Effects Of Coexpression Of KiSS-1 And P27^kip1 Gene On Adult Osteosarcoma MG-63 Cells In Vitro

[Objective]The aims of this study are to construct coexpressive vectors based on adenovirus (pIRES) carrying KiSS-1,P27^kip1,KiSS-1 and P27^kip1 respectively and detect their expressions in MG63 cell lines.To observe the proliferation ability of MG63 cells in vitro after gene transfection for gene therapy of osteosarcoma.[Methods]1. To construct vectors based on coexpressive adenovirus carrying KiSS-1,P27^kip1and both of them.2. To transduct KiSS-1,P27^kip1 and both of them into MG63 cell lines through lipofectamine2000.3. To detect the expression of KiSS-1,P27^kip1mRNA and metastin,P27^kip1 protein in the transfected cell lines by RT-PCR and western blot.4. To examine the effect on proliferation after the transfection by MTT test in vitro.5. To dectct the change of the cell cycle in every groups by flow cytometry after the transfection[Results]1. KiSS-1 and P27^kip1was extracted from the placenta and linked into the vector pIRES. The result was corrected by sequencing.2. The expression of KiSS-1 and P27^kip1between transfected and non-transfected cell lines was observed and considered to be a significant difference.3. The stable expressive cell line ,transfected with pIRES-KiSS-1,pIRES -P27^kip1,pIRES-KiSS-1-P27^kip1 by lipofectamine was picked out by neomycine. 4. The cell proliferation was observed to be slower down in the cell lines with pIRES-KiSS-1-P27^kip1 than the others transfected with single gene vector ;The cell cycle was hindered in G1/S stage.Compared with pIRES MG63 cells,the cell proliferation rate also was observed to be slower in pIRES-KiSS-1,pIRES -P27^kip1 MG63 cells respectively ,Their cell cycle was hinder in G1/S stage and G2/M stage respectively.[Conclusions]1. The way that transferred simultaneously with KiSS-1 and P27^kip1 into the MG63 cell lines by lipofectamine2000 is safe,high-efficiency and low toxicity.2. The pIRES-KiSS-1-P27^kip1 pIRES-KiSS-1,pIRES -P27^kip1 plasmid can express the target gene in high level3. The cell proliferation rate was observed to be much more slower down.in the double gene group4.KiSS-1 and P27^kip1 may exert a synergenic role in repressing the proliferation of the MG63.and hinder the transition of G1/S stage. It would provide some evidence for gene therapy in osteosarcoma.