背景：前期实验证明退变椎间盘内 Notch-1基因高表达，但骨髓间充质干细胞向髓核样细胞转化时 Notch-1的作用尚不明确。 目的：观测Notch-1基因敲除后的骨髓间充质干细胞修复退变椎间盘的作用。 方法：①4只体质量0.4-0.5 kg新西兰兔麻醉后，取股骨骨髓，以密度梯度离心法分离培养骨髓间充质干细胞。②将针对Notch-1的shRNA及无意空质粒shRNA，瞬时转染入骨髓间充质干细胞，转化生长因子β1诱导转染骨髓间充质干细胞分化。③体质量1.0-1.5 kg新西兰兔10只，对兔脊柱L3-4、L4-5、L5-63个椎间盘行穿刺抽吸髓核组织造模，将细胞分为转化生长因子β1诱导转染空质粒组、转化生长因子β1诱导转染shRNA-Notch-1质粒组、转化生长因子β1诱导无转染组细胞，于造模2周后分别移植入L3-4、L4-5、L5-63个椎间盘。 结果与结论：①细胞移植4周后MRI检测发现，L3-4(无转染组)及L5-6(空质粒组)%T2加权像扫描(%ST2WI)值有明显增加，L4-5(shRNA-Notch-1质粒组)%ST2WI值增加最为显著，与其他2组比较差异有显著性意义(P<0.05)。②甲苯胺蓝染色可见L4-5(shRNA-Notch-1质粒组)椎间盘组织内蛋白多糖的表达显著高于L3-4(无转染组)及L5-6(空质粒组)。③RT-PCR和Western blot检测发现L4-5(shRNA-Notch-1质粒组)椎间盘内Ⅱ型胶原及蛋白多糖的表达明显高于 L3-4(无转染组)及 L5-6(空质粒组)，差异有显著性意义。结果提示兔退变椎间盘内移植Notch-1基因敲除兔骨髓间充质干细胞，有效修复了退变的椎间盘组织。%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.
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