| Objective:To observe the inhibitory effect of Pianzihuang on osteosarcoma and pulmonary metastasis in an orthotopic mouse model, and to discuss mechanism of action.Methods:1.Establish pulmonary metastasis mouse model of osteosarcoma:thirty4weeks old female BALB/c nu/nu mice were used. All nude mice were randomly divided into two groups: saline group (n=5) and model group (n=25). All mice were anesthetized with4%chloralic hydras, The UMR106-01cells were aspirated into a lml syringe fitted with a25-gauge needle and100ul of cell suspension were injected (500,000cells/inoculum) in the right tibia of model group, the mice of saline group were injected with same dose NS. Mice of model group were executed by cervical dislocation at1week (n=5) and2(n=5),3(n=5),4(n=5),5(n=5) weeks after inoculation. The right hind limbs and the lungs were immediately dissected and fixed in4%paraformaldehyde in PBS at4℃for24h. The samples were then paraffin embedded for histological study.2.Thirty-two4-week old female BALB/c nu/nu mice were injected (500,000cells/inoculum)in the right tibia, lweek after inoculation, totally nude mice were randomly divide into four groups, including saline group(n=8),MTX group (n=8),Pianzihuang group (n=8), combined therapy group (n=8). To observe totally nude mice general appearance and measure tumor volume every week after inoculation and describe Growth curve of tumor and count the inhibition rate of orthotopic tumor. To count totally nude mice the number of lung surface tumors, the lungs were embedded in paraffin and sectioned at6μm. The sections were stained with Harris’haematoxylin and eosin (H&E) for histological analysis. A standard Immunohistochemistry (IHC) was applied to the tumor sections of all nude mice to detect Survivin, VEGF, MMP-9, and TIMP-1.Results:1.Establishment of an orthotopic mouse model of osteosarcoma:All animals survived and had tumor growth in tongue at1week after inoculation; the lungs appear tumor metastases at3weeks after inoculation, lung metastasis was found macroscopically in any of the mice at4weeks. The tumor of all nude mice showed the typical pathological characteristics of osteosarcoma.2.General condition of every group:the situ tumor volume of NS group, MTX group, Pianzihuang group, combined therapy group is6.3±0.37cm3.4.74±0.35cm3、5.08±0.55cm3、3.59±0.55cm3. the difference of these can not be neglected by comparing with the NS group (P<0.05), and the difference of these can not be neglected by comparing with each other group (P<0.05). The weight of nude mice:The weight of MTX group, Pianzihuang group, combined therapy group is24.00±0.33g、25.50±0.33g、27.20±0.21g, the difference of these can not be neglected by comparing with the saline group (P<0.05), and the difference of these can not be neglected by comparing with each other group (P<0.05).3.The inhibitory rate of osteosarcoma growth:The inhibitory rate of MTX group, Pianzihuang group, combined therapy group is24.8%,19.7%,43%, the difference of these can not be neglected by comparing with the saline group (P<0.05), and the difference of these can not be neglected by comparing with each other group (P<0.05).4.Pulmonary Metastases Formation:The number of surface lung metastases of MTX group, Pianzihuang group, combined therapy group is less than the saline group (P<0.05), The number of surface lung metastases of combined therapy group is44±1.6, the MTX group is50±1.4, the Pianzihuang group is54±1.6, the difference of these can not be neglected by comparing with each other group (P<0.05).5.The protein expression of tumor:The IOD of Survivin, VEGF, MMP-9, of MTX group, Pianzihuang group, combined therapy group is less than the saline group (P<0.05), the difference of these can not be neglected by comparing with each other group (P<0.05). The IOD of TIMP-1of MTX group, Pianzihuang group, combined therapy group is more than the saline group (P<0.05), the difference of these can not be neglected by comparing with each other group (P<0.05).Conclusion:1.Establishment of a spontaneous pulmonary metastasis orthotopic model of osteosarcoma:the characteristics of orthotopic model of osteosarcoma accord with the clinical, the method of building the model is reliable, the maneuverability is strong, spontaneous pulmonary metastases, this is a ideal model of osteosarcoma to research osteosarcoma.2.Pianzaihuang combined with MTX can inhibit the growth of orthotopic tumor.3.Pianzaihuang can inhibit lung metastasis of osteosarcoma, the combined-therapy group showed synergistic effects.4.The inhibition of Pianzaihuang on the growth of osteosarcoma and pulmonary metastases, to have relations of inhibiting the expression of Survivin, VEGF, MMP-9and enhancing the expression of TIMP-1. |
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