TGF-β1 regulate the cells phenotype marker CD24 and extracellular matrix of nucleus pulposus in vitro

Objective To investigate the effects of TGF-β1 regulated on CD24 expression and extracellular matrix synthesis of nucleus pulposus cells with flow cytometry and RT-PCR,which will provide a novel insight to further understand the characteristics of nucleus pulposus cells.Methods The human nucleus pulposus cells were isolated and cultured with enzyme digestion.Flow cytometry and immunohistochemical were combined to identify the nucleus pulposus cells.After the nucleus pulposus cells was stimulate by TGF-β1 in 1 day,3 days,6 days,the percentage of postive antigen CD24 expression change were detected by flow cytometry.The quantification of CD24,SOX9,collagenⅡ,Aggrecan genes was assessed using RT-PCR in different groups.Results In all cases,the morphology of nucleus pulposus cells were polygons or triangle which the immunohistochemical stains ofⅡcollagen type was positive,the proportion was 33%-60%;some cells were spindle,the immunohistochemical stains ofⅡcollagen type was weak positive.The percentage of postive antigen expresstion of CD24 was 25%-56%,there was no statistical difference between the two methods(t=27,P0.05).In the lasting sitmulating nucleus pulposus cells 24 hours,72 hours and 6 days later by TGF-β1,the percentage of CD24 positive cells was respectively 33.9%,76.2%and 99%;RT-PCR results showed that CD24,SOX9,collagenⅡ,aggrecan mRNA express increased significantly than control group after TGF-β1 stimulated.Conclusions Flow cytometry is a novel method which has advantage of rapid,simple,reliable and less demand for cells to identify mature nucleus pulposus cells by detected CD24 expression.TGF-β1 can stimulate CD24 gene expression and protein synthesis,adjusting nucleus pulposus cell to mature.