[Experimental research in vitro of TK/GCV system for osteosarcoma MG-63 cell damage].

OBJECTIVE: To study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects. METHODS: Liposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group. RESULTS: The TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P < 0.05), and in the experimental group 2 that of the 1/10 and 1/7 of concentration ratio of mixed culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased. CONCLUSION: The experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

[Construction of lentivirus vector containing human LIM mineralization protein-1 (LMP-1) and its expression in rat bone mesenchymal stem cells].

OBJECTIVE: To construct a recombiant lentivirus vector of human LMP-1 and detect the expression of LMP-1 in infected rat bone mesenchymal stem cells. METHODS: LMP-1 gene from the cDNA library were extracted by Polymerase Chain Reaction (PCR). The LMP-1 genes were connected into lentiviral vectors pGC-FU-EGFP which was linearized by Age I enzyme to produce recombiant lentivirus vector called as pGC-FU-LMP-1-EGFP,then packaged by 293T cells. The virus supernant congtaining LV-LMP-1-EGFP was harvested, concentrated and titrated. The rat BMSCs were transfected with recombiant lentivirus LV-LMP-1-EGFP at the most appropriate MOI. The mRNA and protein expression of LMP-1 were detected by RT-PCR and Western blot. RESULTS: 1LV-LMP-1I-EGFP was recombined successfully and the titer reached 2x108TU/ml. 2The efficiency of infection was 93.5% ,which was get after LV-LMP-1-EGFP infected rat BMSCs at the most appropriate MOI=100. The expression of LMP-1 gene was confirmed by RT-PCR and Western blot. CONCLUSION: Lentivirus vector containing human LMP-1 gene is constructed successfully,which can transfected efficiently into rat BMSCs,and the infected rat BMSCs can effectively express LMP-1.

[Effects of recombinant gene lentivirus containing LIM mineralization protein-1 on proliferation effect and expression of bone marrow mesenchymal stem cells in rats].

OBJECTIVE: To explore method of recombinant gene lentivirus containing LIM mineralization protein-1 (LMP-1) in transfecting bone marrow mesenchymal stem cells (BMSC), and to observe the effect of gene LMP-1 on proliferation effect and expression of BMSC. METHODS: Six clean SD rats aged 4 weeks were selected, bone marrow mesenchymal stem cells were extracted under sterile conditions and cultured to the third generation, then divided into three groups:control group (the third generation of BMSC), lentiviral vector transfection group (PGC-FU-GFP and Polybrene were injected into the third generation of BMSC) and recombinant gene transfection group (PGC-FU-LMP-1-GFP and Polybrene transfection were injected into the third generation of BMSC). After 48 hours' transfection, fluorescent expression were detected under immuno-fluorescence microscopy; lentiviral transfection efficiency were detected by flow cytometry; effect of lentiviral transfection on BMSC were evaluated by MTT; gene expression of transfected cells were determined by Western Blot. RESULTS: 1) The third generation of BMSC was cultured successfully,and transfected with MOI:100. After 48 hours, green fluorescent expression were detected and transfection efficiency was 67% under immuno-fluorescence microscopy; 2) Compared to control group, there were no statistical differences between control group and other two groups; 3) Western blot teast showed that 72KDa specific band was observed in recombinant gene transfection group and its size was similar to LMP-1 fusion protein (50 kDa+28 kDa=78 kDa). CONCLUSION: There is no effect of recombinant gene lentivirus containing LIM on BMSC, and can effectively influence the expression of LMP-1.