Cepharanthine Promotes Proliferation And Osteogenesis In Human Adipose-derived Stem Cells Via Wnt Signal Pathway

Bone disease has drawn much more attention in recent years.Stem cells has the capacity of self-renew and differentiation into multiple cell lines,which holds the great potential for treating bone disease in tissue engineering.They are regard as effective therapeutical approaches for clinical trials.Human Adipose-derived stem cells(ASCs)are sourced from human adipose tissues with the similar ability as other stem cells.In addition,unlike other stem cells,ASCs are much easier to extract and can be expanded in abundant amount in vitro.Because of the expensive price of stem cell factors,it has been an essential thing to obtain the complimentary.Small molecular from natural products has been shown significant bioactivities in skeletal repaired and regenerative therapies.However it has never been reported that the effect of Cepharanthine on self-renew and osteogenesis of ASCs.Objective: To investigate the action of Cepharanthine on proliferation.,differentiation to osteoblasts.And to elucidate the mechanism of osteogenesis.Methods:(1)To obtain the adipose tissue from lipoaspiration after surgery and isolated by enzymatic digestion with collagenase type I.ASCs will be purified after two passenges.(2)Identification the ASCs that has been extracted by Cell morphological and cell biological characters.Flow cytometry analysis is evaluated the antigen surface marker of ASCs.(3)Its effect on cytotoxicity was determined using MTT assay with concentration of Cepharanthine as 200 μM、100 μM、50 μM、25 μM、12.5 μM、6.25 μM、3.125 μM、1.5625μM.(4)Its effect on proliferation was determined using MTT assay and Real-Time PCR.(5)Osteoblast differentiation was evaluated by alkaline phosphatase activity and the m RNA and protein expression of multiple osteoblast markers.(6)The activation of Wnt/β-Catenin was determined by Real-time PCR,western blot and confocal microscope,respectively.Results:(1)Our results showed that the ASCs which isolation and purification were identified by Flow cytometry analysis.(2)Cells were displayed plastic-adherent ability after culturing overnight.Specific markers were characterized such as CD105+,CD73+,CD90+,CD45-,CD34-,CD14-and HLA-DR-.The pure plastic-adherent ASCs were obtained after being differentiated into multiple cell lines.(3)MTT assay was determined the cytotoxicity of ASCs treating with Cepharanthine.The results showed that concentration below 10 μM of Cepharanthine could be used in the experiments,while about 5 μM has shown the ability of proliferation.(4)Osteogenesis of ASCs could be promoted by Cepharanthine,especially at the concentration of 0.5μM and 0.25μM(5)CNTTBL1 and other osteogenic marker were up-regulated by the effect of Cepharanthine while the expression of protein was increased.Confocal microscope was used for translocation of β-Catenin.Conclusion: Overall these findings demonstrate that Cepharanthine promotes the proliferation and osteoblastic differentiation and mineralization in adipose-derived stem cells via Wnt/β-Catenin pathways.This could be significant contributions to stem cell-based tissue engineering research and regenerative medicine on bone disease.