| Aim:1. The clinical efficacy that BCG treated superficial bladder cancer, furthermore which PPS has synergistic effect, has already been confirmed. But the interactive mechanism remains unknown. In the paper, we studied the interaction locus of recombinant Bacillus Calmette-Guerin (rBCG) in human bladder cancer cell line T24 and the effect of polyporud polysaccharide (PPS) with rBCG. 2. Detecting the effects of polyporus polysaccharide(PPS) on bacillus calmette guerin (BCG) -stimulated toll like receptor4 and CD14 expressions of murine macrophages, studying the immune initiation process participating in by them. 3. To study the effect of Bacillus Calmette-Guerin (BCG) cooperated with polyporus polysaccharide(PPS) on the activation of nuclear factor- κB (NF-κB ) in J774 cell line. 4. To investigate the effects of polyporus polysaccharide (PPS) on bacillus calmette guerin (BCG) -stimulated proinflammatory cytokines secretions of murine macrophages. Methods: 1. With Green fluorescent Protein (GFP) which emits green fluorescence expressed by rBCG as the reporter gene, we used the laser scanning confocal microscope (LSCM) and flow cytometry (FCM) to observe the changes of T24 cells treated with rBCG (&PPS). 2. Murine macrophage J774 cell line was treated with BCG or BCG plus PPS, the expression levels of TLR4 and CD14 were determined by flowcytometery. 3. The expression of NF- k B which strained by indirect immunofluorescence was detected by laser scanning confocal microscopy (LSCM). The change of the expression of NF-κ B in the cytosol and nuclei was analyzed by software. 4. Murine macrophage J774 cell line was treated with BCG or BCG plus PPS, the contents of IL-8, IL-1β and TNF-α were determined by ELISA. Results:1. (1) Compared with the control, in the cytoplasm of T24 cells the green fluorescence (86.335±5.856) was detected after 24h treatment with rBCGbut this change was not remarkable erenow. Meanwhile the results of flow cytometery (FCM) measurements showed in the scattergram the positions of T24 cells treated with rBCG for 24h and 48h shifted to the upper right by comparison to the control on the same voltage. At the same time, considering cells of control group as negative control, we measured there were a certain number of GFP-positivecell[(8. 7 + 1.572)% , (13. 8±2. 31)%]inrBCG group(p<0. 05). (2) Subsequently we have found obvious green fluorescence in the whole cells including the nuclei after treatment with rBCG-PPS over 24h, the green fluorescence intensity of nucleus (Fn) (72. 603 + 1.165) turned much stronger, that of cytoplasm(Fc) (93.06 + 0.958) got weaker, and the ratio (0.78 + 0.005) of Fn to Fc became significantly higher than those of rBCG group(62.832+ 2.909, 105.306+6.393, 0.597+0.012) (p<0.05).2. The levels of TLR4 and CD14 protein increased obviously after BCG or BCG plus PPS stimulation. (l)The expression level of TLR4 increased obviously after BCG (50 ug/ml) stimulation for 0.5 Ik 12 Ik 24 h and 48 h, meanwhile the expression level of TLR4 in macrophages treated with BCG (50 ug/ml) plus PPS (50 ug/ml) was significantly higher than in the groups treated only BCG in 0. 5 Ik 1 Ik 3 Ik 6 h and 24 h. And the expression level of TLR4 increased obviously after BCG (250 ug/ml) stimulation for 0. 5 hu 3 h > 6 h and 48 h, at the same time the expression level of TLR4 in macrophages treated with BCG (250 ug/ml) plus PPS (250 ug/ml) was significantly higher than in the groups treated only BCG in 24h. (2)The expression level of CD14 increased obviously after BCG (50 ug/ml) stimulation for 24 h, meanwhile the expression level of CD14 in macrophages treated with BCG plus PPS was significantly higher than in the groups treated only BCG though 0. 5~48h. The peaks of expression of CD14 and TLR4 at 6h were observed.3. The fluorescent intensity of NF-k B decreased in cytosol, increased in nuclei and the ratio between them were much higher after treatment with BCG or/and PPS in comparison with the control (P<0.05). But there were no significance among the ratios of the fluorescent intensity in groups treated with BCG^ PPS only or BCG plus PPS.4. The contents of IL-1 0 did not increase obviously after BCG or BCG plus PPS stimulation. The IL-13 secretion level of macrophages treated with BCG plus PPS was not significantly higher than in the groups treated only BCG. However, the contents of IL-8 and TNF-a increased obviously after BCG or BCGplus PPS stimulation. And there were no significance among the contents of IL-8 and TNF- a in groups treated with BCG only or BCG plus PPS using the same doses. The peak of content of TNF-a at 12 hour and IL-8 at 24 hour were observed.Conclusions: 1.T24 cells could directly phagocytose rBCG and PPS helped rBCG enter into the nuclei of T24 cells.2. BCG can elevate the expression levels of TLR4 and CD14 in macrophages. And a certain dose of PPS and BCG have synergistic effects on increasing TLR4 and CD14 expression, thus participating in immune initiation process. 3. The activation and internalization of NF-k B can be induced by rBCG or/and PPS, which may consequently activate the signal transduction pathway in immune cells. 4. BCG can elevate the contents of IL-8 and TNF-a in cell medium. But PPS have no synergistic effects on increasing IL~8> IL-1P and TNF-a secretion levels to regulate immunity. It is all indicated that BCG could activate the signal transduction pathway: TLR4 — MyD88- IRAK- TRAF6- NF-kB- cytokines. |
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