Effect Of Overexpression Of M-CSF And RANKL Gene On Related Bone Remodeling Factors

Objective:To investigate respectively the effects of two important factors during osteoclast differentiation:macrophage colony-stimulating factor(M-CSF)and receptor activator of nuclear factor-κB ligand(RANKL)overexpression on osteoclast and osteoblast related factors in bone marrow mesenchymal stem cells(BMSCs).Methods:The whole bone marrow culture was used to culture rat bone marrow mesenchymal stem cells.The overexpression plasmid vector was constructed respectively to upregulate the expression of M-CSF gene and RANKL gene.The enhanced green fluorescent protein(EGFP)was used to determine the optimal transfection dose and the two plasmids were transfected respectively into rat bone marrow mesenchymal stem cells(BMSCs).Specific markers of osteoclast and osteoblast differentiation:M-CSF,RANKL,IL-1,IL-6,TNF-α,ALP,BMP2,Runx2 mRNA and protein expression levels were detected by reverse transcription-polymerase chain reaction and Western-blot.Results:(1)The positive rates of surface markers CD45,CD29 and CD34 of BMSCs were 3.8%,94%and 0.3%,respectively;(2)5μL/porous LTX~?is the best transfection dose;(3)The recombinant overexpression plasmid M-CSF and RANKL were all correctly inserted into the corresponding polyclonal regions;(4)The results of RT-PCR and Western-blot showed that M-CSF,RANKL,IL-1,TNF-αexpression increased and IL-6,ALP,BMP2,Runx2expression decreased in expression group of M-CSF gene(P<0.05);RANKL,M-CSF,TNF-α,ALP,Runx2 expression increased and IL-1,IL-6,BMP2 expression decreased in expression group of RANKL group(P<0.05).Conclusion:The ability of BMSCs to differentiate into osteoclasts was enhanced by transfection of M-CSF and RANKL overexpression plasmids into rat bone marrow mesenchymal stem cells.Both M-CSF and RANKL had effects on differentiation of osteoclast and also on osteoblast differentiation.