The Study of Inhibiting Effects of bFGF Monoclonal Antibody Combined with Radiotherapy on B16 Melanoma in Mice
|Keywords||Melanoma bFGF monoclonal antibody Radiation therapy Vascular endothelial growth factor Tumor microvessel density Radiosensitization rate|
Objective: To investigate the bFGF monoclonal antibody (bFGFmAb) combined with radiotherapy in B16 melanoma bearing mice in vivo antitumor effect and synergy mechanisms. Methods: (1) preparation of ascites bFGFmAb: recovery training in this room has built the strains of of secretion bFGFmAb the hybridoma. Incomplete Freund's adjuvant (IFA) immune BALB / c mice, 7 days after immunization, mice injected with hybridoma cells 2 × 106/7 to 12 days ascites ascites. Protein G affinity chromatography purification bFGFmAb SDS-PAGE electrophoresis detection bFGFmAb purified the purity, BCA assay bFGFmAb protein stock solution concentration, indirect method ESLIA detection antibody titer, -20 ℃ cryopreservation spare. (2) the establishment of B16 melanoma animal models and group therapy: recovery cultured B16 melanoma cells, take the B16 cells, 4 × 105 / inoculated in C57BL / 6 mice right hind limb subcutaneous establish xenograft mouse model of B16. 32 mice were randomly divided into four groups, each group of eight, respectively, for the control group, radiotherapy group, bFGFmAb group, bFGFmAb combination group, bFGFmAb group peritumoral subcutaneous injection bFGFmAb 900μg / only 1/3 days 3 times; radiotherapy group using 6MV X-ray irradiation of the tumor, conventional fractionated radiotherapy 5 times / week, 5Gy / 8, a total dose of 40Gy; combined treatment group first using bFGFmAb treatment, nine days after the joint application of radiotherapy ; Experimental Therapeutics observed for 20 days. (3) detection of tumor: four groups of mice during the experiment, measuring tumor size every two days, calculated tumor volume; 20 days to remove the tumor, known as tumor inhibitory rate was calculated; tumor sent for pathological detection, TUNEL method B16 cells in the detection of tumor apoptosis rate, immunohistochemical SP detected tumors of bFGF, VEGF positive expression rate and tumor microvessel density (MVD). Results: (1) preparation of ascites bFGFmAb 14.5ml, potency 1:1024000. After purification the bFGFmAb titer 1:8192000, by SDS-PAGE electrophoresis Step antibody of high purity, the BCA assay antibody concentration of 9.593mg/ml, and the total amount of antibodies prepared 46.048mg. (2) bFGFmAb group, radiotherapy, tumor inhibition rate of the combined treatment group were 30.49%, 12.17%, 48.76%, and the tumor inhibition rate of the combined treatment group was significantly higher than the other groups (P lt; 0.05); compared with the radiotherapy group, the United Group radiosensitization rate of 2.37. (3) TUNEL staining B16 apoptosis rate in the control group (10.62 ± 1.73)%, bFGFmAb group (28.45 ± 5.47)% in the radiotherapy group (17.21 ± 2.86)%, the combined treatment group (58.56 ± 6.47) %, the combined treatment group significantly increased apoptosis compared the bFGFmAb group and radiotherapy group (P lt; 0.01). (4) The results of immunohistochemistry: Compared with the control group, the the radiotherapy group intratumoral of bFGF, VEGF expression levels decreased bFGF expression level decreased bFGFmAb group combined treatment group bFGF, VEGF expression levels were significantly reduced compared to the other two treatment groups (P lt; 0.01). CD31 staining shows the number of intratumoral microvessel high bFGFmAb group of vascular density, followed by radiotherapy group, at least to the combination therapy group, the proportion of necrotic area of ??microvessels in the control group distribution conglomerates. The Conclusion: bFGFmAb is combined with radiation therapy in B16 melanoma bearing mice produce significant synergies, and the mechanism is by reducing the tumor tissue bFGF, VEGF expression, inhibition of angiogenesis, promoting tumor cell apoptosis, enhanced sensitivity to radiation therapy, inhibitory role. This experiment is to provide experimental evidence for bFGF monoclonal antibody targeted therapy of melanoma.