构建神经再生室培养成年SD大鼠许旺细胞

Construction of neural regeneration chamber to culture Schwann cells of adult SD rats

目的:探讨从成年SD大鼠坐骨神经分离培养获得大量许旺细胞的有效手段。方法:实验于2005-05/11在解放军第四军医大学口腔医学院组织病理教研室和组织工程实验中心完成。①取成年SD大鼠1只截断坐骨神经5mm,缝接入20mm硅胶管中,形成神经再生室。7d后重新打开伤口,刨开硅胶管,截取再生室内神经段置离心管中作为实验组。②取SD仔鼠5只,无菌条件下取双侧坐骨神经混合于离心管中作为对照组。③将两组坐骨神消化培养。通过相差显微镜活细胞计数和S-100细胞化学标记相结合鉴定了许旺细胞增殖和纯化程度。结果:①两组培养的细胞光镜观察结果:实验组的许旺细胞密度高于对照组,特别是在培养4d后细胞的密度差别尤为显著。实验组的许旺细胞密度大于600个/mm,而对照组密度不足200个/mm。②两22组许旺细胞免疫组织化学染色:实验组衬染后计数许旺细胞的纯度为(98.0±1.2%,对照组许旺细胞的纯度为(93.0±2.1)%(P<0.05)③)而。两组细胞的生长曲线:培养7d,实验组细胞数量较对照组明显增加,达到(49.6±3.5)×10L-1,远高于对照组的(31.7±3.9)×10L。77-1结论:从成年SD大鼠坐骨神经分离培养方法能获得大量高纯度的许旺细胞,为自体许旺细胞在修复周围神经缺损中的应用提供了可能。

AIM： To investigate effective means of seperating and culturing a great amount of Schwann cells from sciatic nerve of adult SD rats. METHODS： The experiment was carried out in the Staff Room of Histology and Pathology, Experimental Center of Tissue Engineering, Stomatological College of Fourth Military Medical University from May to November 2005.①The sciatic nerve of an adult SD rat was bridged with 20 mm silicon tube after resected 5 mm, so as to construct the neural regeneration chamber. 7 days after that, the content inside silicon tube were dug out, and nerve segment of regeneration chamber was put into the centrifuge tube as the experiment group. ②Bilateral sciatic nerve of 5 SD rats were mixed in the centrifuge tube as the control group. ③Sciatic nerve of both groups were digested by eollagenase to culture. Schwann cells proliferation and purification were identified by combined using of phasecontrast microscope and S-100 immunocytoehemistry staining. RESULTS： ①The observation result of Schwann cells in the two groups under microscope： Schwann cells density in the experimental group （above 600 cells/mm^2） was higher than that in the control group（below 200 cells/mm^2）, and the difference was especially obvious 4 days alter the culture. ②The immunocytochemistry staining of Schwann cells in the two groups： The purification of Schwann cells after counterstain in the experimental group was （98±1.2）%, while that in the control group was （93±2.1）% （P 〈 0.05）.③Growth curves of Schwann cells in the two groups： The number in the experiment group[（49.6±3.5）× 10^7L^-1 ] after 7-day culture was more than that in the control group [（31.7±3.9）×10^7L^-1. CONCLUSION： Large amount of high-purity Schwann cells can be seperated and cultured from sciatic nerve of adult SD rats, which makes it possible to apply one＇s own Schwann cells in repairing peripheral neurologic defect.