Effects On B7-CD28 Costimulatory Pathway With High Intensity Focused Ultrasound Treating Rat Osteosarcoma

Part one: A model of rat osteosarcoma in rat using orthotopic transplantation and studies on its biological characteristic Objective: To establish a model of rat osteosarcoma in SD rat and to study its biological characteristics, then providing a ture and reliable animal model for next HIFU therapy. Methods: Intact tumor pieces were inoculated into the right tibia of rat, the rats were killed in different time and observed the growth of tumor and metastasis. At same time the ALP in serum would be detected to analyse its change. Results: the tumor growth rate was 91.67%, from first week to second week the tumor grew out from medullary cavity, then it widely destroyed cortical bone and invaded soft tissue on third week but there is no pulmonary metastasis. pulmonary metastasis took place on the fourth week, on fifth week the cortical bone was destroyed more obvious than before and the quantity of tumor in pulmonary metastasis enhanced, and the rat finally died for respiratory failure on sixth or seventh week. The level of ALP began to step up from first week to second week, reached a peak on third week. It fell off from fourth week to sixth week, and approached a platform on seventh week which but was still higher than normal level of same time. Conclusion: The tumor growth rate is high, and the animal is not expensive. so the model can be widely used for science research. the model simulated the clinical process of human osteosarcoma, and could be a satisfied model on studying osteosarcoma. Part two: Effects on B7-CD28 costimulatory pathway with high intensity focused ultrasound treating rat osteosarcoma Objective: We would like to detect the expression of molecular B7 on surface of APC and the corresponding ligand CD28 on T cells in splenocyte as well as the IL-2 level in serum before and after treating osteosarcoma rat by HIFU, so as to investigate whether HIFU therapy could active T cells by affecting B7-CD28 costimulatory pathway. Ultimately the anti-tumor immunological reaction was induced. Methods: We distributed twenty five model rats into five groups(osteosarcoma group, immediate HIFU group, second day after HIFU therapy group, fifth day after HIFU therapy group, fifteenth day after HIFU therapy group) according to randomized block design. Besides fifteen normal rats were distributed into three groups(normal group, normal rat treated by HIFU group1, normal rat treated by HIFU group2 ). The therapy time of HIFU groups and normal rat treated by HIFU groups were respectively 250 s and 90 s. their once therapy time was 10 s. we at the right moment obtained the tumor in situ and lung preparation of osteosarcoma group and all HIFU groups to be checked by light microscope and electron microscope. Each group at the right moment was to detect the expression of CD4+CD28+,CD4+ ,CD80+ and CD86+ in splenocyte by FCM and the level of ALP and IL-2 in serum by ELISA. Results: The tumor in target area revealed coagulation necrosis after HIFU therapy. karyorrhexis And karyolysis can be seen in the necrosis area and eosino-staining enhanced. the osteoblast was necrotic and disappeared in the remanet of bone micro-grain whose structure was still existent. granulation tissue began to repair in the boundary between necrotic area and normal area on the fifth day after HIFU therapy. hyperplasia of lobus intermedius cells could be seen at the boundary of medullary cavity and it began to differentiate to cartilage cells. Hyperplasia of cells could be seenin the remanet of bone micro-grain. The result of FCM and ELISA revealed that comparing with normal group CD4+CD28+ , CD4+ expression decreased and CD80+,CD86+ expression and IL-2 level in osteosarcoma group slightly increased. In comparison with osteosarcoma group, CD4+CD28+ expression of HIFU group obviously increased, there was significant deviation on fifth day or fifteenth day(P<0.05);CD4+ expression of HIFU group obviously increased(P<0.05 or P<0.01);CD80+ expression of HIFU group increased, there was significant deviation on immediate time or fifth day(P<0.05);...