Antitumor Effect And Cooperative Therapy With HD-MTX Of Human Recombinant Monocyte Chemoattractant Protein-1 On Human Osteosarcoma Bearing Nude Mice

【Purpose】To observe the inhibition of recombinant human Monocyte Chemoattractant Protein-1(MCP-1) on implantation and growth of human osteosarcoma cells; To further study its cooperative inhibition of osteosarcoma with high dose of methotrexate(HD-MTX) and provide experimental evidence for clinical use of MCP-1. 【Methods】The levels of samples were investigated by the numbers by using molecular biological, immunological and pathological techniques: (1) pGEX IN/MCP-1 plasmid was transformated into E.coli TG-1 stain, more DNA in the plasmid was exacted and detected by enzyme digestion with EcorH â…  and BamHâ… . (2) IPTG inducing, recombinant MCP-1 was expressed, exacted, purified and detected with SDS-PAGE and Western Blot hybridization. (3) Implanting human osteosarcoma cells on nude mice to set up animal models on tumor. (4) Fifty nude mice were divided into 10 groups. For A_{1～4} groups,the 5 mice in each group were locally injected with doses of 1μg, 10μg, 100μg, 1mg MCP-1 at the same time when osteosarcoma cells were implanted in vivo. For B_{0～4} groups, 5 mice in each group, the injection were given two weeks later when there was the formation of the tumor mass at the dose of 0μg(0.2ml normal saline), 1μg, 10μg, 100μg, 1mg MCP-1 every other day. 5 mice in Group C were injected at dose of 0.2 ml NS as control. Clinical and pathological indexes were detected. (5) Varied doses of MTX were added into the culture medium of osteosarcoma cells, RNA of equal cells were exacted and analysed by electrophoresis. (6) 25 nude mice were divided into 5 groups: tumor-bearing chemotherapy group, tumor-bearing immune therapy group, tumor-bearing controlgroup, tumor-bearing combination therapy group, normal control group. Clinical, pathological and immunological indexes were detected. 【Results】(1) 240bp DNA segment was obtained when pGEX IN/ MCP-1 plasmid was enzyme digested. (2) 34 KD Recombined protein obtained had cross-reaction with anti-huMCP-1 monocolonal antibody. (3) Implantation of osteosarcoma cells were completely prevented among mice of Group A_{2～4},tumor inhibiting effect even in Group A₁ and the tumor inhibition was 69.69%. AFP values in mice of Group A_{1～4} were much lower than those of Group B0(P< 0.01). MCP-1 also expressed tumor inhibition on formatted osteosarcoma, the tumor inhibition rate of Group B₃ reached 84.32%. Pathological examination showed no signal damage in vital organs of the mice in the treated groups. (4) HD-MTX obviously inhabited the growth of osteosarcoma, but the tumors could resume growing in a period. In tumor-bearing combination therapy group, MCP-1 significantly tone up the tumor inhibition of HD-MTX and the regrowth of the tumors were retarded. 【Conclusion】(1) The reconstructed pGEX IN/MCP-1 plasmid contains gene of human MCP-1, it can express recombinant protein with biological activity of human MCP-1. (2) MCP-1 possess significant effect in the prevention of osteosarcoma cells implantation and marked tumor inhibiting . Locally used, MCP-1 would play an important role in clinical therapy of osteosarcoma. Especially it could inhibit the implantation of tumor cells within operation field. (3) MCP-1 possess cooperative inhibition of osteosarcoma with HD-MTX.