Mechanism Study Of Intratumoral Slow Release Drug Therapy Inhibiting The Mouse Tumor

Objective:Ideal method for tumor therapy is not only to keep antineoplastic agent certain density and time in tumor tissue, but also to reduce side effect. Common system chemotherapy is no way to reach effective drug density in tumor, and if increasing the dose, the side effect would raise. So many studies have contacted tumor with high antineoplastic through infused to entity tumor in order to raise the effect for tumor treatment. Based on the above, we use tumor tissue as the drug vector to form delayed release drug depot, and approach the therapy of delayed release in tumor after chemotherapeutics binding with DNP. Though increasing the effect which the drug acted in the tumor, strengthening killer ability to tumor cell and stimulating the immune response, we will achieve the view of tumor treating. Methods:The experiment animals mouse C57BL/6, half of them female and half of them male, all of them about 6 to 8 weeks old and weight about 18g to 22g, breed them in the asepsis environment. Melanoma cell lines B16 was cultivated in RPMI1640 medium with 10% FCS under 37°C, 5% CO2 environment in the incubator and was passage every 2 to 4 days.Choose melanoma cell lines B16 which was in the exponential phase of growth injected into the mouse. Then after 5 to 6 days when the tumor's volume was to fit experiment request, separate the mouse to several group and make the first therapy. (1) negative control group: injected NS into the tumor; (2) Ara-C control group: injected Ara-C into the tumor; (3) only delayed release therapy group: injected Ara-C + fluid delayed release into the tumor; (4) immunology delayed release therapy group: injected Ara-C + fluid delayed release + DNP into the tumor. After four days, take the same therapy and overview the tumor growth in each group. One week later, put the experiment animals to death by casting off cervical vertebrae and dislodge the tumor tissue and the speed to make the next experiment. The next experiment including: (1) measure the volume of the tumor; (2) stain the tumor exemplar by HE and specific fiber labeling; (3) immunohistochemistry detect for tumor tissue: CD4, CD8, ICAM-1; (4) detected the number of T cells by flow cytometry. Through therapies, we can measure gross tumor volume, detect the contents of the three fibers in the interstitial substances of the tumor and the infiltrations of T cells in the tumor and splenic organs. Results:(1) The effect of the therapy of delayed release toward the tumor of the mouse The experiment result showed the two delayed release therapy group both had repress function on the growth of the tumor and it suggest delayed release therapy can repress the tumor growth in mouse. In the two delayed release therapy groups, the immunology delayed release therapy group was better then the only delayed release therapy group.(2) The change on pathology in tumor tissueUnder the microscope, the negative control group: there were a few necrosis domains but a lot of blood vessels and the tumor tissue grow prosperity; the only delayed release therapy group and the immunology delayed release therapy group: there were a lot of necrosis domains, cells were dissolved and inside of the tumor there were a few leukomonocyte.(3) stain the tumor exemplar specific fiberComparable to the control after specific staining, in the two therapy group elastic fiber pachier and the colouration was darker, have more collagen fibers and have derangement and there were more and pachier reticular fibers in the tumor tissue.(4) ICAM-1 immunohistochemistry stain in tumor tissueStain positive signal is buffy in cellular membrane, the result show the number of ICAM-1 positive cells in tumor tissue of the two therapy group raised obviously. In the two therapy group, the immunology delayed release therapy group was better then the only delayed release therapy group.(5) CD4, CD8 immunohistochemistry stain in tumor tissue Stain positive signal is buffy in cellular membrane, the result show the number of CD4~+ or CD8~+ positive cells in tumor tissue of the two therapy group is bigger obviously than NS group. (6)Analysis the number of T cells in the speed by flow cytometryThe detection of FCM showed the efficiency of the speed leukomonocyte labeling CD4~+ and CD8~+ were more than the control group. All the experiment groups, CD4~+ or CD8~+ more than control groups. It suggest that delayed release therapy can change cell's immunology function.Data show that two slow-release therapies can kill tumor more effectually, stimulate the more productions of the fibers in the matrix of tumor and induce the stronger immune response than two control groups. Conclusion:1 The delayed release therapy in tumor can repress tumor's growth apparently, and cause many necrosises of tumor cellular.2 The delayed release therapy can inhibit the shift and diffuse of tumor cell though increasing intercellular adhesion molecule and fibres in tumor position.3 The delayed release therapy may improve immunologic response through increasing the number of CD4 and CD8.