Restraining degeneration of intervertebral discs by transplantation of Notch-1knockout rabbit bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2013.36.004

Abstract

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 cell differentiation of mesenchymal stem cells remains unclear.
OBJECTIVE: To investigate the bone marrow mesenchymal stem cells in suppressing the degeneration of intervertebral discs after Notch1 gene knockout.
METHODS: (1) Bone marrow mesenchymal stem cells 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 cells, and the differentiation of mesenchymal stem cells 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 L_3-4, L_4-5 and L_5-6 were stabbed by a needle, and nucleus pulposus tissue was harvested for modeling. The cells 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 cells were transplanted into L_3-4, L_4-5 and L_5-6, respectively.
RESULTS AND CONCLUSION: (1) Four weeks after the cell transplantation, the signal intensity of T2 weighted images in the L_3-4 (non-transfection group) and L_5-6 (blank plasmid group) discs was increased by magnetic resonance imaging, and the significant difference was found in L_4-5 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 L_4-5 discs (shRNA-Notch-1 plasmid group) was significantly higher than that in L_3-4 (non-transfection group) and L_5-6 (blank plasmid group) discs. (3) Reverse transcription-polymerase chain reaction and western blot assay showed that, the expression of collagen II and proteoglycan mRNA and protein in the L_4-5 discs (shRNA-Notch-1 plasmid group) were significantly increased compared with L_3-4 (non-transfection group) and L_5-6 (blank plasmid group) discs. Transplantation of bone marrow mesenchymal stem cells of Notch-1 gene knockout rabbits can effective restore the degenerated intervertebral discs.

Key words: bone marrow, mesenchymal stem cells, gene knockout techniques, intervertebral disk degeneration

CLC Number:

R394.2

Mori Gele, Shao Zeng-wu. Restraining degeneration of intervertebral discs by transplantation of Notch-1knockout rabbit bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(36): 6403-6408.

Figures/Tables 5

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