Experimental Study On The Ability Of Catalpol To Promote Chondrogenic Differentiation And Migration Of BMSCs In Vitro

Objective1.A large number of bone marrow mesenchymal stem cells(BMSCs)were cultured in vitro efficiently and stably,and BMSCs cells were identified by cell biology.2.To observe the cell proliferation effect of Catalpol on BMSCs,and to investigate the effect of Catalpol on BMSCs to promote chondrogenic differentiation.3.Observe the effect of Catalpol on the migration of BMSCs in vitro,and further study the relationship between Catalpol in cell migration process and Wnt non-classical signaling pathway than Wnt5a/CaMKII.Methods1.The BMSCs of SD rat was cultured with the whole bone marrow adherent wall method,and the cell surface antigen flow cytometry was detected and induced into fat and osteoblast differentiation in the third generation BMSCs.2,CCK-8 methods to detect Catalpol effect on proliferation of BMSCs,RT-PCR detection of Catalpol on BMSCs differentiation into cartilage cells of specific genes COL?,Aggrecan and SOX9 gene expression.3.Transwell method was used to observe the effect of Catalpol on the migration of BMSCs cells and to construct a method of expressing Wnt5 a lentiviral vectors to infect BMSCs.Fluorescence microscopy was carried out to determine the infection effect and the expression of Wnt5 a gene by fluorescent expression of green fluorescent protein.Further Western-blot was used to detect the expression of CaMKII in BMSCs and to observe the effect of Catalpol on its downstream CaMKII protein.Results1.Flow cytometry showed that CD29,CD44,CD90 and positive rates were 99.84%,96.80%,99.99%,and the positive rate was greater than 95%.The marker of hematopoietic cells was only 4.35% and the positive expression rate was less than 5%.In addition,Osteoblast and adipocyte differentiation can be achieved in both osteogenic and adipose induced conditions in vitro are BMSCs,and the reverse identification of cultured cells in vitro was BMSCs.2.BMSCs began to proliferate in 12 h,and the growth rate of cells increased with time.The BMSCs growth rate of Catalpol could reach 86% after 24 h,and the proliferation rate of 36 h cells remained high at a high speed,reaching the highest rate of 130.77%.Different concentration of Catalpol can promote the proliferation of BMSCs in all periods of time,and the proliferation of Catalpol group with 25 mol/L concentration of Catalpol group after BMSCs12 hours was increased.With the increase of Catalpol concentration and the prolongation of the action time,the proliferation effect of BMSCs increased significantly compared with the normal control group(P <0.01).3.RT-PCR results showed that compared with the blank group,the high,medium and low doses of the Catalpol group could improve the mRNA expression of COL1 and SOX9(P < 0.01).The expression of Aggrecan gene can be improved by the high dose of Catalpol(150? mol/L)and the medium dose of Catalpol(100? mol/L)and the classical induction group(P < 0.01).Compared with classic induction group,low,medium dose of positive Catalpol group can improve the COL1 gene expression(P < 0.05),high dose of Catalpol can improve the Aggrecan gene expression(P < 0.05),high,medium and low doses of Catalpol group can improve the SOX9 mRNA expression.Compared with blank group,the expression of Aggrecan mRNA was not statistically significant.4.The transwell experiment showed that compared with the number of cells in the blank control group,the number of cells in the lower chamber cells after the intervention of BMSCs 12 h was significantly increased in 50 and 100 ? mol/L concentrations,and the difference was statistically significant(P < 0.01).In addition,the cells in the lower chamber of the 200 ? mol /L were significantly reduced from the 100 ? mol/L concentration group,indicating that the migration concentration of BMSCs in the optimal Catalpol was 100 ? mol/L,and the difference was statistically significant(P < 0.01).5.Fluorescence microscopy observed the expression of green fluorescent protein fluorescence and RT-PCR to detect the expression of Wnt5 a gene and showed that BMSCs overexpressed Wnt5 a gene.6.Western-blot results showed that the expression of CaMKII protein in Wnt5 a group was significantly increased compared with the blank group(P < 0.01).The expression of CaMKII protein was highest in Wnt5 a group(P < 0.01).ConclusionCatalpol can promote proliferation,chondrogenic differentiation and external migration,and its mechanism may be related to Wnt non-classical pathway Wnt5a/CaMKII.