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

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微镜下犹如“刻在沙地上的文字”一般。S-100染色见激活态许旺细胞成棕色,纯度达95%以上。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:激活态许旺细胞多数生长于胶原几丁糖膜的凹陷中或紧贴膜表面,呈有规律的首尾相接贴附于膜上,胞体呈纺锤形,直径在4～6μm,长度为60～80μm,细胞呈梭形,有许多细小分枝。1周时膜的形态仍然完整。结论:胶原几丁糖膜和高度纯化的激活态许旺细胞有良好的亲和性,以其为工艺材料制成的组织工程支架很有可能在促进周围神经再生中发挥优势作用。

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 microscope . ③ Observation of activated Schwann cells inoculated on Chitosan-collagen film under scanning electron microscope. RESULTS： ① Confirmation of in vitro doubling time ： Concentration of activated Schwann cells inoculated on both Chitosan-collagen and Petri dish was 2×10^7 L^-1, the final concentration was up to 3.0×10^8 L^-1 and 2.0×10^8 L^-1 respectively 2 weeks later. Doubling time of activated Schwann cells cultured on Chitusan-collagen film was 4 days calculated according to DT=（t-t0） lg2/（lgn-lgno）. ②Observation of activated Schwann cells under an inverted microscope ： 24 hours later, the activated Schwann cells inoculated to Petri dish mostly changed from spherical to long shuttle-shape, mutation appeared and most were two-pole shape, fewer were three-pole shape; Morphologically, there was no significant difference between activated Schwann cells inoculated on Chitosan-collagen film and on Petri dish. Activated Schwann cells inoculated to Chitosan-collagen film were like ＂words caved on the sand＂ under phase contrast microscope and the purity was over 95%. ③ Observation of activated Schwann cells inoculated to Chitosan-collagen under scanning electron microscope ： Most of activated Schwann cells grew in the introcession of Chitosan-collagen or closely to surface of Chitosan-collagen, presenting regular head-to-end connection and adhesion to Chitosan-collagen film. The cell body was fusiform, with diameter of 4-6 μm, 60-80 μm in length. Cells were shuttle-shape with some small branches. Morphology of Chitosan-collagen film was still complete at week 1. CONCLUSION： There exists great affinity between Chitosan-colla- gen film and high-purity activated Schwann cell; so tissue-engineering scaffold made of the two components probably promote peripheral nerve regeneration.