Effect of Paxillin gene on the differentiation of bone marrow mesenchymal stem cells to nucleus pulposus-like cells

English Keywords:Bone marrow mesenchymal stem cells  Paxillin  Nucleus pulposus-like cells  Eukaryotic expression vector

English Abstract:

  【Abstract】 Objectives: To investigate the effect of Paxillin on the differentiation of bone marrow mesenchymal stem cells(BMSCs) to nucleus pulposus-like cells. Methods: BMSCs from 3-week-old rat bone marrow were cultured by whole bone marrow adherent method until the third generation. Then, BMSCs surface markers were identified by flow cytometry. The eukaryotic expression vector pcDNA3.1-Paxillin was constructed by using pcDNA3.1(-)/Myc-His B vector. The third generation BMSCs were divided into three groups: experimental group, transfected with pcDNA3.1-Paxillin; the negative control group, transfected with pcDNA3.1(-)/Myc-His B; the control group, no transfection. The experimental group and negative control group were screened by using G418 following cultured for 36h. The mRNA expression levels of Paxillin, aggrecan, collagen type Ⅱ α1(COL2α1), sex determining region Y box protein 9(SOX9), keratin19(KRT19), paired box 1(PAX1) and forkhead box F1(FOXF1) in the three BMSCs groups and NP group were detected by quantitative real-time polymerase chain reaction(qRT-PCR). The protein expression levels of Paxillin, KRT19, PAX1 and FOXF1 were measured by Western blot. Results: The 3rd generation of primary culturing BMSCs was fusiform, connected tightly, evenly distributed with high expressions of CD44(95.6%) and CD90(96.8%), and distributed with low expressions of CD11(2.16%) and CD45(1.82%). The mRNA and protein expressions of Paxillin in the experimental group were higher than those in the negative control group and the control group, the difference was significant(P<0.05). The morphologies of the BMSCs selected for 18d by G418 was similar to the nucleus pulposus cells. The mRNA expression levels of COL2α1, SOX9, Aggrecan, KRT19, PAX1 and FOXF1 in the experiemental were significantly up-regulated compared with negative control group and control group(P<0.05). The protein levels of KRT19, PAX1 and FOXF1 were also obviously increased contrasted with negative control group and control group(P<0.05). Conclusions: Paxillin can promote the differentiation of BMSCs to nucleus pulposus-like cells, providing a novel reference for the treatment by using cell transplantation therapy in degenerative disc degeneration disease.