Restraining degeneration of intervertebral discs by transplantation of Notch-1 knockout rabbit bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Preliminary experiments have demonstrated that the expression of Notch-1 in the degenerated intervertebral disc was increased. However, the role of Notch-1 in the nucleus pulposus-like cel differentiation of mesenchymal stem cel s remains unclear. OBJECTIVE:To investigate the bone marrow mesenchymal stem cel s in suppressing the degeneration of intervertebral discs after Notch1 gene knockout. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated from the femur bone of four New Zealand rabbits weighing 0.4-0.5 kg, under deep anesthesia, and then purified with discontinuous gradient density centrifugation method. (2) Notch1 shRNAs and blank plasmid shRNA were designed, synthesized, and transiently transfected into these mesenchymal stem cel s, and the differentiation of mesenchymal stem cel s was induced with transforming growth factor beta-1. (3) Ten New Zealand rabbits weighing 1.0-1.5 kg were involved in this study. The rabbits’ intervertebral discs in L3-4, L4-5 and L5-6 were stabbed by a needle, and nucleus pulposus tissue was harvested for modeling. The cel s were divided into blank plasmid transfected with transforming growth factor beta-1 group, shRNA-Notch-1 plasmid transfected with transforming growth factor beta-1 group, and non-transfected treated with transforming growth factor beta-1 group. Two weeks later the treated cel s were transplanted into L3-4, L4-5 and L5-6, respectively. RESULTS AND CONCLUSION:(1) Four weeks after the cel transplantation, the signal intensity of T2 weighted images in the L3-4 (non-transfection group) and L5-6 (blank plasmid group) discs was increased by magnetic resonance imaging, and the significant difference was found in L4-5 discs (shRNA-Notch-1 plasmid group) in comparison with other two groups (P<0.05). (2) Toluidine blue staining showed that, the expression of proteoglycan in the L4-5 discs (shRNA-Notch-1 plasmid group) was significantly higher than that in L3-4 (non-transfection group) and L5-6 (blank plasmid group) discs. (3) Reverse transcription-polymerase chain reaction and western blot assay showed that, the expression of col agen II and proteoglycan mRNA and protein in the L4-5 discs (shRNA-Notch-1 plasmid group) were significantly increased compared with L3-4 (non-transfection group) and L5-6 (blank plasmid group) discs. Transplantation of bone marrow mesenchymal stem cel s of Notch-1 gene knockout rabbits can effective restore the degenerated intervertebral discs.