Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Nucleus Pulposus-like Cells Induced By GDF-5and Dexamethasone

Objective: To investigate the NP-like cells differentiation of rat bone marrowmesenchymal stem cells induced by GDF-5and Dexamethasone.Methods：BMSCs were collected from Sprague Dawley rats and the feasibilityof isolating and culturing rats nucleus pulposus cells (NPCs)by the sequential enzymemethod. the use of flow cytometry to identification BMSCs phenotype with CD90,CD45, CD44, CD11antibody, and by type Ⅱ collagen immunohistochemicalstaining NPCs and purity analysis.Cells at passage3were used and divided intocontrol group,group of BMSCs+Dex,group of BMSCs+GDF-5,group ofBMSCs+Dex+GDF-5,group of NPCs,then the cellular morphology was observedunder microscope and induced for21days.Alcian Blue stain showed the changes ofproteoglycan,RT-PCR detected expression COL1，ACAN,SOX9,COL2and positivegenes of nucleus pulposus cells for KRT19,PAX1,FOXF1.Western blot was used todetect COL2and COL1.Results：BMSCs present a single long spindle,grew with whirlpool-like;NPCspresent spherical, characteristics of island growth. BMSCs identified by flowcytometry shows: CD90and CD44expression was high trend, respectively,96.76%and98.09%. The CD45and CD11marker showed low expression of thehematopoietic system, respectively,8.25%and2.91%. The positive rate of NPCsis95.35±1.02%by collagen Ⅱ immunohistochemical. BMSCs becamecircles,triangles changes,then,cells shrinkaged and the cytoplasmic color of cellsgradually deepened as compared with the control group.The content of proteoglycanwas increased obviously in Co-induction.The relative expression levels of genes ofACAN,SOX9,COL2were gradually increased,and the proportion of whichACAN/COL2showed an increasing trend. The mRNA expression of COL1showed adecreasing trend. The positive genes of NPCs for KRT19,PAX1,FOXF1weresignificant different as compared with control group(P＜0.05).The expression ofCOL2protein with GDF-5group was increased significantly as compared withcontrol group (P＜0.05),then the expression of COL1protein was minimum in Co-induction.Conclusions： GDF-5may induce rat BMSCs to differentia into NP-like cellsdifferentiation,and dexamethasone plays a promotion effect in the differentiation ofinduction, and provides a theoretical basis for BMSCs transplantation in the treatmentof intervertebral disc degeneration.