Establishment Of Osteosarcoma Resistance Cells Line Stably Transfected By Limk1 Gene And Effective Of Theproliferation And Invasion On The Cell

Objective:Establish a new osteosarcoma of multi-drug resistance cell lines MG63/VCR of stable express up-regulate 、 down-regulate and empty carrier of LIMK1 kinase actively, comparative analysis of cell proliferation and invasion and other biological characteristics, so as to lay a foundation for further study the role of LIMK1 in the regulation of human osteosarcoma resistance cell line. Methods:1. In MG63 cells adding vincristine, initial concentration of 500ng/ml, MG63 containing vincristine(500ng/ml) were cultured in stablely growth, the induction process will be consuming about six months. Finally, the drug-resistant osteosarcoma cells cang growth in contains 500 ng /ml concentration of VCR stablely, called the MG63/VCR. Then we will be use the containing vincristine final concentration of 100ng/ml culture, to maintain resistant strains of phenotype, computing MG63 and MG63/VCR cells’ IC50 and RI to common chemotherapy drugs.2. Establishment of MG63/VCR cell line of stably transfected by LIMK1 gene.The initial concentration of screening by G418 the determine screening of concentration. Using to obtain LIMK1 gene recombinant plasmid of express up-regulate、down-regulate and empty carrier of LIMK1 kinase actively.Transfect LIMK1 into human osteosarcoma of multi-drug resistance cell lines MG63/VCR by cationic lipofectin medium. Then, to observe the cell morphological changes by microscope and q RT-PCR assay to detection transfection efficiency. Immune deposition、Western blot assay verification the activity of kinase. After screened the G418, we establish osteosarcoma of multi-drug resistance cell lines stable upregulate LIMK1 kinase actively Named for CFP-LIMK1-WT, short for up-regulate group) and down-regulate LIMK1 kinase actively(Named for YFP-LIMK1-DA,short for downregulate group) and empty carrier LIMK1 kinase actively(Named for MG63/VCR-E, short VI for empty carrier group). According to kit for extraction and reverse transcription of LIMK1, designed and synthesized the primer of LIMK1 and GAPDH gene. The relative expression of m RNA was analyzed using software. After made sure the standard curve and melting curve, we compare LIMK1 gene expression level in the experimental group.3. Validation the biological characteristics of LIMK1 gene effect on the cell proliferation、invasion、cell cycle、cell adhesiveness in stable transfection cell group. Using CCK-8 methods to observe growth, and draw growth curve in three groups cell.Study the proliferation ability of each group. Using cell wound scratch assay methods to observe the invasion of effects in three groups cell. The flow cytometry assay test the cell cycle changes and ELISA methods tests the human FN for cell adhesiveness. Results:1. Osteosarcoma multidrug resistant cell MG63/VCR to vincristine(VCR) have higher tolerance and display moderate resistant cells. Similarly to paclitaxel, imidacloprid showed a higher degree of cross resistance, the resistance index respectively 377.67, 32.85, and the resistance index for 1.2 gemcitabine insensitive. Proved moderately resistant cells of osteosarcoma cell MG63/VCR resistance, and has characteristics of multi drug resistant.2. Through the initial concentration of screening by G418,we ultimately determine 400μg/ml of screening of concentration(P < 0.05).3. Through q RT-PCR assay, the stably transfected efficiency of up-regulate、downregulate and empty carrier of LIMK1 gene actively group is 70%. Result of q RT-PCR show that: compared to the normal group, LIMK1 expression level of up-regulate group and down-regulate were significantly increased(P < 0.05), but compared to the empty carrier group with MG63/VCR cell group have no significant difference(P > 0.05).4. Immune deposition and Western Blot assay results showed that: compared with the MG63/VCR cells group, up-regulated and down-regulated group expression were significantly increased(P < 0.05); The kinase activity of protein significantly enhanced in up-regulated and decreased in down-regulated group;While the protein expression in empty group with no significantly difference(P > 0.05).5. The results of CCK-8 assay show that: osteosarcoma of multi-drug resistance cell MG63/VCR’s IC50 is 450.4±36.4, RI is 390.63 of moderately resistant,at the same time,have multi-drug resistance ability. Seven day growth curve indicate that compared to the MG63/VCR cell group, proliferation ability of up-regulate group significantly increased(P < 0.05),ut the down-regulate group was decreased(P < 0.05),indicated the down-regulate expression of LIMK1 kinase actively in osteosarcoma cells has the effect of inhibiting the proliferation of tumor cells.6. Cell scratch assay results show that the up-regulate has a significant role in promoting migration; While the down-regulate is able to effectively reduce the ability of migration(P < 0.05).7. Cell cycle assay results showed that down-regulate group cell cycle was affected, the cells in G0 / G1 phase of the proportion of the increased to 55.637 ± 1.24, up-regulate group of cells in G0 / G1 phase cell ratio decreased to 42.535± 1.07, empty carrier group and MG-63/VCR group was only 46.183± 1.13% of and 46.564±1.23. There was no significantly difference; S phase cell decreased in MG63/VCR group was 39.593 ± 1.21, down-regulate group decreased to 31.908 ±1.33, up-regulate group increased to 42.671 ± 0.93(P < 0.05).8. The ability of cell adhesion assay results showed that compared with the control group, down-regulate group adhesion capability has been markedly enhanced, up-regulate adhesion ability significantly reduced,empty group had no significant difference. At the same time, the ELISA assay to detect human cell fibronectin FN expression. The results showed that: up-regulate group fibronectin FN expression increased, up-regulate FN expression decreased, further illustrates the differences of each group adhesion ability(P < 0.05). Conclusions:1. We successfully establish three types of osteosarcoma multi-drug resistance cell lines MG63/VCR of stable up-regulate、down-regulate、empty carrier of Ll MK1 kinase actively,offer the cell model in subsequently of gene regulation.2. LIMK1 kinase activity have an important role on regulation of MG63/VCR cell proliferation、migration、adhesion 、invasion and other biological characteristics.