Differentiation of rat bone marrow mesenchymal stem cells into nucleus pulposus-like cells induced by GDF-5 and Dexamethasone
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Objective To investigate the NP-like cells differentiation of rat bone marrow mesenchymal stem cells induced by GDF-5 and Dexamethasone .Methods：BMSCs were collected from Sprague Dawley rats and the feasibility of isolating and culturing rats nucleus pulposus cells (NPCs)by the sequential enzyme method.Cells at passage 3 were used and divided into control group,group of BMSCs+Dex,group of BMSCs+GDF-5,group of BMSCs+Dex+GDF-5,group of NPCs ,then the cellular morphology was observed under microscope and induced for 21 days.Alcian Blue stain showed the changes of proteoglycan,RT-PCR detected expression COL1，ACAN,SOX9,COL2 and positive genes of nucleus pulposus cells for KRT19,PAX1,FOXF1.Western blot was used to detect COL2 and COL1.Results ：BMSCs present a single long spindle ,grew with whirlpool-like;NPCs present spherical, characteristics of island growth. BMSCs became circles,triangles changes ,then ,cells shrinkaged and the cytoplasmic color of cells gradually deepened as compared with the control group.The content of proteoglycan was increased obviously in Co-induction.The relative expression levels of genes of ACAN,SOX9,COL2 were gradually increased ,and the proportion of which ACAN/COL2 showed an increasing trend. The mRNA expression of COL1 showed a decreasing trend. The positive genes of NPCs for KRT19,PAX1,FOXF1 were significant different as compared with control group(P＜0.05).The expression of COL2 protein with GDF-5 group was increased significantly as compared with control group (P＜0.05),then the expression of COL1 protein was minimum in Co-induction.Conclusions ： GDF-5 may induce rat BMSCs to differentia into NP-like cells differentiation ,and dexamethasone plays a promotion effect in the differentiation of induction, and provides a theoretical basis for BMSCs transplantation in the treatment of intervertebral disc degeneration.