The Study Of Inhibiting Effects Of BFGF Monoclonal Antibody Combined With Radiotherapy On B16 Melanoma In Mice

Objective:To investigate the synergistic effect of bFGF monoclonal antibody combined with radiotherapy on B16-transplanted melanoma and the synergistic mechanism.Methods:(1)Preparation of bFGFmAb producing from ascites:Hehybridomas cells secreting bFGF monoclonal antibodies, which had been cryopreservated by our lab,were recoveried and cultured, BALB/c mice were immunized by injecting incomplete Freund's adjuvant(IFA) intraperitoneally. on the 7th day after immunity, the 2×106 hehybridomas cells per mouse were injected intraperitoneally. On the 7 to 12th day after the injection, the ascites containing the bFGFmAb was produced and extracted. bFGFmAb was purified from the ascites through the Protein G-Sepharose affinity column, Indirect method ESLIA was used to detect the bFGFmAb titration before and aftter purification. The concentration of purified bFGFmAb stock solution was assayed by BCA standard solution. The bFGFmAb purity was determined by SDS-PAGE electrophoresis. The bFGFmAb solution was cryopreservated at -20℃for use.(2) Establishing model of C57BL/6 mice bearing melanoma and treating differently according groups:B16 melanoma cells were recoveried and cultured; the mouse model bearing melanoma were established through injection subcutaneously of 4×105B16 melanoma cells per mouse into the right-hind limb of C57BL/6 mice,32 mice model were randomly divided into four groups with 8 mice per group, respectively control group, radiaotherapy group, bFGFmAb treatment group, the bFGFmAb treatment combined with radiaotherapy group(combined treatment group). Radiaotherapy group were treated with 6MV, X-ray, conventional fractionated radiotherapy,5 times weekly,5Gy/day/time, eight times totally,40Gy total dose; bFGFmAb treatment group were injected subcutaneously bFGFmAb 900μg per mouse around tumour, once per 3 days,3 times totally; Combined treatment group were treated with bFGFmAb first, on the 9th day combined with the radiotherapy; Four groups were treated and observed for 20 days.(3) Detection of melanoma tumors:The transplanted melanoma tumors were measured the size changes every two days,20 days after treatment were collected, weighted and sent to pathologic- cal test in groups. TUNEL staining was used to detect apoptosis rate of B16 cell, immunohisto-chemical methods was used to detect positive expression rate of bFGF and VEGF; CD31 staining was used to detect microvessel density(MVD) in the B16 transplanted tumor tissue.Results:(1) 14.5ml ascites containing bFGFmAb was prepared, the bFGFmAb titration was 1:1024000,while the titration of purified bFGFmAb solution was 1:8192000. The result of SDS-PAGE electrophoresis revealed the high purfity of purified bFGFmAb solution.The Concen-tration of purified bFGFmAb solution was 9.593mg/ml, the total weight of bFGFmAb was 46.048mg.(2) The inhibiting rate of tumor growth of the combined treatment group was significant higher than those of radiotherapy group, bFGFmAb treatment group(48.76% VS 12.17%,30.49%, P<0.05). Radiotherapy sensitization enhancement ratio of the combined treatment group was 2.37 times than that of radiotherapy group.(3) TUNEL staining revealed apoptosis rate of the combined treatment group increased significantly compared with the bFGFmAb treatment group and radiotherapy group, which of control group was lowest.(4) Immunohistochemisty results:Compared with control group, expression level of intratumor bFGF and VEGF decreased significantly in radiotherapy group. In bFGFmAb treatment group, expression level of bFGF down-regulated significantly(P>0.05); expression level of Both bFGF and VEGF decreased significantly in combined group(P<0.05) than that in the other treatment groups. CD31 staining was used to show intratumor MVD, in bFGFmAb treatment group MVD was high, in Radiotherapy group lower, least in combined treatment group with high proportion of necrotic area; MVD in control group was highest and distributed closely and excessively.Conclusions:bFGFmAb treatment combined with radiotherapy on B16 transplanted melanoma in C57BL/6 mice was used to produce significant synergistic effects, the antitumor mechanism was that combined treatment decreased expression level of bFGF,VEGF, inhibited angiogenesis, promoted tumor cell apoptosis, improved the sensitivity of radiotherapy. This study generally provided an important experimental research on targeted therapy of melanoma.