The Effect And Mechanism Study Of Icariin In Regulating Proliferation And Differentiation Of Adipose-derived Stem Cells

Part I The effect of icariin on the proliferation and osteogenic differentiation of adipose-derived stem cellsObjective This study was to investigate the potential of icariin on the proliferation and osteogenic differentiation of ASCs and confirm the optimal concentration of icariin for proliferation and osteogenic differentiation of ASCsMethods 1. Adipose tissue was isolated from SD rats, and ASCs were isolated with Collagenase Type I digestion methods. 2. ASCs were cultured with different concentrations of icariin(0mol/L 10-8mol/L 10-7mol/L 10-6mol/L 10-5mol/L),the proliferation related gene and protein were investigated with RT-PCR, WESTERN BLOT and CCK8 methods 3. ASCs were cultured with different concentrations of icariin(0mol/L 10-8mol/L 10-7mol/L 10-6mol/L 10-5mol/L),the osteogenic ability was investigated with RT-PCR and alizarin red staining methods. Results 1. ASCs showed a homogenous morphology, most of which were fibroblast-like shape with many hands.2. Results of WESTERN BLOT, RT-PCR and CCK8 revealed that icariin had a dose-dependent effect on the proliferation of ASCs and the most appropriate concentration is 10-7M. 3. Results of RT-PCR revealed that icariin promoted the expression of osteogenic related genes and the effect was dose-dependent Conclusion Icariin had a dose-dependent effect on the proliferation and osteogenic differentiation of rat ASCsPart II Icariin influenced the expression and subcellular localization of YAP in ASCsObjective This study tried to investigate the molecular mechanism which regulates the proliferation and osteogenic differentiation of ASCsMethods 1. ASCs were cultured with icariin and YAP subcellular localization of ASCs was investigated. 2. ASCs were cultured with different concentrations of icariin(0mol/L 10-8mol/L 10-7mol/L 10-6mol/L 10-5mol/L),and the expression YAP and downstream genes were investigated. 3. ASCs were cultured with optimal concentration of icariin for different time courses, the phosphorylation of YAP and the expression of CTGF were investigated Results 1. Fluorescence microscope revealed that YAP expression was upregulated and mainly distributed in the nucleus when ASCs were cultured with icariin. YAP was also mainly distributed in the nucleus in the control group 2. Icariin had a dose-dependent effect on the YAP pathways of ASCs. The expression of YAP, TAZ and CTGF reached a peak when the concentration of icariin was 10-7mol/L. 3. Icariin promoted YAP dephosphorylation Conclusion icariin could promote the expression and dephosphorylation of YAP, YAP was localized in the nucleus and the expression of downstream genes were promoted.Part III Investigation on the effect of icariin induced 3D cultivated ASCs on the repair of calvarial defectsObjective To explore the effect of ASCs which were cultivated in bioglass and icariin in repairing the critical sized calvarial defectsMethods 1. Critical sized bone defects were constructed in rat calvaria 2. ASCs were cultured in bioglass material and then induced with icariin in vitro 3. The icariin-BG-ASCs scaffold was filled in the rat calvarial defect and observed the effect of repair. Results 1. Rat critical sized calvarial defect was a round defect with a diameter of 8 mm and it wound not self-repair 2. Bioglass scaffold was porous with many pores, ASCs grew well in the BG scaffold 3. The experiment in vivo demonstrated that icariin could promote the repair of rat critical sized calvarial defect Conclusion ASCs grew well in the bioglass scaffold, Icariin promoted the effect of 3D cultivated ASCs on the repair of the bone defect.