Effects Of Recombinant HIFN-α-2B-BCG On The Regulation Of The TLR4Signaling Pathway In Hpbmc And Its Mechanism In Cancer Immunotherapy

It is important assistance means that intravesical BCG administeration for preventing and curing urine bladder tumor.But due to local and systemic complications of bladder makes the application of intravesical BCG therapy subject to certain limit. Recombinat human IFN-α-2b-BCG that successfully manufactured by ourselves is a new germ bacterial vaccine,comparison with wild BCG, in addition to reserving basic characteristics of wild BCG, its advantage consist in keeping on secreting IFN-α-2b, BCG and IFN-α-2b simultaneous working can produce the best immunity effect, its anti-tumor effect is more obviously, side effect less takes place, more suitable for patients with long-term perfusion.The anti-tumor mechanism of BCG is currently considered more related to the role of the immune system. Toll-like receptor which is an important immune system components by identifying and the appropriate combination of pathogen associated molecular pattern start signal transduction pathway, induceing the expression of certain immune effector molecules and mediating TLRs tumor immunotherapy by signaling pathways.Objective:The previous studies of our laboratory have confirmed that recombinant human IFN-a-2b-BCG anti-tumor biological effect is more prominent than wild BCG. And we also preliminary investigation on the expression and significance of TLR4in the anti-tumor effects of BCG induced hPBMC. To further study of recombinant human IFN-α-2b-BCG anti-tumor mechanisms, we conducted the following two studies:The first part mainly discussed the dose-effect relationship of recombinant human IFN-α-2b-BCG induced hPBMC secreting hTNF-α, hIL-12, hIFN-γ. In the second part we applied functional-specific TLR4blocking antibody to study the regulation of TLR4signaling pathway in recombinant human IFN-a-2b-BCG, wild BCG and interferon induced hPBMC and the effects on the expression of TNF-a, IL-12, IFN-y.Experimental methods:Part Ⅰ:We applied recombinant human IFN-a-2b-BCG to stimulate different cell concentrations of, and analysed the expression of TNF-α, IL-12, IFN-γ by ELISA over time; After that we use different concentrations of rBCG to stimulate hPBMC to analyse dose-effect relationship; experimental blank control group (hPBMC+PBS), experimental group hPBMC+rBCG.Part II:Application of TLR4functional blocking antibodies to intervene the TLR4signaling pathway of hPBMC, and then we apply recombinant human IFN-a-2b-BCG, wild BCG, interferon, and PBS to stimulate the hPBMC of blocking group and non blocking group, comparing of the changes of TNF-α, IL-12, IFN-γ between the blocking group and the non blocking group by ELISA, and using RT-PCR to detect the changes of NFκB, MyD88which are the key regulator in TLR4signaling pathway; Experimental blank control group (hPBMC+PBS), experimental group hPBMC+rBCG; hPBMC+wBCG; hPBMC+hIFN-a-2b. Results:Part Ⅰ:When the concentration of hPBMC is less than1.6×106/ml rBCG (final concentration0.01OD) does not enhance the expression of hTNF-α、hIL-12、 hIFN-γ; when the concentration of hPBMC is more than1.6×106/ml rBCG (final concentration0.01OD) can effectively increase the expression of hIFN-γ、hTNF-α、 hIL-12, and with the increase of hPBMC cells concentration, the effect of promoting is strengthening. Different concentrations of rBCG effectively stimulate the hPBMC and each concentration group compare with control group has significant differences (p<0.05). The expression of hTNF-α, hIL-12, hIFN-γ changed significantly after24hours due to the change of rBCG concentration,which in accordance with dose effect relationship. Different cytokine has its optimal inducing concentrations,according to this experiment the best concentrations of rBCG induced hPBMC to express hTNF-α, hIL-12, hIFN-γ were0.02OD,0.04OD and0.0025OD after24hours.Part Ⅱ:Recombinant human IFN-a-2b-BCG, wild BCG, interferon, and PBS respectively stimulating hPBMC after3hours,we ascertained the RNA expression of NFκB, MyD88was significantly higher than6h and12h,which prompted the RNA expression peak of NFκB, MyD88at6h ago. After TLR4blocking3hours, the RNA expression of NFκB, MyD88was significantly inhibited, but there was no significant difference between TLR4blocking groups and non blocking groups in the RNA expression of NFκB, MyD88at6h and12h. The comparison of NFκB, MyD88expression after stimulating3hours:rBCG group> wBCG group> IFN group> PBS group. After respectively stimulating with rBCG、wBCG、IFN、PBS, The expression of hIFN-γ, hTNF-α, hIL-12in TLR4blocking groups were significantly inhibited than in non blocking groups, at6h,12h,48h and72h(p<0.05). rBCG can significantly increase the expression of hTNF-a, and the role is better than wBCG, IFN-a-2b and the control group (p<0.05); The role of rBCG upregulating hlFN-y was no significant difference with wBCG, but both of them caught significantly better than IFN-a-2b and the control group (all p<0.05). rBCG, wBCG and IFN can significantly enhance the NFκB, MyD88expression of hPBMC,and which were significantly inhibited by the TLR4antibody, this change in accord with the expression of hIFN-γ, hTNF-α, hIL-12. Conclusion:There is dose-effect relationship in the role of Recombinant human IFN-a-2b-BCG inducing hPBMC to secrete hTNF-α, hIL-12, hlFN-γ; TLR4signaling pathway plays an important part in recombinant hIFN-a-2b-BCG mediated immunotherapy, and the results suggest rBCG regulate the secretion of Thl cytokines through the TLR4-MyD88-NFκB signaling pathway.