中国临床康复
中國臨床康複
중국림상강복
CHINESE JOURNAL OF CLINICAL REHABILITATION
2006年
29期
173-176,封面
,共5页
刘铖%阙海萍%舒翠莉%刘少君%吴祖泽
劉鋮%闕海萍%舒翠莉%劉少君%吳祖澤
류성%궐해평%서취리%류소군%오조택
组织培养技术%胶原%脊髓%神经再生%培养基%肝细胞生长因子
組織培養技術%膠原%脊髓%神經再生%培養基%肝細胞生長因子
조직배양기술%효원%척수%신경재생%배양기%간세포생장인자
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.
揹景:肝細胞生長因子可促進體外培養的皮層神經細胞突起生長,在無血清條件下支持皮層神經元的存活,因此被認為是一種新髮現的神經營養因子.目的:採用半固體培養基繫統培養脊髓組織塊,原位觀察脊髓組織塊神經突起再生及肝細胞生長因子對組織塊神經突起再生的作用.設計:觀察對比實驗.單位:解放軍軍事醫學科學院放射醫學研究所實驗血液學研究室.材料:實驗于2004-08/2005-05在解放軍軍事醫學科學院放射醫學研究所實驗血液學研究室和基礎醫學研究所神經生物學研究室完成.選用40隻Wistar胚胎大鼠,孕期14-16 d,由解放軍軍事醫學科學院動物中心提供.鼠尾膠原取自成年250±50 g雄性Wistar大鼠.方法:①用自備的鼠尾膠原製作半固體培養基繫統,無菌條件下快速分離齣孕期14-16 d胚鼠的脊髓,剪成0.5-1.0 mm3的小塊,置于半固體培養基中作為原代培養,組織塊生長5 d時,將髮齣的神經突起在距離脊髓組織塊約200mm處離斷,併在離斷遠耑將約2 mm2大小的鼠尾膠去除,用2 mL鼠尾膠原重新鋪缺損區,待新鋪鼠尾膠原固化後加液體培養基作為二代培養,在離斷後0,1,6,12和24h,通過顯微鏡原位連續觀察神經突起再生.②將突起離斷後的培養基改為含0.5%的N3條件培養基,以加入10μg/L肝細胞生長因子作為實驗組,加入含0.5%N3的無血清的DMEM培養基作為對照組,用每箇組織塊再生突起最長的3根突起長度均值代錶這箇組織塊神經再生情況,每組分彆觀察12箇組織塊,24 h後觀察計算兩組神經再生突起長度,比較兩組神經突起再生情況.主要觀察指標:①原位神經突起再生情況.②對照組與實驗組神經突起再生狀況比較.結果:①原位神經突起再生情況:剛離斷時在離斷部位兩側神經突起開始崩解,持續時間約1-2 h,在離斷近耑延伸的距離約20mm.此後近耑神經突起逐漸變得增粗、腫脹,神經突起停止崩解併開始嚮外延伸,神經再生開始,而且再生速度在劃傷12 h後明顯加快.再生的神經纖維較劃傷前的纖維分支增多.②對照組與實驗組神經突起再生情況:實驗組神經再生突起長度明顯大于對照組[(375±96)mm,(200±75)mm,(P＜0.05)].結論:①建立瞭一種原位觀察神經組織塊突起再生的方法,此方法簡單、經濟.②肝細胞生長因子體外能促進脊髓組織塊神經突起再生.
배경:간세포생장인자가촉진체외배양적피층신경세포돌기생장,재무혈청조건하지지피층신경원적존활,인차피인위시일충신발현적신경영양인자.목적:채용반고체배양기계통배양척수조직괴,원위관찰척수조직괴신경돌기재생급간세포생장인자대조직괴신경돌기재생적작용.설계:관찰대비실험.단위:해방군군사의학과학원방사의학연구소실험혈액학연구실.재료:실험우2004-08/2005-05재해방군군사의학과학원방사의학연구소실험혈액학연구실화기출의학연구소신경생물학연구실완성.선용40지Wistar배태대서,잉기14-16 d,유해방군군사의학과학원동물중심제공.서미효원취자성년250±50 g웅성Wistar대서.방법:①용자비적서미효원제작반고체배양기계통,무균조건하쾌속분리출잉기14-16 d배서적척수,전성0.5-1.0 mm3적소괴,치우반고체배양기중작위원대배양,조직괴생장5 d시,장발출적신경돌기재거리척수조직괴약200mm처리단,병재리단원단장약2 mm2대소적서미효거제,용2 mL서미효원중신포결손구,대신포서미효원고화후가액체배양기작위이대배양,재리단후0,1,6,12화24h,통과현미경원위련속관찰신경돌기재생.②장돌기리단후적배양기개위함0.5%적N3조건배양기,이가입10μg/L간세포생장인자작위실험조,가입함0.5%N3적무혈청적DMEM배양기작위대조조,용매개조직괴재생돌기최장적3근돌기장도균치대표저개조직괴신경재생정황,매조분별관찰12개조직괴,24 h후관찰계산량조신경재생돌기장도,비교량조신경돌기재생정황.주요관찰지표:①원위신경돌기재생정황.②대조조여실험조신경돌기재생상황비교.결과:①원위신경돌기재생정황:강리단시재리단부위량측신경돌기개시붕해,지속시간약1-2 h,재리단근단연신적거리약20mm.차후근단신경돌기축점변득증조、종창,신경돌기정지붕해병개시향외연신,신경재생개시,이차재생속도재화상12 h후명현가쾌.재생적신경섬유교화상전적섬유분지증다.②대조조여실험조신경돌기재생정황:실험조신경재생돌기장도명현대우대조조[(375±96)mm,(200±75)mm,(P＜0.05)].결론:①건립료일충원위관찰신경조직괴돌기재생적방법,차방법간단、경제.②간세포생장인자체외능촉진척수조직괴신경돌기재생.
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 experimental 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].CONCLUSION: ① We establish a simple, economic model to evaluate neurite regeneration. ② By this model, we prove that HGF can promote neurite regeneration.
