest intensity of cell-associated crystallization was observed when the number of bacteria within the urothelium remained stable during the time of incubation. These results show that P. mirabilis has an ability to form crystals inside the host cells. Under these conditions bacteria are protected from antibiotic killing, which leads to persistent and recurrent infections. We also suspect that this phenomenon may be an important stage of kidney stones formation.     

影响肺炎克雷伯菌粘附上皮细胞作用的研究

目的:研究不同理化及生物因素对肺炎克雷伯菌粘附宿主细胞的影响,探讨其粘附宿主细胞的可能机制。方法:用肺炎克雷伯菌临床分离株K.pnueumoniae 03183(K.p03183)建立粘附宿主细胞模型,通过结晶紫染色及扫描电镜观察其粘附效率;以不同理化及生物因素预处理K.p03183,平板菌落计数法观察上述因素对K.p03183粘附细胞的影响;用基因芯片技术,检测HMGN2蛋白预处理细菌对于其粘附相关基因表达的影响。结果:K.p03183可粘附至A549、T24、HeLa等3种上皮细胞系,其粘附率与E.coli 25992接近;氯化锂和盐酸胍、高碘酸钠及热预处理可明显降低K.p03183对于A549和T24的粘附率(P0.05);同时,胃蛋白酶及HMGN2蛋白预处理K.p03183,也可抑制其对A549和T24的粘附(P0.01);但胰蛋白酶预处理却能增加细菌对于A549细胞的粘附(P0.05);同时,HMGN2还可通过上调dksA基因表达,从而干扰细菌粘附细胞。结论:肺炎克雷伯菌可能借助细胞壁及其表面蛋白和碳水化合物等成分粘附至宿主上皮细胞,通过非共价键结合表面蛋白或消除碳水化合物成分,抑制细菌粘附至宿主细胞。     

苹果酸舒尼替尼抑制膀胱癌T24细胞系的体外增殖

 摘要：目的：探讨苹果酸舒尼替尼对人膀胱癌T24细胞系体外增殖的影响。方法：取人膀胱癌T24细胞系悬液, 分别加入浓度为0.625、1.25、2.5、5、10 和 20μmol/L的苹果酸舒尼替尼,分别于24、48、72 h进行处理,用细胞毒抑制实验（MTT法）检测药物对其生长的影响。结果：苹果酸舒尼替尼可浓度依赖性抑制人膀胱癌细胞系T24细胞增殖,当药物浓度>5μmol/L时,苹果酸舒尼替尼与顺铂比较,对T24细胞系具有更强的抑制作用（P<0.05）。这种抑制作用随着药物培育时间延长（24、48、72ｈ）而增强,表现为IC50值显著降低, 但其抑制能力与顺铂比较无显著差别（P>0.05）。结论：苹果酸舒尼替尼可呈浓度和时间依赖地抑制膀胱癌T24细胞系增殖。