肝细胞生长因子促进脊髓组织块离断神经突起再生的体外观察

肝细胞生长因子促进脊髓组织块离断神经突起再生的体外观察刘铖;阙海萍;舒翠莉;刘少君;吴祖泽
【期刊名称】《中国组织工程研究》
【年(卷),期】2006(010)029
【摘要】BACKGROUND: Hepatocyte growth factor (HGF) promotes neurite outgrowth from neocortical explants, and supports neuronal survival under serum-free condition. Thus, HGF can mediate neurotrophic function as a novel neurotrophic factor.OBJECTIVE: To establish an in vitro injury model with a semi-solid culture system for the purpose of improving the evaluation of neurite regeneration of transected spinal cord neurons from rat embryo, and investigate the effect of HGF on neurite regeneration.DESIGN: Randomized controlled study.SETTING: Hematology Laboratory of Radian Medical Institute of Academy of Military Medical Sciences of PLA.MATERIALS: The experiment was carried out at the Hematology Laboratory of Radian Medical Institute of Academy of Military Medical Sciences of PLA from August 2004 to May 2005. Wistar fetal rats of 14-16 days old were provided by Animal Center of Academy of Military Medical Sciences of PLA. Tail collagen was extracted from adult male Wistar rats with body mass of (250±50) g.METHODS: ① Rat tail type Ⅰ collagen substrate was prepared and spread on a culture dish, cut into about 0.5-1.0 mm3 slices, then spinal cord slices of 15-day-old fetal Wistar rat were explanted on the primary culture. Five days later, the outgrowing
processes were severed, then a block of collagen, with the surface area of 2 mm2 and 200 μm away from the slice, was removed and the vacancy was replaced with a fresh collagen block of 2 μL after aspirating the medium. The fresh collagen block could be solidified and then fresh liquid medium was added as the secondary culture. The regeneration of neurite was observed by microscopy at 0, 1, 6,12 and 24 hours after severing. ② The medium was changed with 0.5% N3-conditioned medium. 10 μg/L HGF was added in the experimental group, and 0.5% N3-conditioned medium was added in the control group.The status of regeneration was evaluated by the average value of 3 longest regenerative neurites for each slice. There were 12 slices in each group.The status of neurite regeneration was calculated and was evaluated 24 hours later.MAIN OUTCOME MEASURES: ① neurite regeneration in situ; ②comparisons of neurite regeneration between control group and experiment al group.RESULTS: ① Neurite regeneration in situ: The neurites disintegrated near the severing line immediately following the transection injury. This process persisted about 1-2 hours and the distance away from the severing line was about 20 μm. Then the proximal end of neurites would swell and thicken. At this time neurites stopped collapsing and neurite regeneration began. Their regenerating rate would quicken at 12 hours after severing. Regenerating neurites were more branching and curlier as compared with original neurites. ② Comparisons of neurite regeneration between control group and experimental group: The average length of regenerative neurites was more in the experimental group than that in the control group [(375±96)
μm, (200±75) μm, P ＜ 0.05].CO NCLUSION: ① We establish a simple, economic model to evaluate neurite regeneration. ② By this model, we prove that HGF can promote neurite regeneration.%背景:肝细胞生长因子可促进体外培养的皮层神经细胞突起生长,在无血清条件下支持皮层神经元的存活,因此被认为是一种新发现的神经营养因子.目的:采用半固体培养基系统培养脊髓组织块,原位观察脊髓组织块神经突起再生及肝细胞生长因子对组织块神经突起再生的作用.设计:观察对比实验.单位:解放军军事医学科学院放射医学研究所实验血液学研究室.材料:实验于2004-08/2005-05在解放军军事医学科学院放射医学研究所实验血液学研究室和基础医学研究所神经生物学研究室完成.选用40只Wistar胚胎大鼠,孕期14-16 d,由解放军军事医学科学院动物中心提供.鼠尾胶原取自成年250±50 g雄性Wistar大鼠.方法:①用自备的鼠尾胶原制作半固体培养基系统,无菌条件下快速分离出孕期14-16 d胚鼠的脊髓,剪成0.5-1.0 mm3的小块,置于半固体培养基中作为原代培养,组织块生长5 d时,将发出的神经突起在距离脊髓组织块约200mm处离断,并在离断远端将约2 mm2大小的鼠尾胶去除,用2 mL鼠尾胶原重新铺缺损区,待新铺鼠尾胶原固化后加液体培养基作为二代培养,在离断后0,1,6,12和24h,通过显微镜原位连续观察神经突起再生.②将突起离断后的培养基改为含0.5%的N3条件培养基,以加入10μg/L肝细胞生长因子作为实验组,加入含0.5%N3的无血清的DMEM培养基作为对照组,用每个组织块再生突起最长的3根突起长度均值代表这个组织块神经再生情况,每组分别观察12个组织块,24 h后观察计算两组神经再生突起长度,比较两组神经突起再生情况.主要观察指标:①原位神经突起再生情况.②对照组与实验组神经突起再生状况比较.结果:①原位神经突起再生情况:刚离断时在离断部位两侧神经突起开始崩解,持续时间约1-2 h,在离断近端延伸的距离约20mm.此后近端神经突起逐渐变得增粗、肿胀,神经突起停止崩解并开始向外延伸,神经再生开始,而且再生速度在划伤12 h后明显加快.再生的神经
纤维较划伤前的纤维分支增多.②对照组与实验组神经突起再生情况:实验组神经再生突起长度明显大于对照组[(375±96)mm,(200±75)mm,(P＜0.05)].结论:①建立了一种原位观察神经组织块突起再生的方法,此方法简单、经济.②肝细胞生长因子体外能促进脊髓组织块神经突起再生.
【总页数】5页(P173-176,封面)
【作者】刘铖;阙海萍;舒翠莉;刘少君;吴祖泽
【作者单位】解放军军事医学科学院放射医学研究所实验血液学研究室,北京市,100850;解放军军事医学科学院基础医学研究所神经生物学研究室,北京
市,100850;解放军第三○二医院肝病研究所,北京市,100039;解放军军事医学科学院基础医学研究所神经生物学研究室,北京市,100850;解放军军事医学科学院放射医学研究所实验血液学研究室,北京市,100850
【正文语种】中文
【中图分类】R6
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