The Study Of Regulation To The Expression Of Toll-like Receptors On Peripheral Blood Immunocyte And Anti-tumour Function Of Recombinat Human IFN-α-2b-BCG

It is important assistance means that intravesical BCG administeration for preventing and curing urine bladder tumor. It was already confirmed that good result in the aspects of curing the superficial bladder tumor, preventing its relapsing and keeping malignant degree in tumor from increasing highly.But its anti-tumor mechanism is still not definite, thinking now much that it have something to do with BCG to the function of the immune system. Toll-like receptors that is a kind of trans-mmebrane protein discovered in the last few years rises important function in immune system, its corresponding ligand can activate trans-membrane signal transmittion iter mediated by Toll-like receptors,further activating nuclear transcription factor, regulating the expression of cytokines, creating IL-1,IL-6,IL-12, IFN-γ ,TNF- α and so on, finally activating immunity cell to kill tumor cell or cause directly the apoptosis of tumor cell.The current clinical medication is wild BCG, clinical proof support the obvious result in the aspects of preventing and cure urine bladder tumor, but it exsits some problems: 50% patients do not respond to BCG, obvious side effect, they seriously influence therapeutic result. 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, even suits the long-term course of treatment, is a kind of vaccine containing very big social worth.The topic applies some kinds of technique method including flow cytometer, immunity fluorescence and MTT to compare recombinat hIFN- α -2b-BCG with wild BCG on regulating the expression of Toll-like receptor 4 on human immunitycell surface and immunity cell's expressing Toll-like receptor4 anti-tumor effect to expound hlFN- a -2b-BCG anti-tumor mechanism, providing theories supports for clinical application, putting forward at the same time a new way of thinking for prevention and cure of urine bladder tumor - toll-like receptor enhancer and recombinat hIFN-a-2b-BCG will together be applied to treatment of bladder tumour.The research consists of three parts, the first part compares recombinat hlFN- a -2b-BCG with wild BCG on regulating the expression of Toll-like receptor 4 on human immunity cell surface.Extracting lymphoid cell from healthy human peripheral blood , the same amount of hlFN- a -2b-BCG and wild BCG to stimulate, lymphocyte as blank, flow cytometer measure the expression of toll-like receptor4 on lymphocyte surface. Result: both of them have positive accommodation on expression of toll-like receptor4, recombinat hlFN- a -2b-BCG regulates stronger ( P=0.0\3).The second part discusses the possible reason why recombinat hlFN- a -2b-BCG different from wild BCG Extraction lymphocyte from healthy human peripheral blood, the same amount of hlFN- a -2b-BCG supernatant, wild BCG supernatant and same equivalent interferon to stimulate, lymphocyte as blank, flow cytometer measure the expression of toll-like receptor 4 on lymphocyte surface in different groups.ResulthlFN- a -2b-BCG supernatant and same equivalent interferon have similar positive accommodation on expression of toll-like receptor 4(P=0.091);wild BCG supernatant has no effect on expresson of toll-like receptor 4 (P=0.702).Study from this part we can push to know the reason why hlFN- a -2b-BCG has stronger positive accommodation than wild BCG may be hlFN- a -2b-BCG can secrete IFN- a -2b to last, and IFN- a -2b itself can up-regulate the expression of toll-like receptor 4 resulting in the accommodation action of hlFN- a -2b-BCG better than that of wild BCG.The third part approachs anti-tumor effect of lymphocyte expressing th*4 in different groups.We place bladder tumor cell (5637 cell-line) in the logarithmic phase as target cell, lymphocyte excited by different agents as effect cell, different agents include recombinant IFN- a -2b-BCQ wild BCG, hlFN- a -2b-BCG supernatant, wild BCG supernatant, interferon.We set up nature release group and the maximum kill group with 10% Trionx-100, MTT method compare anti-tumor effect of diffetent groups.Result: lymphocyte of different sets all kill bladder tumor cell and killing degree different, from strong to weak in order: rBCG set, wBCG set, rBCG supernatant set, interferon set, wild BCG supernatant set, blank set;Among the total, rBCG set is 1.3 fold than wBCG set (P=0.003), rBCG supernatant set is 2.88 fold than wBCG supernatant set (P=0.000), rBCG supernatant set and interferon set difference only a few (/M).897).We inquires into anti-tumor mechanism of hlFN- a -2b-BCG, compares hlFN- a -2b-BCG with wild BCG on anti-tumor effect, announcing to public that trans-membrane signal transmittion iter mediated by Toll-like receptors may play an important role on rBCG anti-tumor function, it supplies rationale for further go deeper into researching rhIFN- a -2b-BCG anti-tumor mechanism and clinical application.