| Objective:To establish the model of adherence of host cells by uropathogenic E.coli (UPEC) 132 for studies on the relationship between bacteria and cells, and observe the gene expression spectrum changes of bladder carcinoma cell (EJ cells) adhered by UPEC132.Methods:1. The passage cells of green monkey kidney cell line (Vero cells), human renal carcinoma line (Ketr-3 cells) and bladder cancer cell line (EJ cells) were cultured to compare the ability of adherence by UPEC132. The morphological changes of host cells were observed and the adhesion rates and indexes were calculated after bacterial adherence. Bacterial invasion into host cells was tested by gentamicin protection assay and the invasion indexes of above three cells were calculated. Nonfimbrial strain E.coli K-12 p678-54 was used as negative control in the adherence and invasion tests.2. The P pili receptors on above three cells were detected by indirectimmunofluorescence. The primary antibody was anti-P1 (IgM) monoclonal antibody and the second antibody was FITC-labeled rabbit anti-mouse IgM antibody. The fluorescence on cells was observed by fluorescence microscope to compare the receptor distribution on different cells.3. The gene expression spectrum changes of EJ cells adhered by UPEC132 wereobserved by DNA microarrays. The total RNA of infected and uninfected cells was extracted respectively by using Trizol reagent. cDNA was synthesized with total RNA and labeled with fluorescent dye (Cy5 and Cy3-dCTP). The array was hybridized at 42℃overnight and washed with two consecutive washing solutions. Arrays were scanned with LuxScan 10K/A scanner, and the obtained images were analyzed with LuxScan3.0. Those genes whose alteration tendency was above or below two fold were selected as differentially expressed genes. Results:1. After infected for 1 hour by UPEC132, the above three cells showed significant morphologic changes. Intercellular space became larger, cytoplasm retracted or ruptured, and the ability of adherence to culture flask weakened. The adhesion rates of Vero, Ketr-3 and EJ cells by UPEC132 were(61.44±3.21) %, (55.22±4.09) % and (58.67±5.12) % respectively without statistical differences. But the adhesion indexes of these cell lines were 1.44±0.06, 1.74±0.09 and 2.27±0.18 respectively with statistical difference ( p<0.05 ). The invasion indexes of EJ and Ketr-3 cells by UPEC132 were (3.25±0.20)×10-3 and(3.00±0.34)×10-3, and both of which were significantly higher than that of Vero cells[(2.61±0.32)×10-3, p<0.05].E.coli K-12 p678-54 did not adhere and invade tested cells.2. The P1 monoclonal antibody bound to P pili receptor specifically. The cells ofexperiment group showed a clear outline using a fluorescent microscope. The distribution of fluorescent material was continuous on cell surface. But the cells of control group could not show fluorescence.3. Data from the gene expression profile indicated that UPEC132 infection altered the expression of 29 genes, of which 28 were up-regulated and one down-regulated. These differentially expressed genes were mainly related to cell growth and proliferation, inflammation, apoptosis, the stress response, signal transduction and so on. It was indicated that UPEC132 interacted with host cells in multi-targets, at multi-levels and through multi-channels.Conclusion:In this study we established the model of adherence of host cells by UPEC132, analyzed the ability of adherence and invasion of UPEC132, and observed the gene expression spectrum changes of host cells infected by UPEC132. The findings provided a good foundation for further research on the pathogenesis of UPEC. |
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