Effect Of MT01/PEN Complexes On The Expression Of OPG And RANKL In MG63 Cell

Oligodeoxynucleotide( ODN) MT01 is a kind of 27-mer single stranded DNA molecules with a design based on human mitochondrial DNA.It promotes the ossification of osteoblasts and stimulates the differentiation of bone marrow mesenchymal stem cells(BMSCs) into osteoblasts.In addition,MT01 inhibits orthodontic tooth movement in rat.As a kind of synthetic nonviral vectors, cationic polymers PEN,a derivative of polyethylenimines modified by N-isopropylacrylamide via Michael addition has advantages for their potential safety,low production cost,definite physicochemical properties and large gene loading capacity.Because natural ODNs with only a phosphodiester backbone are easily degraded by deoxyribonuclease(DNase) in serum, the low transfection efficiency inhibites the clinical application of ODNs.Chemical modification of Cp G ODN with a phosphorothioate backbone is an effective technique to improve the stability of ODNs and protect against degradation by DNase.However,the negative charge on the surface of ODNs still inhibites cellular uptake,moreover several severe side effects caused by the modification of DNA backbone have been reported.The appearance of PEN provides a new means to solve the delivery problem of MT01.PEN with positive charge and MT01 with negative charge can bond together through electrostatic interaction.In previous study,PEN show good binding affinity to MT01 and build PEN/MT01 delivery system with MT01 successfully.In the present study, we investigate the effect of complexes on the expression of OPG and RANKL in MG63 cell and intend to confirme that MT01 can enhance its effect on the promotion of ossification by establishing the delivery system with PEN.Objective:To synthesize MT01/PEN complexes and investigate the effect of complexes on the expression of OPG and RANKL in MG63 cell.Methods:MG63 cell were transfected by MT01/PEN complexes formed at three different mass ratio(1:2,1:4,1:6) of MT01 to PEN.backbone-modified MT01(MT01-s) and unmodified MT01 were positive control.PEN was negative control.Blank control wasdesigned. ELISA and Real time PCR were performed to estimate the amount of OPG and RANKL released into the culture media and in MG63 at 24、48、72h.Results:The MG63 responded to MT01/PEN complexes by significantly upregulating the OPG on protein and m RNA levels(P<0.05);The protein and m RNA levels of RANKL were lower in most of the groups with complexes,and the OPG/RANKL ratio were higher(P<0.05);MG63 were affected by the MT01/PEN complexes with different mass ratios,particularly when the ratio was 1:6.Conclusion:MT01 can enhance its effect on the promotion of ossification by establishing the delivery system with PEN;The MG63 responded to MT01/PEN complexes by upregulating the OPG/RANKL ratio,confirmed that MT01 can enhance its effect on the promotion of ossification on protein and gene level by establishing the delivery system with PEN,particularly when the MT01/PEN mass ratio was 1:6.