Co-Modification of Beta-Catenin and Sex Determining Region Y-Box9 Genes Regulates the Chondrogenesis of Human Adipose Tissue Derived Stem Cells

Abstract

132 *Address for correspondence E-mail: [email protected] Beta catenin gene can inhibit the chondrogenic differentiation of human adipose tissue derived stem cells, while sex determining region Y-box9 gene can promote the chondrogenic differentiation of human adipose tissue derived stem cells. However, it is still necessary to study the role of the combination of the two genes in the early chondrogenic differentiation of human adipose tissue derived stem cells. The purpose of this study was to investigate whether the knockdown of Beta catenin gene and overexpression of sex determining region Y-box9 gene combined with modification of human adipose tissue derived stem cells could promote the early chondrogenic differentiation of stem cells. Therefore, human adipose tissue derived stem cells was divided into four groups with knockdown or overexpression lentivirus vectors, including negative control group (empty vector), Beta-catenin knockdown group (lentivirus-RNA interference-Betacatenin), sex determining region Y-box9 overexpression group (lentivirus-FLAG-sex determining region Y-box9) and union group (lentivirus-RNA interference-Beta-catenin+lentivirus-FLAG-sex determining region Y-box9). After 24 h of transfection, quantitative reverse transcription Polymerase Chain Reaction and Western blot were used to detect the knockdown efficiency of Beta catenin gene and the overexpression efficiency of sex determining region Y-box9 gene. Furthermore the pellets were cultured in chondrogenic differentiation medium for 21 d and the tissue was stained with safranin-O-green and toluidine blue. The messenger RNA expression of cartilage related genes was detected by quantitative reverse transcription polymerase chain reaction. Quantitative reverse transcription polymerase chain reaction and Western blot results showed that both Beta catenin knockdown gene and sex determining region Y-box9 overexpression gene were successfully transfected and expressed (p< 0.05). quantitative reverse transcription polymerase chain reaction results showed that type-II collagen alpha 1 chain and aggrecan messenger RNA expression levels in the union group were higher than those in the first three groups (p<0.05), while type-X collagen alpha 1 chain and type-I collagen alpha 1 chain in the union group were lower than in the negative control group (p<0.05). The results of staining showed that the absorbance of union group was higher than that of the former three groups (p<0.05). Immunohistochemical results showed that the protein expression of type-II collagen alpha 1 chain and aggrecan in the union group was higher than that in the first three groups (p<0.05). Therefore, the combination of Beta-catenin gene knockdown and sex determining region Y-box9 overexpression can promote the early chondrogenic differentiation of human adipose tissue derived stem cells.