Establishment Of Human Vincristine-induced Human Osteosarcoma Cell Line And Initial Mechanism Study Of LIMK1

Background:Osteosarcomas are the more frequent one than the other malignant bone tumors,often happened to the teenagers. Predilection sites lie in far end of femur, proximal tibia, the metaph of proximal humerus and so on. The rate of pulmonary metastasis is so high that the prog is rather bad.The current therapy is complex therapy that Preoperative and Postoperative chemotherapy combined with the operation.In recent,we found that multidrug resistance(MDR) to chemotherapeutic agents become a major barrier to the successful therapy of Osteosarcomas.And establishing a cell model of multidrug resistance(MDR) to chemotherapeutic agents is an good choice to study the multidrug resistance mechanism and to avoid the occurrence of MDR in vitro,which can make chemotherapy become effective.To establish a multidrug resistance cell model anti- vincristine of human osteosarcoma from the MG-63 cell line is very essential for testing its biological attributes,drug-resistance index,cell growth curve,cell shape and so on.Objective: By estbalishing a drug resistant cell line of humman osteosacroma MG63 to vincristine we can study its characterization and analysis the drug resistance correlation with LIMK1 .Methods: The humman Osteosarcoma MG63 cells were exposed to vincristine gradually increasing dose of the VCR 5 months or so to establish drug resistnace cell model MG63/VCR.IC50(50% inhibiting concentration) of drug resistant cells was measured by CCK-8 assay.Moprhology was obsevred by optical microscopy and Wright's-Giemsa staining.The ability ofcell proliferation was measured through growth curve testing by CCK-8 assay. In additon, RT-PCR and Western blot were used to detect the expression of the gene of MDR1, LIMK1 and the protein of p-Cofilin.Results: The resistance index of MG63/VCR is 7.007 measured by CCK-8 assay, and drug resistance belongs to midrange. Disordered structure of MG63/VCR was observed by optical microscopy and Wright's-Giemsa staining.We detected the remarkable increasing expression of MDR1 and LIMK1 from MG63/VCR of the gene level, we found the same tendency in these two gene, and the increasing expression of p-Cofilin of protein level which suggests LIMK1 has been activated. In addition, the cell transfection demonstrates hyper-expression LIMK1 increased the drug resistance to VCR of MG63/VCR , while the siRNA of LIMK1 decreased the drug resistance to VCR. above-mentioned results can prove that LIMK1 could regulate the drug resistance of MG63 cells.Conclusion: Drug resistant cell line (MG63/VCR) from humman osteosarcoma cell line MG63 was successfully estbalished to some extent.It will provide a ideal experimental model for our experiments to the further study of mechanism of drug resistance to vincristine,and provide scientific theoretical evidence for clinical chemotherapy.