Establishment Of Nude Mousemodels Of MG63 And MG63/ADR Osteosarcoma Cell Lines And The Significance Of Expression Of VEGF And NF-κB1

Osteosarcooma is the most common primary malignant bone tumor affecting children and adolescents. Despite obvious improvements of surgery and neoadjuvant chemotherapy in osteosarcoma therapy over the last 30 years, a considerable proportion of patients have a poor prognosis and a lower survival rate because of multidrug resistance. There is an urgent need for further study of drug resistance mechanisms of osteosarcoma. Our previous work using gene chip technology detected VEGF and NF-κB1 genes probably associated with drug resistance. In this study, we established animal models of MG63 and MG63/ADR Osteosarcoma Cell Lines firstly. Using immunohistochemistry and RT-PCR technology we furtherly verify the expression of VEGF and NF-κB1 gene in osteosarcoma MG63 parental cell lines and resistant cell lines, which provide experimental basis of exploring to the further resistance mechanisms of osteosarcoma and seeking more effective therapeutic target to osteosarcoma.Part 1 Establishment of Nude Mouse Models of MG63 and MG63/ADR Osteosarcoma Cell LinesOBJECTIVE: Construct MG63 and MG63/ADR osteosarcoma nude mouse model.METHODS: An adriamycin-resistant human osteosarcoma cell subline (MG63/ADR) was established in vitro using gradually increased concentration of adriamycin(ADM) in culture. BALB/C nude mice were divided into two groups: MG63 and MG63/ADR group. The animal model of osteosarcoma xenografts were constructed by subcutaneous inoculation of osteosarcoma MG63 and MG63/ADR cells respectively.RESULTS: Compared to MG63, the number of MG63/ADR cells in G0/G1-phase was significantly decreased (10.61%) while those in S-phase increased 15.06%. Furthermore, MG63/ADR was resistant to many anti-tumor agents, and its IC50 of ADM was 9.09 times higher than that of parent cell line MG63. Significant overepression of mdr, bcl-2, cyclinD1 and survivin protein were detected. After injected MG63 and MG63/ADR cells approximately one week, all nude mice formed tumor at the injected site.CONCLUSION: Animal model of MG63 and MG63/ADR was constructed successfully, providing an experimental model for further research of osteosarcoma drug resistance.Part 2 The significance of expression of VEGF and NF-κB1 in MG63 and MG63/ADR osteosarcoma nude mouse modelOBJECTIVE: To observe significance of gene expression differences of VEGF and NF-κB1 in MG63 and MG63/ADR osteosarcoma nude mouse model.METHODS: The animal model of osteosarcoma xenografts were constructed by subcutaneous inoculation of osteosarcoma MG63 and MG63/ADR cells respectively. Two group mice were killed at 6 weeks and gene expression differences of VEGF and NF-κB1 in the tumor formation of two groups were detected using immunohistochemistry and RT-PCR method.RESULTS: Both VEGF and NF-κB1 expression in MG63/ADR group was significantly higher than MG63 group(P<0.05).CONCLUSION: The upregulation of VEGF and NF-κB1 may be an important reason of drug resistance in osteosarcoma, providing an experimental basis for exploring the molecular mechanism of drug resistance in osteosarcoma and finding more effectively targets of osteosarcoma treatment.