| Objective: To observe the changes of angiogenesis before and after excision of primary tumor on the nude mice bearing osteosarcoma, and to study the effects on pulmonary metastasis, providing the experimental basis for the mechanism of concomitant tumor resistance (CTR).Methods: The models of nude mice bearing osteosarcoma were constructed by cell suspension. The left anterior flank of each nude mouse was subcutaneously injected by 0.2ml osteosarcoma cell suspension (2.0×10~6 cells). All the subjects were grouped after 2 weeks. The osteosarcoma formed in the left anterior flank was regarded as the primary tumor. 1. Effects of primary tumor excision on the angiogenesis in nude mice.①The changes of VEGF and ES before and after excision of primary tumor. The normal group (20 mice), the control group (20 mice) and the test group (20 mice) were involved. Each 5 mice from 3 groups were killed and blood collected on the 7th, 14th and 21st day after the tumor was developed. The rest 5 test mice were performed the excision of left anterior flank (primary tumor excision) on the 21st day while the 5 control ones were performed the excision of right anterior flank, leaving the normal ones untreated. All the 15 mice were killed and blood collected one week later. ELISA was used to detect the serum concentration of ES and VEGF.②The changes of hemoglobin (HB) in the Matrigel before and after primary tumor excision. The normal group (20 mice), the control group (20 mice) and the test group (20 mice) were involved. Each 5 mice from 3 groups were subcutaneously injected 0.5ml Matrigel gel (which contained 200ng bFGF and 50u Heparin) on the 7th, 14th and 21st day, respectively, of the tumor formation and the gel was taken out one week right after the injection. The rest of the control and test group were treated the same operation as the above on the 21st day and all the mice including normal, control and test groups were subcutaneously injected gel at the same time. All the 15 mice were killed and gel taken out one week later. HiCN was used to detect the hemoglobin (HB) in the gel. 2. Effects on osteosarcoma with pulmonary metastasis. The normal group (20 mice), the control group (20 mice) and the test group (20 mice) were involved. The test mice were excised left anterior flank 2 weeks after the tumor injection while the control ones excised right anterior flank, leaving the normal ones untreated. Two weeks later, all the nude mice were dissected and analyzed whether the pulmonary metastasis existed or not. The counting of pulmonary metastasis nodus and the grading were carried out if the result was positive.Results: 1. No significant differences of serum ES and VEGF was obtained on the 7th, 14th or 21st day in both the normal and the control group. Nor was the concentration of HB in the Matrigel gel pro- and post-operation. The serum ES and VEGF obviously decreased in the primary tumor excision group compared to the normal and the control one (ES: 40.77±5.41ng/ml vs. 123.18±5.94ng/ml, 128.06±4.52ng/ml, P<0.01 ; VEGF: 71.43±9.15pg/ml vs. 115.81±4.38pg/ml, 111.68±12.26pg/ml, P<0.01); The HB concentration of Matrigel gel obviously increased in the primary tumor excision group compared to the normal and the control one (36.55±2.35g/L vs. 16.84±1.15g/L, 16.29±1.10g/L, P<0.01). 2. The incidence rate of pulmonary metastasis in the primary tumor excision group was obviously higher than that in the un-excision one (80% vs. 40%, 35%, P<0.05).Conclusion: 1. After the primary tumor of nude mice bearing osteosarcoma being removed, both ES and VEGF obviously decreased. However, since the reducing extent of ES was much greater than that of VEGF, it enhanced the relative ratio of VEGF/ES, which stimulated the systematic angiogenesis, resulting in the accelerated formation of the new vessels of tumor. 2. The speed of pulmonary metastasis from osteosarcoma was evidently accelerated after the primary tumor of nude mice bearing osteosarcoma was removed.
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