| Objective: Through the intervention of serum containing musk bone marrow mesenchymal stem cells(Bone mesenchymal stem cells,BMSCs),study Ing from the perspective of the signaling pathways,To explore the correlation between signaling pathway and stem cell migration theory and the guide theory of traditional Chinese medicine,as the study provides a new idea for the study of drug delivery and provides a theoretical basis for its migration mechanism.Methods: The rats were given high,medium and low dose of musk and preparation of different concentrations of serum,serum with equal volume of saline were prepared as the blank control group.By using adherent method in vitro cultured rat BMSCs cultured to the third generation,using cell surface antigen was detected by flow cytometry.After BMSCs was induced successful by training osteogenesis and lipid,using different concentrations of the preparation of musk drug-containing serum concentration of 5% medicated serum intervention with the third generation BMSCs.the proliferation of BMSCs was examined by MTT method,Alkaline phosphatase(alkaline phosphatase,ALP)was qualitatively and quantitatively detected after osteoblasts induction,I-type collagen was performed by immunofluorescence staining,the number of calcified nodules was measured.Transwell chamber was used to detect the changes of BMSCs cell mobility by adding different signal pathway blockers,the differences between groups in different time periods were compared.Statistical analysis was carried out to investigate the effects of different concentrations of Musk on proliferation and osteogenic differentiation of BMSCs in rats,and to explore the effects of different concentrations of musk mediated signaling pathway on BMSCs migration.Results:BMSCs isolated and cultured by whole bone marrow adherence were spindle-shaped or vortex-like,adherent growth,good growth condition,high purity and good activity;Phenotypic characterization results showed that P3 generation of BMSCs CD45 and CD34 negative antigen,CD44 and CD90 positive antigen;BMSCs could be osteogenic and adipogenic differentiation after adding osteogenic and adipogenic inducers;MTT results showed that first days the concentration of medium musk group promoted the proliferation of BMSCs significantly,compared with the control group(P<0.05),In the rest of the time,the low concentration musk group had the best effect on BMSCs proliferation(P<0.05),there was no difference between groups in different time periods(P>0.05);different concentrations of musk group could promote BMSCs osteogenic in different period of time,specifically:(1)compared with the control group,the treated group can improve ALP activity and positive staining number in different time periods(P<0.05),with a low concentration group has obvious effects(P<0.01),that is no difference in the different times group(P>0.05);(2)Compared with the control group,the number of calcified nodules in different concentrations of musk group was significantly increased(P<0.05),with a low concentration of musk group significantly(P<0.01);(3)Compared with the control group,the positive expression of?type collagen was significantly increased(P<0.05),with a low concentration of musk group was best(P<0.01);experimental results show that the migration in Transwell:(1)compared with the control group,different concentration of musk increased BMSCs migration in 24 h,48h,72h(P<0.05),musk promote BMSCs migration effect is negatively correlated with the concentration and positively correlated with time,and the effect was the best in the low concentration group(P<0.01),and there was no significant difference between different time groups(P>0.05);(2)after joining blockers PD98059,musk group compared with control group,low concentration group increased the number of musk BMSCs migration in different time(P<0.05),low concentration musk group + PD intervention group is decreased with its migration quantity significantly over time,different time periods within the group is no statistical significance(P> 0.05);(3)after joining blockers GF109203 X,compared with the control group,low concentration promote BMSCs migration quantity increased significantly within 24 h,48 h,72 h(P< 0.05),low concentration of musk group +GF intervention group that the migration quantity is significantly reduced in different period,different time periods within the group to compare have no statistical significance(P>0.05);(4)after joining blockers LY294002,According to the different concentrations of musk group compared with the control group,low concentration group musk promote BMSCs migration quantity increases with prolonged(P<0.05),The number of migration of low concentration musk group and LY intervention group was negatively correlated with time.The mechanism of musk promoting BMSCs migration is related to activation of MAPK/ERK1/2,PKC and PI3K/AKT signaling pathway.Conclusions:Musk can promote the proliferation of BMSCs and participate in the process of osteogenesis differentiation.Musk can promote the migration of exogenous BMSCs,its mechanism may be related to MAPK/ERK1/2,PI3K/AKT and PKC pathway activation,this approach has important significance for musk to promote the migration of BMSCs to the bone defects and to explain guiding action. |
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