The Experimental Study For Superficial Bladder Cancer By HIL7-SA And MIL21-SA Fusion Protein Jointly Anchored The Treatment

BACKGROUND Worldwide, bladder cancer ranked men the most common solid tumor in the fourth and women ranked seventh, newly diagnosed patients with bladder cancer each year more than350,000. In China, bladder cancer is still the most common malignant tumor of the urinary system, urinary system tumors, the morbidity and mortality ranks first. In recent years, the incidence of bladder cancer in some cities in China showed a trend of steadily. Domestic large cities such as Beijing, Shanghai, Tianjin, bladder cancer incidence rates have ranked sixth place in male common malignant tumors, while the mortality rate ranked seventh. More than90percent of bladder cancer in patients with transitional cell carcinoma, Of which about75%for superficial bladder cancer. The main treatment is transurethral bladder tumor resection,2/3cases will recurrence and tumor stage in5years, so the postoperative routine supplemented by intravesical instillation therapy to prevent recurrence. Drug perfusion use of chemical drugs MMC, ADM. intravesical instillation of chemotherapy in the postoperative treatment of superficial bladder cancer has played an important role, but because in some tumor chemo therapy resistance and is not sensitive to chemotherapeutic drugs and to explore new treatments for this type of tumor, is the focus of the study. Biological treatment through with the role of biological agents to mobilize the body’s defense mechanism to regulate the body’s biological response, thus inhibiting or preventing tumor growth to become the fourth therapy. The biological immune agents BCG in clinical studies show that there are still30%to40%No Answer, in the effective treatment of cases, the side effects and further restricted its broader application. Therefore, to find other immunosuppressive agents recurrence of the tumor, has become the focus of attention in the prevention of tumor recurrence. In recent years a large number of clinical studies have shown that IFN-a, IFN-y, GM-CSF, TNF-a, IL-2for bladder instillation for the treatment and achieved certain good results. However, these cytokines in the body, often due to short half-life, lesions around the effective concentration is difficult to time to maintain, while reducing its anti-tumor effect.The Institute used a new protein anchoring technology, the main principle:take full advantage of the amino easy biotinylation of cell surface proteins and biotin and streptavidin biotin efficient and powerful almost irreversible combination of these two characteristics. This protein anchoring technology platform, rapid surface modification in superficial bladder cancer, immune-stimulating factor rapidly anchored on the cell surface, and make these cytokines is still anchored to maintain its biological activity, slow down the speed of their metabolism, improve local tumor tissue cytokine concentrations, to stimulate the body’s anti-tumor immune response.Interleukin7(IL-7) found in1988that bone marrow stromal cells, target cells, mainly lymphocytes, both to promote growth and activity of B progenitor cells, thymocytes and peripheral mature T cells, the regulation of lymphocyte andmacrophage differentiation, proliferation, activation. The main biological functions, including to promote the proliferation of pre-B cells to stimulate the activated T cell proliferation and regulate the thymus DN cell differentiation, induction of macrophage maturation, promote CTL and LAK proliferation, differentiation, and enhance their killing activity. After bone marrow transplantation, IL-7and stem cell factor (stemcellfactor SCF) combination therapy and found that IL-7can accelerate the recovery of bone marrow-derived lymphoid progenitor cell regeneration and immune function against a variety of cellular and molecular immune suppression network to improvethe immune capacity. At the same time, the number of studies have confirmed that IL-7with anti-tumor immunotherapy.Interleukin21(IL-21) was discovered in2000, belongs to the IL-2superfamily, mainly produced by activated CD4+T cells secrete to regulate B cell proliferation and differentiation of T cells, NK cells and dendritic cells. The main biological functions, including the synergistic stimulation to promote T cell proliferation and differentiation of anti-CD3monoclonal antibody to stimulate NK cell proliferation and differentiation by enhancing the response of IgG antibodies inhibit IgE synthesis and regulating the normal humoral immunity, through the adjustment of CD8+T cell and NK cell activity, removal of the tumor cells. Studies have reported that IL-21in the process of anti-tumor, increasing the number of tumor invasion of CD8+T cells, increase the number of tumor-specific CD8+T cells, protecting the individual to undergo the same kind of tumor attack.In this study, hIL7-SA mIL21--SA dual function fusion protein jointly anchored mice treated with superficial bladder cancer, and explore the efficacy of the treatment and prevention of superficial bladder cancer, especially IL-7and IL-21jointly anchor a synergistic effect. In this study, divided into two parts. Chapter1expression, purification, refolding and identification of hIL7-SAand mIL21-SA Bifunctional fusion protein1.1OBJECTIVEThe preparation hIL7-SA and mIL21-SA bifunctional fusion protein, study its biological activity.1.2METHODS1) The original expression plasmid hIL7-SA-pET24a transforming into E. coli BL21(DE3) competent cells. Filter out the best induction conditions (expression, optimum temperature, etc.). Expansion of a large number of bacteria, harvested, ultrasound sonicated extract of inclusion bodies, Ni-NTA separation and purification, the gradient method refolding. The same principle, preparation mIL21-SA fusion protein.2) MTT were used to detect hIL7-SA, mIL21-SA fusion protein on the proliferation of murine thymocytes. Flow cytometry analysis hIL7-SA, mIL21-SA fusion protein on biotinylated MB49cells anchored to the modified rate and anchored modification rate.1.3RESULTS1) Get hIL7-SA dual function fusion protein optimum inducing conditions:37℃induced4h. hIL7-SA recombinant protein in E. coli to achieve a highly efficient expression of target protein accounting for about15%of bacterial proteins. Determine the expression of fusion protein in inclusion bodies, the purpose protein was more than90%. The refolded fusion protein was mainly in the form of polymer. Western blot results confirmed that the monomers and polymers of the refolded hIL7-SA can bind with the hIL7-SA antibody binding, and can occur the color reaction.2) Get mIL21-SA dual function fusion protein optimum inducing conditions:37℃induced4h. mIL21-SA recombinant protein in E. coli to achieve a highly efficient expression of target protein accounting for about30%of bacterial proteins. Determine the expression of fusion protein in inclusion bodies, the purpose protein was more than95%. The refolded fusion protein was mainly in the form of polymer. Western blot results confirmed that the monomers and polymers of the refolded mIL21-SA can bind with the mIL21-SA antibody binding, and can occur the color reaction.3) hIL7-SA fusion protein can efficiently anchored on the surface of tumor cells after biotinylation, the anchor was99.10%. hIL7-SA fusion protein can promote the proliferation of murine thymocytes, and a dose-dependent relationship, hIL7-SA fusion protein activity is1×106U/mg.4) mIL21-SA fusion protein can efficiently anchored on the surface of tumor cells after biotinylation, the anchor was98.74%. mIL21-SA fusion protein can promote the proliferation of murine thymocytes, and a dose-dependent relationship, mIL21-SA fusion protein activity is1×104U/mg.5) hIL7-SA and mIL21-SA fusion protein can anchor on the surface of tumor cells after biotinylation simultaneously, the anchor was97.69%1.4CONCLUSION1) The hIL7-SA fusion protein we get has a bifunctional activity, which not only has the cativity of IL7but also can combine with the biotin efficiently and powerfully.2) The mIL21-SA fusion protein we get has a bifunctional activity, which not only has the cativity of mIL21but also can combine with the biotin efficiently and powerfully.Chapter2the experimental study of the treatment of bladder cancer in mice use hIL7-SA and mIL21-SA dual function fusion protein jointly anchored.2.1OBJECTIVETo evaluate the anti-tumor effect of hIL7-SA and mIL21-SA in the mouse MB49bladder cancer model, by immobilization of the fusion protein on the mucosal surface.2.2METHODS1) Take females of C57BL/6mice were125, were randomly divided into5groups (n=25), four groups of which the establishment of the MB49mouse bladder cancer model, namely, PBS negative control group, hIL7-SA anchored treatment group, mIL21-SA anchored treatment group, hIL7-SA and mIL21-SA joint anchoring treatment group. The last group in the control group for the secondary attack. The second attack in the control group, normal feeding60days after the joint anchoring treatment group, hIL7-SA and mIL21--SA accept the MB49cell bladder instillation.2) PBS negative control group, hIL7-SA anchored treatment group, mIL21-SA anchored treatment group, hIL7-SA and mIL21-SA joint anchoring treatment group on the day after the perfusion MB49bladder cancer cells, respectively, to give the PBS hIL7-SA mIL21-SA, hIL7-SA mIL21-SA bladder infusion once every three days, six times in a row.3) Modeling after60days, hIL7-SA mIL21--SA joint anchoring After treatment, the survival of the mice and the second attack in the control group again intravesical instillation of MB49cells, observed in mouse tumor and survival time.4) After19days Treatment,100ul of each group were randomly selected five mice, and100ul mix of whole blood via the tail vein added to the flow cytometry tube. Content of CD4+T and CD8+T cells by flow cytometry.5) After60days of Modeling, whichever is the hIL7-SA anchoring treatment group, mIL21-SA anchoring treatment group, hIL7-SA mIL21--SA joint anchoring treatment group, the normal mouse spleen, as effector cells for tumor-specific destruction experiments detection, calculation of the CTL6)7days after the last intravesical instillation, each randomly selected a mouse, removal of the bladder, the production of bladder cancer tissue sections. Detection of CD4+T and CD8+T lymphocytes secrete.2.3RESULTS1) PBS control group in the modeling of the first8to10days can reach the bladder small mass, gross hematuria in15to16days, weight loss diet to reduce the19days the mice began to appear of death; hIL7-SA anchored treatment group45days, mIL21-SA anchored treatment group44days of death, hIL7-SA and mIL21-SA, anchored to the treatment group died50days.2) Observation to modeling after60days, the PBS control group all died; hIL7-SA anchored treatment group of nine mice survived mIL21-SA anchored treatment group,10mice survived, hIL7-SA and mIL21-SA joint anchoring treatment group,13mice survived. PBS group, survival time and the other three groups compared to the difference was statistically significant (P<0.05), But hIhI7-SA anchored treatment group, mIL21-SA anchored treatment group, hIL7-SA mIL21-SA anchored between treatment groups was no significant difference (P>0.05). Again the hIL7-SA and the mIL21-SA joint anchoring treatment group survival of13mice and13normal untreated mice, the perfusion of bladder cancer MB49cells after60days, hIL7-SA and mIL21-SA joint anchoring treatment group still8mice survived, compared with the control group difference was statistically significant (P<0.05).3) Flow cytometry was used to detect peripheral blood of CD4+, CD8+cell levels, IL7-SA anchored treatment group, mIL21-SA, anchor of treatment group, hIL7-SA mIL21-SA joint anchoring treatment group was significantly higher than the PBS group. Note mIL2, hIL7can promote immune function. hIL7-SA and the mIL21-SA anchored treatment group than hIL7-SA, mIL21-SA alone anchored treatment group. 4) Will hIL7-SA, mIL21-SA anchored treatment group, the hIL7-SA and mIL21-SA jointly anchored treatment group, the normal mouse spleen cells stimulated with MB49cells inactivated, MB49are killing effect. The results showed that normal mice in the effective target than the killing rate1:1,25:1,505%7%8%; hIL7-SA anchored treatment group,10%,21%,39%; mIL21-SA anchored treatment group,10%,24%,38%; hL7-SA mIL21--SA joint anchoring treatment group,12%,27%,42%.5) Using immunohistochemical detection of bladder tumors in mice of CD4+and CD8+lymphocytes secret. In the PBS group tumor of CD4+, CD8+lymphocytes rarely, while the opposite hIL7-SA anchored treatment group, mIL21-SA anchored treatment group, hIL7-SA and mIL21--SA joint anchoring treatment group can obviously detect more number of CD4+and CD8+lymphocytes. In particular, the hIL7-SA and mIL21--SA joint anchoring treatment group.2.4CONCLUSIONThe hIL7-SA and mIL21-SA anchoring the treatment of bladder cancer, can effectively inhibit the growth of superficial bladder cancer, prolong the survival time of mice, the anti tumor effects, and can induce homologous tumor-specific immune response. But hIL7-SA and mIL21-SA joint anchoring the treatment effect is not significantly better than hIL7-SA or mIL21-SA, a separate anchor treatment.