Experimental studies on peripheral nerve regeneration through the bio-nerve with 3D cultured Schwann cell

3D培养雪旺细胞生物神经再生周围神经的实验研究

• 批准号: 14571401
• 负责人: NAKAO Yasushi
• 金额: $ 2.3万
• 依托单位: KEIO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571401/
• 关键词: Schwann cell; artificial nerve; nerve graft; 組織工学; 再生医学; 末梢神経; ティッシュエンジニアリング

Schwann cells were initially isolated P7 Wister rat sciatic nerve and dorsal root ganglia. The neurotrophic factor cDNA was inserted into multiple cloning site of shuttle vector. The adenoviral constructs were transfected into 293 packaging cell line. The cultured Schwann cells were infected with concentrated adenoviruses. The expression of the target neurotrophic factor gene was evaluated by Western blot analysis. Neurons from rat DRGs were cultured in a medium obtained from the transferred Schwann cell culture at 3days, 3 weeks, 8 weeks after infection. Neurite outgrowth was measured by immunostaining of axons with monoclonal antibody RT97. A 20mm nerve gap was bridged with a bioabsorbable polymer conduit containing a mixture of the transferred Schwann cells and Pluronic gel. 12 weeks later, histological evaluation was performed at the midportion of the grafted lesion. The transferred Schwann cells lysates showed the higher expression of the transgene product of 30 Kilo Dalton level by Western blot analysis. Histological evaluation demonstrated that, contrary to the poor regeneration in the control group, the genetically modified group showed not only well-regenerated axons, but also formations of vessels and epineurium-like tissue observed.

将P7 Wistar大鼠坐骨神经和背根神经节中的雪旺细胞分离出来。将神经营养因子基因插入穿梭载体的多克隆位点。将重组腺病毒载体导入293包装细胞系。用浓缩腺病毒感染培养的雪旺细胞。Western印迹分析检测靶神经营养因子基因的表达。分别于感染后3d、3周、8周将大鼠背根神经节细胞接种于雪旺氏细胞培养液中培养。用单抗RT97免疫组织化学染色检测轴突生长情况。用生物可吸收的聚合物导管修复20 mm的神经缺损区，该导管含有被转移的雪旺细胞和Pluronic凝胶的混合物。12周后，在移植皮损的中段进行组织学评估。Western印迹分析表明，转基因雪旺细胞裂解产物在30Kilo Dalton水平上有较高的表达。组织学评估显示，与对照组再生不良相反，转基因组不仅轴突再生良好，而且还观察到血管和神经外膜样组织的形成。

项目成果

Komiyama T, Nakao Y, Toyama Y, Vacanti CA, Vacanti MP, Asou H.: "A novel technique to isolate adult Schwann cells for an artificial nerve conduit."Journal of Neuroscience Methods. 122. 195-200 (2003)

Komiyama T、Nakao Y、Toyama Y、Vacanti CA、Vacanti MP、Asou H.：“一种分离成人雪旺细胞用于人工神经导管的新技术。”神经科学方法杂志。

N Okuyama, Y Nakao: "The Effect of the Number of Fascicle on Axonal Regeneration in Cable Grafts"Microsurgery. (in press). (2004)

N Okuyama，Y Nakao：“束束数量对电缆移植中轴突再生的影响”显微外科。

Komiyama T, Nakao Y, Toyama Y, Vacanti CA, Vacanti MP, Ronald Ignotz: "A novel technique for peripheral nerve reconstruction in the absence of an artificial conduit."Journal of Neuroscience Methods. 139. 133-140 (2004)

Komiyama T、Nakao Y、Toyama Y、Vacanti CA、Vacanti MP、Ronald Ignotz：“一种在没有人工导管的情况下重建周围神经的新技术。”《神经科学方法杂志》。

Komiyama T: "A novel technique for peripheral nerve reconstruction in the absence of an artificial conduit."J.Neuroscience Methods. 134. 133-140 (2004)

Komiyama T：“一种在没有人工导管的情况下重建周围神经的新技术。”J.神经科学方法。

栩木弘和: "シュワン細胞の3次元培養を応用した人工神経の開発"整形災害外科. 46. 247-251 (2003)

Hirokazu Tokigi：“应用雪旺细胞三维培养的人工神经的开发”骨科外科。 46. 247-251 (2003)