Primary Study On The Adhesion Of Uropathogenic E.coli To Culture Cells In Vitro

Objective: To establish the culture method of normal human renal pelvis epithelial cells, study on the adhesion of uropathogenic Escherichia coli (UPEC) and research the toxicity of the UPEC virulence factors to host cells. Methods: The virulence genes papC, hly and cnfl of UPEC 132 were detected by PCR and multiplex PCR. The hemolysin of UPEC 132 was determined by hemolytic experiment. In addition, primary culture of normal human renal pelvis transitional epithelial cells was performed to compare the culture results of the two main methods ("tissue cubes" & "enzyme digestion") and that of two different medium (RPMI 1640 with FBS & KSFM with EGF and BPE) by observing, counting, HE dying, identification, calculation the success rate and polluted rate so as to choose an ideal primary culture system. After the UPEC 132 adhesion experiment and Giemsa dying, the shape changings of host cells (normal human renal pelvis epithelial primary cells, Vero cells and EJ cells) were observed by microscopy. At the same time, both the adhesion rates and the indexes were calculated in different period between 15 minutes and 180 minutes. At last the UPEC 132 adhesion to the 3 kinds of cells were compared. In the test, standard stain UPEC J96 was set as the positive control and non-pilus stain E.coli K-12p678-54 as the negative control. The test data were dealt with statistical analysis (t-test & x~2-test) by SPSS. Results: Gelelectprophoresis results show PCR products of UPEC 132 papC, hly and cnfl genes positive. Thehemolysin of UPEC132 was present. The KSFM medium system success rate is higher than that of RPMI1640.(x2-test, PO.05) The success rate of "tissue cubes" method is higher than "enzyme digestion" method success rate.(x2-test, PO.01) The primary cells are flat irregulate multi-argle under light microscope, connecting with each other and growing as a whole, which look like "paving stones" and in line with the shape of epithelial cells. They show positive through the immunocytochemistry dying of keratin antibodies, which defines the transitional epithelium. The average adhesion rates and indexes of UPEC132 with 3 kinds of cells stable at 30 minutes are: 68.00&20.27, 45.60&16.55, 69.60&32.28. After the adhesion experiment of UPEC132, the shapes of the cells change obviously. The cells shrink, loose, bubble change and their cytoplasmic convexities decline or vanish under the oil lens. Some UPEC on the surface, some into the cytoplasma, and others in the nuclei. Nevertheless, both the adhesion rate and the index of E.coli K-12p678-54 with 3 kinds of cells are 0. Conclusions: UPEC 132 has papC, hly and cnfl virulence genes. Normal human kidney renal pelvis epithelial primary cells can be cultivated successfully by "tissue cubes" method using KSFM with EGF and BPE. UPEC 132 can adhere to all the 3 kinds of cells within 30 minutes. P pilus is the essential factor of the adhesion to host cells. Hemolysin and cytotoxic necrotising factor 1 have the virulence effect to the host cells.