Research On The Mechanism Of Immunne Startup By Injection With Bacillus Calmette-Guerin And Polyporud Polysaccharide

【Objective】: 1. To investigate the effect of injection with PPS and/or BCGon the expression of TLRs, adhesion molecules and costimulatory molecules onmouse Peritioneal macrophage. 2. To investigate the effect of injection withPPS and/or BCG on the activation of NF-k B in mice peritoneal macrophages. 3.To investigate the effect of injection with PPS and/or BCG on the inflammatoryfactors expression by mice peritoneal macrophages, spleen and peripheralblood. 4. To investigate the site of BCG interacted with T24 cells and effecton ultra-structure and the coordinated interaction with polyporuspolysaccharide (PPS)【Method】: The mice were divided into four groups(6 in a group), then sometreatment were given:①one mouse in control group underwent the subcutaneousinjection with salt 0.1ml.②one mouse in BCG group underwent the subcutaneousinjection with BCG 0.1ml.③one mouse in B+P group underwent the subcutaneousinjection with BCG 0.1ml, and intraperitondal injection with PPS 2mg/0.2ml.④one mouse in PPS group underwent intraperitondal injection with the PPS2mg/0.2ml. Two hours or 12h, 24h after treatment, 6 of mice in each group weresacrificed, and the murine peritoneal macrophages were collected.1. The immune mice peritoneal macrophages were labeled with direct fluorescentmethod. Thus, the effects of PPS,BCG on the expression of TLR2,TLR4 and CD14were studied at different time points, such as 2h,12h and 24h.2. The immune mice peritoneal macrophages were labeled with direct fluorescentmethod. Thus, the effects of PPS,BCG on the expression of CD11 and CD40 werestudied at different time points, such as 2h,12h and 24h. 3. The miceperitoneal macrophages were with in-direct fluorescent method. Then thechanges of expression level and transfer of nuclear transcription factor-kB, NF-kB were studied incytoplasm and karyon, which also need Laser ScanningConfocal Microscope technique. 4. IL-1β,IL-12 and IL-10 were detected in thesupernatants of mice peritoneal macrophages which were immunized by PPS andBCG with ELISA method, so is the determination of IL-2,IL-4 in spleensupernatant and the amount of TNF-αin peripheral blood. 5. The site of BCGacted in T24 cells and its effect on the ultra-structure of T24 cell and thecoordinated interaction with PPS were studied with transmission electronmicroscope and scanning electron microscope. 6. The PEMS 3.0 Statisticalsoftware was used for the compare of the experimental results.【Result】: 1. (1). TLR2 expression of mice peritoneal macrophages was higherthan control (P＜0.05) after adding PPS 12h, while start to fall down after24h. TLR2 expression of mice peritoneal macrophages after adding B and Ptogether 2h were higher than either adding PPS or BCG (P＜0.05), while startto fall down after 12h. (2). There was no obvious change of TLR4 expressionin mice peritoneal macrophages at 3 different time points, more, the amountthe expression were lower than control at the time of 12h and 24h (P＜0.05).TLR4 expression of mice peritoneal macrophages were higher after adding B andP together 2h than either adding PPS or BCG (P＜0.05), while start to fall downafter 12h. (3). There was no obvious change of CD14 expression in miceperitoneal macrophages at 3 different time points, more, the amount theexpression was lower than control (P＜0.05). CD14 expression of mice peritonealmacrophages after adding B and P together 2h were higher than either addingPPS or BCG (P＜0.05), while start to fall down after 12h.2. (1) CD11b expression of mice peritoneal macrophages were higher than control(P＜0.05) after injection with PPS 12h, while start to fell down after 24h.CD11b expression of mice peritoneal macrophages after injection with B andP together 2h and 12h were higher than either adding PPS or BCG (P＜0.05),while start to fall down next. (2) There was no obvious change of CD40expression on mice peritoneal macrophages at 3 different time points, more,the amount the expression were lower than control (P＜0.05). CD40 expressionof mice peritoneal macrophages after injection with B and P together 2h werehigher than with PPS.3. (1) the fluorescence intensities of NF-κB increased slowly within the threetime points. However, all of them were lower than control (P＜0.05); Thefluorescence intensities of NF-κB nuclear transcription factor of B+P group was higher than PPS group (P＜0.05) and increased obviously 12h, which wasStatistically different from control. (2) The fluorescence intensities of NF-κB in the plasm increased slowly within the three time points. However, allof them were lower than control (P＜0.05); The fluorescence intensities of NF-κB of B+P group was higher than PPS group (P＜0.05) at 2h and 12h, whichincreased obviously at the time of 12h. (3). The fluorescence intensities ofNF-κB in the plasm showed downtrend within the three time points and lowerthan control at the time of 24h (P＜0.05); The fluorescence intensities ofNF-κB of B+P group was higher than PPS group and control group (P＜0.05),which increased obviously at 24h,4. (1) the amount of IL-1βreleased by mice peritoneal macrophages of PPS groupwas higher than control (P＜0.05) after 2h interaction. The amount ofIL-1βrelease dby mice peritoneal macrophages of B+P group was lower than PPSgroup, which were Statistically consistent at the timepoints of 2h and 24h(P＜0.05). (2) No obvious changes of IL-4 contained in the supernatant ofmice spleen were found at three time points, more, the amount of IL-4 was lowerthan control but it does not make significant difference. The amount of IL-4contained in B+P group was very similar with that of PPS group, but thedifference was not statistically consistent. (3) Generally, no expression ofIL-12, IL-10 by mice peritoneal macrophages were detected, neither does IL-2in spleen nor TNF-αin peripheral blood.5. Distinct changes of cancer cell morphologs were detected, such as thesurface texture was seriously damaged, microvilli and cytoplasmic processesdisappeared.Based on the above research, we got some viewpoints. 1. The expression of TLRmolecular could not be induced by injection with PPS alone, while combinedwith BCG induces higher expression of TLRs. 2. Injection with PPS and BCG wouldinduce higher expression of CD11b on macrophage. 3. NF-κBp65 can be activatedand transfer towards nucleus from cytoplasm after injection with PPS and BCG24h. 4. During the initial period of injection with PPS plus BCG (24h), theinflammatory factors can not be induced to obviously release. 5. As BCG wasswallowed into cytoplasm of T24 cell, the structures of cell membranes andorgans were damaged and the damage of tumor cell can be stimulated moreseriously with the coordinated interaction with PPS.【Conclusion】: Totally, the expression of CD14,TLR2,TLR4 and adhesion molecule CD11b on macrophage can be induced in the primary period afterinjection with PPS and BCG, and NF-κBp65, TLR molecular and signal channelcan be activated, which may be one of the most important pathway triggeringthe human immune system and inducing the anti-tumor effect.