激活态许旺细胞在胶原几丁糖膜上的生长规律(英文)  

Growth rule of activated Schwann cells cultured on Chitosan-collagen film

作　　者：何继银[1] 劳杰[1] 顾玉东 蒋良福[1] 李继峰[1] 

机构地区：[1]复旦大学附属华山医院手外科,上海市200040

出　　处：《中国临床康复》2006年第33期158-160,i0006,共4页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助(30170965);国家"九七三"创伤基础研究资助(G1999054202)~~

摘　　要：背景:新型组织工程材料和许旺细胞扩增后置入生物合成管内去修复周围神经的缺损,是人工生物材料管的两大进展。目的:以胶原几丁糖为支架,以激活态的许旺细胞为种子细胞,观察二者的亲和性以及激活态的许旺细胞在胶原几丁糖膜上的生长规律,为人工神经的预构做准备。设计:开放性实验。单位:复旦大学附属华山医院手外科。材料:实验于2003-07/2003-12在卫生部手功能重建重点实验室完成。选取清洁级雄性SD大鼠4只。胶原几丁糖膜(上海其胜生物材料技术研究所提供),许旺细胞激活液(自制)。方法:大鼠麻醉后坐骨神经切断预变性7d,再次麻醉后引颈处死,迅速切取双侧坐骨神经,置于含青霉素和链霉素的D-HANK’S液中,剔除神经外膜,剪碎成1mm的小段,移入盛有质量浓度为5g/L的胰蛋白酶和0.6g/L的胶原酶的离心管中,每2mL液体中加入激活液0.5mL,复合酶分步消化法获激活态许旺细胞,以浓度为2×107L-1激活态的许旺细胞200μL接种于胶原几丁糖膜上和培养皿上,2周后通过相差显微镜和扫描电镜观察细胞生长情况,S-100染色鉴定细胞的纯化程度。主要观察指标:①绘制细胞生长曲线,确定体外倍增时间。②激活态许旺细胞倒置相差显微镜下观察结果。③激活态许旺细胞接种于胶原几丁糖膜上扫描电镜观察结果。结果:①体外倍增时间的确定:激活态的许旺细胞接种于胶原几丁糖膜和培养皿上时的浓度均是2×107L-1,2周后细胞浓度分别达到30×107L-1和20×107L-1。根据DT=(t-t0)lg2/(lgn-lgn0)算出激活态许旺细胞在胶原几丁糖膜上的倍增时间为4d。②激活态许旺细胞倒置相差显微镜下观察结果:接种于培养皿上的激活态许旺细胞24h后大多数由圆球形变成长梭形,有突起,多为双极,也有的呈三极状;接种于膜上的激活态许旺外形上和培养皿中无明显差异,但膜上的激活态许旺细胞在相差显BACKGROUND： New-type tissue engineering materials and post-proliferation Schwann cells are implanted into biosynthesis tube for repairing peripheral nerve defect, which are two great developments in the field of artificial biomaterial tube. OBJECTIVE： Taking chitosan-collagen as scaffold, activated Schwann cells as seed cells, we are in attempt to observe the affinity between them as well as growth rule of activated Schwann cells on Chitosan-collagen, so as to provide basis for pre-construction of artificial nerve. DESIGN： Open experiment. SETTING： Department of Hand Surgery, Huashan Hospital Affiliated to Fudan University. MATERIALS： This experiment was conducted at the Key Laboratory of Hand Function Reconstruction, Ministry of Public Health from July 2003 to December 2003. Four male SD rats, of clean degree, were used in this experiment. Chitosan-collagen film was made in Qisheng Biomaterial Technique Institute, Shanghai, Schwann cells activator solution was made in our laboratory （self-made）. METHODS： After rats were anaesthenia, the sciatic nerve was cut off to perform predegeneration for 7 days. Another anaesthenia later, the rats were euthanized. Both sides of sorciatic nerves were cut off quickly and put in the D-HANK＇s solution containing penicillin and streptomycin. Epineurium was eliminated and chipped into 1 mm pieces, then put in the centrifuge tube containing 5 g/L trypsinase and 0.6 g/L collagenase. 0.5 mL activator solution every 2 mL liquid was added and the activated Schwann cells were harvested with the way of two-step enzymolysis. 2×10^7 L^-1 activated Schwann cells in 200 μL were inoculated to Chitosan-collagen film and Petri dish . Two weeks later, cellular growth was observed under phase contrast microscope and scanning electron microscope. Cellular purity was identified with S-100 staining. MAIN OUTCOME MEASURES： ① Drawing cell growth curve and con- finning in vitro doubling time. ②Observation of activated Schwann cells under an inverted phase contrast

关 键 词：生物医学工程 生物相容性材料 许旺氏细胞/细胞学 周围神 壳多糖/类似物和衍生物 显微镜检查 电子 扫描 显微镜检查 相差 

分 类 号：R318[医药卫生—生物医学工程]