中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
12期
2364-2368
,共5页
安庆祝%朱巍%汪洋%毛颖%张荣%周良辅
安慶祝%硃巍%汪洋%毛穎%張榮%週良輔
안경축%주외%왕양%모영%장영%주량보
神经干细胞%脑出血%大鼠%移植
神經榦細胞%腦齣血%大鼠%移植
신경간세포%뇌출혈%대서%이식
背景:外源性神经干细胞具有神经修复作用,可能对脑出血后的神经功能恢复起到一定的作用.目的:观察胎鼠神经干细胞的体外生长、分化及移植到脑出血大鼠后的存活、迁徙、分化情况,探讨神经干细胞对脑出血模型大鼠受损神经功能的修复作用.设计:完全随机分组设计,对照动物实验.单位:复旦大学附属华山医院神经外科材料:选用健康雄性成年SD大鼠18只为受体,体质量280～320 g,由中国科学院上海实验动物中心提供.实验用鼠抗BrdU为Neomarkers产品,鼠抗胶质纤维酸性蛋白和兔抗微管相关蛋白2为Chemicon产品.方法:实验于2006-02/12在复旦大学附属上海医学院解剖组胚实验室完成.从胎龄14 d的胎鼠海马中分离、培养、鉴定神经干细胞.16只受体SD大鼠被随机分为3组:对照组,PBS组和移植神经干细胞组.均通过尾状核内注射自体动脉血制作大鼠脑出血模型.移植NSC组在造模后30min在血肿腔周围四点分别移植浓度为2X1011L-1神经干细胞悬液5μ L;PBS组于相同时间点在脑内相同部位注射PBS:PBS和神经干细胞的移植方法同自体血的移植方法.对照组大鼠在造模后30 min只造成四点损伤,不注射任何物质.主要观察指标:在造模后立即,1,3,5,14,21.28 d采用前肢评分和转身评分对大鼠神经功能进行评估.大鼠于造模后28 d麻醉后取脑,并通过双标胶质纤维酸性蛋白、微管相关蛋白2、BrdU免疫组化来检测移植入脑的神经干细胞在体内的分化情况.结果:①神经功能评分:造模后5 d,各组差异无显著性意义(P＞0.05).造模后14～28 d,干细胞移植组较其他3组明显改善(P＜0.05).②脑组织切片双免疫组织学双标染色结果:干细胞移植组血肿周围凋亡细胞少于PBS组.受体大鼠脑组织切片显示有BrdU,微管相关蛋白2,胶质纤维酸性蛋白阳性细胞,说明神经干细胞可以在宿主脑内存活、迁徙和分化,可以分化为神经元样细胞和神经胶质样细胞.结论:神经干细胞移植可能通过分化为神经元样细胞和神经胶质细胞促进大鼠脑出血的神经功能恢复.
揹景:外源性神經榦細胞具有神經脩複作用,可能對腦齣血後的神經功能恢複起到一定的作用.目的:觀察胎鼠神經榦細胞的體外生長、分化及移植到腦齣血大鼠後的存活、遷徙、分化情況,探討神經榦細胞對腦齣血模型大鼠受損神經功能的脩複作用.設計:完全隨機分組設計,對照動物實驗.單位:複旦大學附屬華山醫院神經外科材料:選用健康雄性成年SD大鼠18隻為受體,體質量280～320 g,由中國科學院上海實驗動物中心提供.實驗用鼠抗BrdU為Neomarkers產品,鼠抗膠質纖維痠性蛋白和兔抗微管相關蛋白2為Chemicon產品.方法:實驗于2006-02/12在複旦大學附屬上海醫學院解剖組胚實驗室完成.從胎齡14 d的胎鼠海馬中分離、培養、鑒定神經榦細胞.16隻受體SD大鼠被隨機分為3組:對照組,PBS組和移植神經榦細胞組.均通過尾狀覈內註射自體動脈血製作大鼠腦齣血模型.移植NSC組在造模後30min在血腫腔週圍四點分彆移植濃度為2X1011L-1神經榦細胞懸液5μ L;PBS組于相同時間點在腦內相同部位註射PBS:PBS和神經榦細胞的移植方法同自體血的移植方法.對照組大鼠在造模後30 min隻造成四點損傷,不註射任何物質.主要觀察指標:在造模後立即,1,3,5,14,21.28 d採用前肢評分和轉身評分對大鼠神經功能進行評估.大鼠于造模後28 d痳醉後取腦,併通過雙標膠質纖維痠性蛋白、微管相關蛋白2、BrdU免疫組化來檢測移植入腦的神經榦細胞在體內的分化情況.結果:①神經功能評分:造模後5 d,各組差異無顯著性意義(P＞0.05).造模後14～28 d,榦細胞移植組較其他3組明顯改善(P＜0.05).②腦組織切片雙免疫組織學雙標染色結果:榦細胞移植組血腫週圍凋亡細胞少于PBS組.受體大鼠腦組織切片顯示有BrdU,微管相關蛋白2,膠質纖維痠性蛋白暘性細胞,說明神經榦細胞可以在宿主腦內存活、遷徙和分化,可以分化為神經元樣細胞和神經膠質樣細胞.結論:神經榦細胞移植可能通過分化為神經元樣細胞和神經膠質細胞促進大鼠腦齣血的神經功能恢複.
배경:외원성신경간세포구유신경수복작용,가능대뇌출혈후적신경공능회복기도일정적작용.목적:관찰태서신경간세포적체외생장、분화급이식도뇌출혈대서후적존활、천사、분화정황,탐토신경간세포대뇌출혈모형대서수손신경공능적수복작용.설계:완전수궤분조설계,대조동물실험.단위:복단대학부속화산의원신경외과재료:선용건강웅성성년SD대서18지위수체,체질량280～320 g,유중국과학원상해실험동물중심제공.실험용서항BrdU위Neomarkers산품,서항효질섬유산성단백화토항미관상관단백2위Chemicon산품.방법:실험우2006-02/12재복단대학부속상해의학원해부조배실험실완성.종태령14 d적태서해마중분리、배양、감정신경간세포.16지수체SD대서피수궤분위3조:대조조,PBS조화이식신경간세포조.균통과미상핵내주사자체동맥혈제작대서뇌출혈모형.이식NSC조재조모후30min재혈종강주위사점분별이식농도위2X1011L-1신경간세포현액5μ L;PBS조우상동시간점재뇌내상동부위주사PBS:PBS화신경간세포적이식방법동자체혈적이식방법.대조조대서재조모후30 min지조성사점손상,불주사임하물질.주요관찰지표:재조모후립즉,1,3,5,14,21.28 d채용전지평분화전신평분대대서신경공능진행평고.대서우조모후28 d마취후취뇌,병통과쌍표효질섬유산성단백、미관상관단백2、BrdU면역조화래검측이식입뇌적신경간세포재체내적분화정황.결과:①신경공능평분:조모후5 d,각조차이무현저성의의(P＞0.05).조모후14～28 d,간세포이식조교기타3조명현개선(P＜0.05).②뇌조직절편쌍면역조직학쌍표염색결과:간세포이식조혈종주위조망세포소우PBS조.수체대서뇌조직절편현시유BrdU,미관상관단백2,효질섬유산성단백양성세포,설명신경간세포가이재숙주뇌내존활、천사화분화,가이분화위신경원양세포화신경효질양세포.결론:신경간세포이식가능통과분화위신경원양세포화신경효질세포촉진대서뇌출혈적신경공능회복.
BACKGROUND: Exogenous neural stem cells (NSCs) can repair nerve and promote recovery of neurofunction following cerebral hemorrhage.OBJECTIVE: To observe the growth and development of NSCs in vitro, to evaluate the survival, migration and differentiation of transplanted NSCs surrounding hematoma and the possible recovery function of NSCs, and to investigate the repairing effect of NSCs on damaged neurofunction in cerebral hemorrhage model rats.DESIGN: Completely randomized grouping design and controlled animal study,SETTING: Department of Neurosurgery, Huashan Hospital, Fudan University.MATERIALS: Eighteen adult healthy male SD rats weighing 280-320 g were provided by Shanghai Animal Center of Chinese Science Academy. BrdU was provided by Neomarkers Company; rat-anti-glial fibrillary acidic protein (GFAP) and rabbit-anti-microtubule-associated protein-2 (MAP-2) by Chemicon Company.METHODS: This study was performed at the Laboratory of Anatomy and Histology & Embryology, Shanghai Medical College,Fudan University from February to December 2006. The NSCs was isolated, cultured, and evaluated from hippocampus of day E14fetal SD rats. Eighteen rats were randomly divided into control group, PBS group and NSC transplantation group. Cerebralhemorrhage rat models were established via injection of autoiogous arterial blood in caudate nucleus. Thirty minutes after model establishment, 5 μ L NSC suspension with the concentration of 2×1011 L-1 was transplanted at four points surrounding hematoma cavity in the NSC transplantation group. Transplantation of PBS and NSCs was the same as autoblood transplantation. Thirty minutes after model establishment, injuries at the four points were performed, and nothing was injected in the control group.MAIN OUTCOME MEASURES: Neurofunction was evaluated with forward limb scale and turning scale just soon after transplantation and at 1, 3, 5, 14, and 28 days after transplantation. Brain was colleted by anesthesia 28 days after model establishment.Differentiation of transplanted NSCs was detected through testing GFAP, MAP-2, and BrdU by using immunohistochemistry.RESULTS: ①Neurofunction scores: There was no significant difference 5 days after model establishment (P＞0.05). However, the scores were significantly improved in the NSC transplantation group 14-28 days after model establishment (P＜0.05).②lmmunofluorescent double labeling: Apoptosis ceils around hemotoma in the NSC transplantation group were less than those in the PBS group. BrdU and MAP-2 or GFAP-positive ceils were observed in cerebral tissue sections, and this suggested that NSCs could survive, migrate and differentiate in host brain and differentiate into neurons or astrocytes.CONCLUSION: NSC Transplantation contributes to the recovery of neurofunction in cerebral hemorrhage rats through differentiation into neurons or astrocytes.
