| Objective: Ab, as one of the inspiratory opportunistic pathogen,the course of pathogenesis was not clear until now. There is only a few of articles about Ab adhere to epithelial cells. The immune defense, as the initial step of the body defense, can be evoked by adherence and invasiveness of microorganisms to epithelial cells. Hence the correlation between pathogen and epithelial cells become focal point in the molecule bacterial and cell microbiology. It is reported that Ab possessed one of the toxic factors was ability of mucus production. In recent years, our laboratory gradually isolated Ab of mucosal phenotype from clinic, it is different from traditional phenotype. So we observe the adherence of different phenotype to epithelia cells, and the condition about expression of cytokine under stimulation of live bacterial suspension, bacterium spent culture supernatant and bacterium spallation. The aim was to observe Ab can induce epithelia cells express cytokine directly or not, and there exists difference on ability of expression cytokine by epithelial cells stimulated by bacterial between mucosal phenotype and common phenotype.Methods: The first part is the adherence of Ab, we establish the standard curve of optical density and the quantity of bacterial. According to it, we made live bacterial suspension of 2×109cfu/ml, 2×108cfu/ml, 2×107cfu/ml. We incubate human lung epithelial cells A549. 3ml of 1×105/ml cell susupension was added to six-pores-plate with coverslips, then incubated 12h. After that, in the place of culture medium, 3ml live bacterial suspension of different concentration was added to it. Hence cell and bacterial were incubated together for 30min, 60min, 90min. coverslips were washed by disinfectioned NS three times, and then were stained by Gimsa. We observe the number of bacterial adhered to 50 cells (averge) with optical microscope.2. The second part was the inducement of COX-2 and IL-8 in human lung epithelial cells by live bacterial suspension, spent culture supernant, bacterium spallation. According to standard curve we made the live bacterial susupention of 2×109cfu/ml, 2×108cfu/ml, 2×107cfu/ml. Among them 2ml the concentration of 2×109cfu/ml was added to LB liquid culture medium (250ml), then was cultured for 72h succussion. The liquid was centrifuged and flitered. 20ml of different concentrations live bacterial suspension were transoniated; with 5000Hz for 30min, then flitered.Untill cells grew fully in culture bottle about 25cm2, 4ml of live bacterial susupention and bacterium spallation on different concentration and spengt culture supernant were added into culture bottle. After cells were incubated with all bove for 6h, 12h, 24h, total RNA of cell was taken and RT-PCR. The product of PCR was electrophoresed in 1% agarose gelatum. The result was anlysed by chromatography. Statistical analysis was performed by SPSS13.0 software.Results:1 the study of adherence: (1)When the live bacterial susupention of Ab at 2×109cfu/ml, 2×108cfu/ml, 2×107cfu/ml of different phenotype was incubated with A549 for 30min, the number of adherence to cell reched the peak. And the number of adherence gradually decreased with the time prolongerment. There exits significant statistical difference (P<0.01). (2) When the live bacterial susupention of Ab at 2×109cfu/ml, 2×108cfu/ml, 2×107cfu/ml of different phenotype was incubated with A549 for 30min, 60min, 90min, the number of adherence gradually decreased with the decreasement of the concentration. There exits significant statistical difference (P<0.01). In other words the number of adherence was positive correlation to the concentration of live bacterial susupention and negative correlation to the time prolongerment. (3) At the same time point and concentration, the number of adherence of musocal Ab was higher than non-musocal Ab. There exits significant statistical difference (P<0.01).2 The expression of COX-2, IL-8 in the human lung epithelial cells: (1) When the live bacterial suspension, spent culture supernant, bacterium spallation of Ab at different concemtration incubated with A549 together for different time, COX-2 was only expressed in cells at 12h. When the live bacterial suspension, spent culture supernant of Ab at different concemtration incubated with A549 together for different time, L-8 was only expressed in cells at 24h. The bacterium spallation of musocal Ab can induce cells express IL-8 at 2×108cfu/ml but not at 2×109cfu/ml and 2×107cfu/ml. While the bacterium spallation of non-musocal Ab can not induce cells express IL-8 at each concentration. (2) The ablity of expression COX-2, IL-8 in the cells induced by live bacterial susupention of musocal Ab strengthened first weakened later with the decreasement of concentration, reached the peak at 2×108cfu/ml. There exits significant statistical difference (P<0.01). The ablity of expression COX-2, IL-8 in the cells induced by live bacterial susupention of non-musocal Ab strengthened with the decreasement of concentration. There exits signifiacnt statistical difference (P<0.01). (3) The ability of expression of COX-2 induced by bacterium spallation strengthened with the decreasement of concentration. There exits signifiacnt statistical difference (P<0.01). (4)There existed no difference between the musocal Ab and non musocal phenotype of live bacterial susupension at 2×109cfu/ml on the ability of IL-8 induction (P>0.05). The ability of expression COX-2 induced by live bacterial susupention, spent culture, bacterium spallation of musocal Ab was stonger than non-musocal Ab. While the ability of expression COX-2 induced by live bacterial susupention, spent culture, bacterium spallation of musocal Ab was weaker than non-musocal Ab. There exits signifiacnt statistical difference (P<0.01). (5) The ability of expression COX-2 induced with live bacterial susupention was stronger than bacterium spallation by different phenotype at the same concentration. The ability of expression IL-8 induced with live bacterial susupention was stronger than bacterium spallation by musocal Ab at 2×108cfu/ml. There exits signifiacnt statistical difference (P<0.01).Conclusions: The adherence number of Ab to lung epithelial cells was positive correlation to the concentration of bacterium susupengtion and was negative correalation to the time. The peak of adherence index was at 30 minetes. The adherence index of common phenotype was more than mucosal phenotype (P<0.01). The difference on phenotype could influence the expression of cytokine in lung epithelial cells. It was likely due to the contens of certain material in bacterial or the binding ability to receptors. SCS and bacterium spallation also can induce the expression of cytokine. It was suggested that a few contens of secrotory and cell wall was pathogenic. |
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