摘要
目的 探讨从成年大白鼠坐骨神经中培养出纯的许旺细胞的方法。 方法 采用以下几种培养方法 :(1)常规方法进行大白鼠坐骨神经组织块培养 (A法 )。 (2 )应用常规的胰酶消化方法进行坐骨神经细胞培养 (B法 )。 (3 )是经过改良的细胞培养方法 (C法 )。实验组 :从坐骨神经束抽取单条神经纤维 ,剪碎后用高浓度的双酶 (0 5 %胰蛋白酶和 0 0 6%胶元蛋白酶 )经 80～ 90min的消化后进行细胞培养。对照组 :把抽取神经纤维后剩余的神经束膜和神经鞘膜 ,常规胰酶消化后进行细胞培养。用S10 0抗体和成纤维细胞抗体对培养物进行鉴定 ,并用MTT测定和H3 胸腺嘧啶核苷检测细胞的增殖能力。 结果 在A ,B法的培养物中 ,虽然有少量许旺细胞 ,但同时出现大量的成纤维细胞 ;用C法分离培养的细胞经鉴定证实全为许旺细胞 ,MTT和H3 测定证实生长良好 ,而对照组的培养物均全部为成纤维细胞。 结论 在C法中使用的抽取单条神经纤维和高浓度双酶长时间消化技术 ,能彻底清除成纤维细胞 。
Objective Attempt to find out the method of isolated purified Schwann cells from sciatic nerve of adult SD rat Methods Three methods of culturing Schwann cells from sciatic nerve of adult rats were compared: (1) Conventional primary explant technique in tissue culture (Method A) (2) Conventional enzymatic disaggregating technique for cell culture (Method B) (3) A modified method of cell culture (Method C): Test group: we dissected very carefully and separated the sciatic nerve into individual fibers under surgical microscpe, individual fibers were cut into 0 5～1 mm pieces and treated with enzymatic disaggregating by twoenzymes (0 5% Trpsin and 0 06% Collagenase) in 37℃ for 80～90 min Control group: a application of conventional enzymatic disaggregating technique for culturing the nerve adventital tissues, which werethe remaining procedures of Test group We applied immunohistochemistry method with S100 antibody and Fibroblast antibody to identify Schwann cells Function of these Schwann cells proliferated were assessed by MTT assay and H 3 Thymidine incorporation assay. Conclusion In method C, a pure culture of Schwann cells was obtained, where the two techniques were used: careful separation of the sciatic nerve into individual fibers and use of higher concentrations of two enzymes for longer time to digest the nerve tissues These two technigues can cleanly remove and inhibit the fibroblasts, and enable healthy and normal proliferation of Schwann cells
出处
《中华显微外科杂志》
CSCD
北大核心
2002年第3期197-200,共4页
Chinese Journal of Microsurgery
