Effect Of RANKL Gene Transfection On RANKL?OPG Expression In Bone Marrow Mesenchaymal Stem Cells

OBJECTIVE:To investigate the RANKL?OPG expression of bone marrow mesenchymal stem cells?BMSCs?transfected with receptor activator of NF-?B Ligand?RANKL?gene in vitro,explore the feasibility of enhancing the ability of BMSCs to induce osteoclastogenesis.And provide the basis for the mechanism of RANKL gene transfection promoting the degradation of bone substitute materials.METHODS:SD rat BMSCs were isolated and purified by the whole bone marrow adherence method in vitro,and identificated by morphology,osteogenetic and adipogenic differentiation potentiality at 3^rd passage;RecombinantretroviruspBABE-RANKL-puroconstructionand identification;The cultured passage 3 BMSCs were set up in control group?RANKL-transfected group and empty-vector group.And the expression level of RANKL of three groups were verified by realtime-PCR?RT-qPCR?and western blot;Puro was used to screening for stably transfected BMSCs wich obtained puro resistance cability from retrovirus vector.The cell proliferation viability and osteogenic potentiality were detected by MTT assay,alkaline phosphatase?ALP?staining,alizarin red staining and ALP activity assay,respectively.On days 3,7,14 and 21 after transfection,The expression of RANKL?OPG at mRNA and protein levels were detected by RT-qPCR and western blot respectively.Immunohistochemical staining and double-labeling immunofluorescence stain were used as auxiliary inspection to verified the expression of RANKL?OPG.RESULTS:1.Results of isolation,cultivation,purification and identification of BMSCs:BMSCs isolated from 1 week old SD rats were showed in similar morphology at 3^rd passage:spindle-shaped.And they showed the ability differentiating into osteogenic cells and adipocytes under osteogenic and adipogenic induction:alizarin red and oil red-Ostaining has an positive result.2.identification results of recombinant retrovirus pBABE-RANKL-puro transfected BMSCs:pBABE-RANKL-puro recombinant retrovirus vectors were identified by restriction enzyme digestion and sequencing assays.The relative expression of RANKL mRNA and protein was significantly increasing in RANKL-transfected group,72 hours after trasfection?P<0.01?;Empty-vector group shows no difference in RANKL mRNA and protein expression compared with control group?P>0.05?.3.Result of proliferation,osteogenic potentiality and RANKL/OPG expression detection after RANKL gene transfer:Growth curve revealed that proliferative activity showed no significant difference among the three groups during detected period?P>0.05?.After cultured in osteogenic induced medium for 14d,RANKL-transfected group and osteogenic-induced-control group shared the similar postive result in ALP staining;Whlie RANKL-transfected group showed a significant increment in ALP activity as compared with blank-control group?P<0.01?and osteogenic-induced-control group?P<0.05?.After osteogenic induced for 21d,mineralized nodules was observed both in RANKL-transfected group and osteogenic-induced-control group,mineralized nodules in RANKL-transfected group were smaller,but a litter bit more in quantity.The expression of RANKL mRNA in RANKL-transfected group significantly elevated at 3^rdd day after transfection?P<0.01?,and continue grown in a stable and persistent trend,lasted for at least 21d;OPG mRNA in RANKL-transfected group was transiently suppressed at 3^rdd day after transfection?P<0.05?,and soon recovered to the same leve as control group at7^rdd day?P>0.05?;There was no statistical difference between empty-vector group and control group in RANKL and OPG mRNA expressions?P>0.05?.As for the expression of RANKL protein,RANKL-transfected group show a significant increment compared with control and empty-vector group?P<0.05?,and raised up over time under observation;The expression of OPG protein in RANKL-transfected group was slightly decreased at 3^rdd day after transfection?P>0.05?,and then increased during the period of observation;The expression of RANKL?OPG protein in empty-vector group was similar to control group?P>0.05?.Immunohistochemistry and double-labeling immunofluorescence stain auxiliary inspection results showed that RANKL specific coloration was stronger in RANKL-transfected group than control and empty-vector group.The OPG specific coloration among three group had no significant difference?P>0.05?.The variation of RANKL/OPG ratio showed a correspondence tendency at gene and protein level.RANKL-transfected group had a high RANKL/OPG ratio at the 3^rd,7^th and 14^th day after transfection,and reached summit at the 7^thh day,then slowly declined subsequently;CONCLUSION:1.Recombinant retrovirus pBABE-RANKL-puro transfection can successfully increase RANKL expression at mRNA and protein level in BMSCs,which had no impact on OPG expression.2.RANKL gene transfer by p BABE-RANKL-puro doing no harm to proliferation viability and osteogenic-induced differentiation potentiality of BMSCs,BMSCs^RANKLANKL overexpressed RANKL still have osteogenic differentiation ability.