骨髓间充质干细胞移植治疗血管性痴呆模型鼠的实验研究
|
摘要
第一部分大鼠骨髓间充质干细胞的分离、培养和鉴定
【目的】体外分离培养大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs),观察其生物学特性并对其进行鉴定和标记。
【方法】采用全骨髓贴壁法在体外分离培养大鼠MSCs,相差显微镜观察MSCs细胞形态,透射电镜观察MSCs超微结构并绘制生长曲线,分别使用成骨细胞诱导液和脂肪细胞诱导液诱导其向成骨细胞和脂肪细胞两个方向分化。用BrdU对MSCs进行体外标记,通过免疫组化染色观察并计算BrdU阳性细胞标记率。
【结果】分离培养的MSCs为贴壁生长的成纤维样细胞,传代后的细胞均一性好。超微结构显示MSCs核质比大,胞浆可见较丰富的粗面内质网、线粒体等,细胞表面有大量微绒毛。第3代MSCs生长曲线呈S形,经历潜伏期、对数增殖期和停滞期。经诱导的MSCs能分化为成骨细胞和脂肪细胞,茜素红S和油红O染色阳性。10μmol/L BrdU体外孵育48h后标记率为90%。
【结论】体外MSCs易于分离、培养和扩增。体外培养的MSCs有独特的超微结构特点。MSCs具有多向分化潜能,可通过诱导向成骨细胞和脂肪细胞分化。用BrdU标记MSCs的浓度为10μmol/L,时间为48h。第二部分大鼠骨髓间充质干细胞对血管性痴呆大鼠学习记忆功能及对BDNF表达的影响
【目的】建立2-VO血管性痴呆大鼠模型。探讨MSCs在VD大鼠脑组织中的存活情况,MSCs对VD大鼠空间学习记忆功能、病理改变及脑组织内BDNF表达的影响。
【方法】①将11～13月龄的SD大鼠随机分成3组:假手术组(n＝10),暴露双侧颈总动脉但不结扎;模型对照组(n＝15),结扎双侧颈总动脉(2-VO),30d后脑立体定位将10μl PBS注入大鼠海马内;MSCs治疗组(n＝15),2-VO 30d后脑立体定位将1×10~6 MSCs移植入大鼠海马内。②饲养4w后以Morris水迷宫检测各组大鼠空间学习记忆能力。③HE染色后光镜下观察各组大鼠海马区和额叶的病理变化。④免疫组织化学染色观察BrdU标记的MSCs在VD大鼠脑组织中的存活情况及BDNF在各组大鼠脑组织中的表达情况并进行分析。
【结果】①造模前各组大鼠Morris水迷宫检测成绩差别无统计学意义,海马定位移植4w后,模型对照组与假手术组比较,平均逃避潜伏期长(P＜0.01),且60s内在原平台象限滞留时间短(P＜0.01)。而MSCs治疗组测试成绩优于模型对照组。②光镜下可见模型对照组细胞核固缩,胞质密度增高,细胞脱失。而MSCs治疗组上述病理改变减轻。③MSCs移植4w后可在海马及额叶观察到BrdU标记的MSCs。④海马定位移植4w后,模型对照组海马及额叶的BDNF表达较假手术组少(P＜0.05),而MSCs治疗组BDNF表达较模型对照组高(P＜0.05)。
【结论】①MSCs移植4W后,VD大鼠空间学习记忆能力得到改善。②BrdU免疫组化染色证实MSCs能够在移植鼠脑内存活并迁移。③MSCs可以增加VD大鼠脑内BDNF的表达,提示MSCs可能通过增加BDNF的表达改善VD大鼠空间学习记忆能力。
Part 1 Isolation,cultivation and identification of rat bone marrow-derived Mesenchymai Stem Cells(MSCs)
[Objective]To study the isolation,purification of rat MSCs in vitro. Detecte the biological features of MSCs.Identify and label the MSCs in the study.
[Methods]We selected suitable culture medium and FBS concentration, separated and purified the MSCs with adherent method.The morphology of MSCs was observed with invert-microscopy constantly.The ultrastructure of MSCs was observed by transmission electron microscope.MSCs'curve of growth was depicted.MSCs were induced to differentiate into osteoblasts and adipocytes in suitable induction medium.MSCs were marked in vitro by BrdU. Immunohistochemical stain and light microscope were used for identification BrdU marker ratio.
[Results]MSCs could be isolated and proliferated by adherent method in vitro.The appearence of MSCs were fibroblast-like cells and its growth curve was like shape of S.Under the electron microscope MSCs had plentiful rough endoplasmic reticula.The ratio of the nucleus to cytoplasm was high,and the surface of MSCs had a lot of microvilli.MSCs could be differentiated into osteoblasts and adipocytes in vitro.The incubation with 10μmol/L BrdU for 48h achieved over 90%of the labeling rate.
[Conclutions]MSCs can be isolated,pured and expanded easily by adherent method.MSCs have the characters of earlier immature cells.MSCs have mutipotency which can be induced to differentiate into osteoblasts and adipocytes.The optimal BrdU labeling concentration and time period are 10μmol/L and 48h.
Part 2 Transplantation of MSCs improved cognition of VD rats and enhanced the expression of BDNF
[Objective]To construct the model of VD rats.To observe of MSCs improve cognition of VD rats.To observe the pathology and the expression of BDNF in rats' brain tissues after transplantation of MSCs.
[Methods]①The rats were randomly divided into a sham group(n＝10);a model group(n＝15),10μ1 PBS was injected into rats'hippocampus at 30 days after 2-VO;a treatment group(n＝15),1×10~6 MSCs was transplanted into rats' hippocampus at 30 days after 2-VO.②The performance of the rats in Morris water maze were tested at 4 weeks after transplantation.③Hematoxylin eosin staining was used to observe the pathology of rats' brain tissues.④Immunohistochemical staining was used to identify MSCs in VD rats'brains and analysesed the expression of BDNF in brain tissues of rats in all groups.
[Results]①There was no marked difference in the sham group,model group and treatment group before 2-VO.4w after transplantation,compared with the sham group,the average escape latency of model group was longer(P＜0.01),and the lingering time in platform area was shorter(P＜0.01).But the performance of treatment group had obvious improvement compared with the model group.②Light microscope assessment showed that the brain tissue in the model group had the pathology changes such as pyknosis and condensation of the cytoplasm.These damage was lighten in the treatment group compared to the model group.③BrdU positive cells were found in the brain tissue of rats in the treatment group by immunohistochemical assessment.④4w after transplantation,the number of BDNF positive cells in model group was less than sham group(P＜0.05).But BDNF increased in the treatment group(P＜0.05)
[Conclutions]①MSCs which was transplanted into VD rats' hippocampus can live and migrate in VD rats'cerebral,and improve cognition of VD rats at 4w after translpantation.②The transplanted MSCs could enhance the expression of the BDNF.It suggests that the improvement of cognition of VD rats is probably due to the increased BDNF.
【目的】体外分离培养大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs),观察其生物学特性并对其进行鉴定和标记。
【方法】采用全骨髓贴壁法在体外分离培养大鼠MSCs,相差显微镜观察MSCs细胞形态,透射电镜观察MSCs超微结构并绘制生长曲线,分别使用成骨细胞诱导液和脂肪细胞诱导液诱导其向成骨细胞和脂肪细胞两个方向分化。用BrdU对MSCs进行体外标记,通过免疫组化染色观察并计算BrdU阳性细胞标记率。
【结果】分离培养的MSCs为贴壁生长的成纤维样细胞,传代后的细胞均一性好。超微结构显示MSCs核质比大,胞浆可见较丰富的粗面内质网、线粒体等,细胞表面有大量微绒毛。第3代MSCs生长曲线呈S形,经历潜伏期、对数增殖期和停滞期。经诱导的MSCs能分化为成骨细胞和脂肪细胞,茜素红S和油红O染色阳性。10μmol/L BrdU体外孵育48h后标记率为90%。
【结论】体外MSCs易于分离、培养和扩增。体外培养的MSCs有独特的超微结构特点。MSCs具有多向分化潜能,可通过诱导向成骨细胞和脂肪细胞分化。用BrdU标记MSCs的浓度为10μmol/L,时间为48h。第二部分大鼠骨髓间充质干细胞对血管性痴呆大鼠学习记忆功能及对BDNF表达的影响
【目的】建立2-VO血管性痴呆大鼠模型。探讨MSCs在VD大鼠脑组织中的存活情况,MSCs对VD大鼠空间学习记忆功能、病理改变及脑组织内BDNF表达的影响。
【方法】①将11～13月龄的SD大鼠随机分成3组:假手术组(n＝10),暴露双侧颈总动脉但不结扎;模型对照组(n＝15),结扎双侧颈总动脉(2-VO),30d后脑立体定位将10μl PBS注入大鼠海马内;MSCs治疗组(n＝15),2-VO 30d后脑立体定位将1×10~6 MSCs移植入大鼠海马内。②饲养4w后以Morris水迷宫检测各组大鼠空间学习记忆能力。③HE染色后光镜下观察各组大鼠海马区和额叶的病理变化。④免疫组织化学染色观察BrdU标记的MSCs在VD大鼠脑组织中的存活情况及BDNF在各组大鼠脑组织中的表达情况并进行分析。
【结果】①造模前各组大鼠Morris水迷宫检测成绩差别无统计学意义,海马定位移植4w后,模型对照组与假手术组比较,平均逃避潜伏期长(P＜0.01),且60s内在原平台象限滞留时间短(P＜0.01)。而MSCs治疗组测试成绩优于模型对照组。②光镜下可见模型对照组细胞核固缩,胞质密度增高,细胞脱失。而MSCs治疗组上述病理改变减轻。③MSCs移植4w后可在海马及额叶观察到BrdU标记的MSCs。④海马定位移植4w后,模型对照组海马及额叶的BDNF表达较假手术组少(P＜0.05),而MSCs治疗组BDNF表达较模型对照组高(P＜0.05)。
【结论】①MSCs移植4W后,VD大鼠空间学习记忆能力得到改善。②BrdU免疫组化染色证实MSCs能够在移植鼠脑内存活并迁移。③MSCs可以增加VD大鼠脑内BDNF的表达,提示MSCs可能通过增加BDNF的表达改善VD大鼠空间学习记忆能力。
Part 1 Isolation,cultivation and identification of rat bone marrow-derived Mesenchymai Stem Cells(MSCs)
[Objective]To study the isolation,purification of rat MSCs in vitro. Detecte the biological features of MSCs.Identify and label the MSCs in the study.
[Methods]We selected suitable culture medium and FBS concentration, separated and purified the MSCs with adherent method.The morphology of MSCs was observed with invert-microscopy constantly.The ultrastructure of MSCs was observed by transmission electron microscope.MSCs'curve of growth was depicted.MSCs were induced to differentiate into osteoblasts and adipocytes in suitable induction medium.MSCs were marked in vitro by BrdU. Immunohistochemical stain and light microscope were used for identification BrdU marker ratio.
[Results]MSCs could be isolated and proliferated by adherent method in vitro.The appearence of MSCs were fibroblast-like cells and its growth curve was like shape of S.Under the electron microscope MSCs had plentiful rough endoplasmic reticula.The ratio of the nucleus to cytoplasm was high,and the surface of MSCs had a lot of microvilli.MSCs could be differentiated into osteoblasts and adipocytes in vitro.The incubation with 10μmol/L BrdU for 48h achieved over 90%of the labeling rate.
[Conclutions]MSCs can be isolated,pured and expanded easily by adherent method.MSCs have the characters of earlier immature cells.MSCs have mutipotency which can be induced to differentiate into osteoblasts and adipocytes.The optimal BrdU labeling concentration and time period are 10μmol/L and 48h.
Part 2 Transplantation of MSCs improved cognition of VD rats and enhanced the expression of BDNF
[Objective]To construct the model of VD rats.To observe of MSCs improve cognition of VD rats.To observe the pathology and the expression of BDNF in rats' brain tissues after transplantation of MSCs.
[Methods]①The rats were randomly divided into a sham group(n＝10);a model group(n＝15),10μ1 PBS was injected into rats'hippocampus at 30 days after 2-VO;a treatment group(n＝15),1×10~6 MSCs was transplanted into rats' hippocampus at 30 days after 2-VO.②The performance of the rats in Morris water maze were tested at 4 weeks after transplantation.③Hematoxylin eosin staining was used to observe the pathology of rats' brain tissues.④Immunohistochemical staining was used to identify MSCs in VD rats'brains and analysesed the expression of BDNF in brain tissues of rats in all groups.
[Results]①There was no marked difference in the sham group,model group and treatment group before 2-VO.4w after transplantation,compared with the sham group,the average escape latency of model group was longer(P＜0.01),and the lingering time in platform area was shorter(P＜0.01).But the performance of treatment group had obvious improvement compared with the model group.②Light microscope assessment showed that the brain tissue in the model group had the pathology changes such as pyknosis and condensation of the cytoplasm.These damage was lighten in the treatment group compared to the model group.③BrdU positive cells were found in the brain tissue of rats in the treatment group by immunohistochemical assessment.④4w after transplantation,the number of BDNF positive cells in model group was less than sham group(P＜0.05).But BDNF increased in the treatment group(P＜0.05)
[Conclutions]①MSCs which was transplanted into VD rats' hippocampus can live and migrate in VD rats'cerebral,and improve cognition of VD rats at 4w after translpantation.②The transplanted MSCs could enhance the expression of the BDNF.It suggests that the improvement of cognition of VD rats is probably due to the increased BDNF.
引文
[1]Pittenger MF,Mackay AM,Beck SC,ef al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284(5411):143-147.
[2]Hakuno D,Fukuda k,Makino S,et al.Bone marrow-derived regenerated cardiomyocytes(CMG cells) express functional adrenergic and muscarinic receptors[J].Circulation,2002,105(3):380-386.
[3]江逊,崔鹏程,陈文弦,等.人骨髓间充质干细胞体外培养及生物特性的观察[J].第四军医大学学报,2003,24(4):351-353.
[4]Koc ON,Day J,Nieder M,et al.Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukody strophy(MLD) and Hurler syndrome(MPS-IH).Bone Marrow Transplant,2002,30(4):215-222.
[5]Le Blanc K,Gotherstrom C,Ringden O,et al.Fetal mesenchymal stem cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta.Transplantation,2005,79(11):1607-1614.
[6]Dennis JE,Carbillet JP,Caplan AI,et al.The STRO-1~+ marrow cell population is multipotential[J].Cells Tissues Organs,2002,170(23):73-82.
[7]Tondreau T,Lagneaux L,Dejeneffe M,et al.Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection:phenotype,proliferation kinetics and differentiation potential.Cytotherapy,2004,6(4):372-379.
[8]张本斯,王凡,邓力,等.大鼠骨髓间充质干细胞的分离纯化与初步鉴定[J].中国组织化学与细胞化学杂志,2003,12(2):161-165.
[9]路艳蒙,付文玉,朴英杰,等.人骨髓间充质干细胞超微结构[J].电子显微学报,2002,21(4):373-376.
[10]Minguell JJ,Erices A,Conget P.Mesenchymal stem cells.Exp Biol Med,2001,226(6):507-520.
[11]Bianco P,Riminucci M,Gronthos S,et al.Bone marrow stromal stem cells:nature,biology and potential applications.Stem Cells,2001,19(3):180-192.
[12]Dominici M,Le BK,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[13]Gratzner HG.Monoclonal antibody to 5-bromo and 5-iodo-deoxyuridine.A new reagent for detection of DNA replication[J].Science,1982,218(4571):474-475.
[14]高东,周中和,王景周.实验性血管性痴呆的模型制作问题[J].国外医学(老年医学分册),2002,23(2):59-61.
[15]Pulsinelli WA,Brierley JB.A new model of bilateral hemispheric ischemia in the unanestheized rat[J].Stroke,1979,10(3):267-272.
[16]刘红梅,高天明.急性脑缺血动物模型实验研究进展[J].第一军医大学学报,1999,19(4):368-370.
[17]王蕊,杨秦飞,唐一鹏,等.大鼠拟“血管性痴呆”模型的改进[J].中国病理生理杂志,2000,16(10):914-916.
[18]Tsuchiya M,Sako K,Yura S,et al.Local cerebral glucose utilisation following acute and chronic bilateral carotid artery ligation in wistar rats:relation to changes in local cerebral blood flow[J].Exp Brain Res,1993,95(1):1-7.
[19]刘汇波,叶翠飞,李斌,等.双侧颈总动脉结扎对大鼠学习记忆功能和海马组织形态学的影响[J].基础医学与临床,1998,18(4):54-58.
[20]Savitz SI,Dinsmore JH,Wechsler LR,et al.Cell therapy for stroke[J].Neuro Rx.2004,1(4):406-414.
[21]孙莉,吴江,王守春,等.慢性前脑缺血大鼠学习记忆功能的研究[J].中风与神经疾病杂志,2002,19(1):20-22.
[22]Barbash IM,Chouraqui P,Baron J,et al.Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium:feasibility,cell migration,and body distribution[J].Circulation,2003,108(7):863-868.
[23]Lee JB,Kuroda S,Shichinohe H,et al.A pre-clinical assessment model of rat autogeneic bone marrow stromal cell transplantation into the central nervous system[J].Brain Res Protoc,2004,14(1):37-44.
[24]Chen J,Li Y,Wang L,et al.Caspase inhibition by Z-VAD increases the survival of grafted bone marrow cells and improves functional outcome after MCAO in rats[J].J Neurol Sci,2002,199(1-2):17-24.
[25]Zhao LR,Duan WM,Reyes M,et al.Human bone marrow stem cells exhibit neural phenotypes and meliorate neurological deficits after gafting into the ischemic brain of rats[J].Exp Neurol,2002,174(1):11-20.
[26]刘伟国,陈向阳,郑学胜,等.不同数量神经干细胞移植治疗颅脑外伤疗效的对比研究[J].全科医学临床与教育.2005,3(4):230-232.
[27]Guillermo ME,Akiva JM,Thomas MC,et al.Adult bone marrow stromal cells in the embryonic brain:engraftrment,migration,differentiation,and long-term survival[J].Neuroscience,2004,24(19):4585-4595.
[28]Gage FH.Mammalian neural stem cells[J].Science,2000,287(5457):1433-1438.
[29]Lowenstein DH,Parent JM.Brain,heal thyself[J].Science,1999,283(5405):1126-1127.
[30]高唱,王景周,陈曼娥.骨髓间充质干细胞对血管性痴呆 BDNF 和NGF 的影响[J].中国临床康复,2003,7(25):3434-3435.
[31]James M,Julie C,Hiao Yan,et al.Distribution of BDNF protein and mRNA in the normal adult rat CNS[J].J Neurosci,1997,17(7):2295-2313.
[32]Castillo DV,Figueroa-Guzman Y,Escobar ML.Brain-derived neurotrophic factor enhances conditioned taste aversion retention[J].Brain Res,2006,1067(1):250-255.
[33]von Bohlen und Halbach O,Krause S,Medina D,et al.Regional-and-age-dependent reduction in trkB receptor expression in the hippocampus is associated with altered spine morphologies[J].Biol Psychiatry,2006,59(9):793-800.
[34]Gingrich JA,Hen R.The broken mouse:The role of development,plasticity and environment in the interpretation of phenotypic changes in knockout mice[J].Curr Opin Neurobiol,2000,10(1):146-152.
[35]李爱丽,杨洪发,王彦红,等.老龄大鼠全脑缺血再灌注部分脑区NGFBDNF的表达[J].中风与神经疾病杂志.1999,16(4):214-216.
[36]Majumdar MK,Thiede MA,Mosca JD,et al.Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs) and stromal cells[J].J Cell Physiol,1998,176(1):57-66.
[37]Suzumura.The cytokines and their functions on neural cells[J].Tanpakushitsu Kakusan Koso,1995,40(6):691-699.
[38]Brandoli C,Sanna A,De Bernardi MA,et al.Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA neurons receptor function in cerebellar granule cells[J].J Neurosci.1998,18(19):7953-7961.
[39]Lindvall O,Kokaia Z,Bengzon J,et al.Neurotrophins and brain insults[J].Trends Neurosci,1994,17(11):490-496.
[40]Hempstead BL,Martin-Zanca D,Kaplan DR,et al.High-affinity NGF binding requires coexpression of the trk protooncogene and the low-affinity NGF receptor[J].Nature,1991,350(6320):678-683.
[41]Kim DH,Zhao XR.BDNF protects neurons following injury by modulation of caspase activity[J].Neurocritical Care,2005,3(1):71-76.
[42]Horch HW,Katz LC.BDNF release from single cells elicits local dendritic growth in nearby neurons[J].Nat Neurosci,2002,5(11):1177-1184.
[43]Schabitz WR,Berger C,Kollmar R,et al.Effect of brain-derived neurotrophic factor treatment and forced arm use on functional motor recovery after small cortical ischemia[J].Stroke,2004,35(4):992-997.
[44]Marmigere F,Rage F,Tapia-Arancibia L,et al.Regulation of brain-derived neurotrophic factor transcripts by neuronal activation in rat hypothalamic neurons[J].J Neurosci Res,2001,66(3):377-389.
[45]Pang PT,Teng HK,Zaitsev E,et al.Cleavage of pro-BDNF by tPA/plasmin is essential for long-term hippocampal plasticity[J].Science,2004,306(5695):487-491.
[1]Endres M,Fan G Hirl L,et al.Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene espression[J].J Cereb B100d Flow Metab,2000,20(1):139-144.
[2]Castillo DV,Figueroa-Guzman Y,Escobar ML.Brain-deriVed neurotrophic factor enhances conditioned taste aversion retention[J].Brain Res,2006,1067(1):250-255.
[3]von Bohlen und Halbach O,Krause S,Medina D,et al.Regional-and-age-dependent reduction in trkB receptor expressiOn in the hippocampus is associated with altered spine morphologies[J].Biol Psychiatry,2006,59(9):793-800.
[4]Hall J,Thomas KL,Everitt BJ.Rapid and selective induction of BDNF expression in the hippocampus during contextual learning[J].Nat Neurosci.2000,3(6):533-535.
[5]James M,Julie C,Hiao Yan,et al.Distribution of BDNF protein and mRNA in the normal adult rat CNS:evidence for anterograde axonal transport[J].J Neurosci,1997,17(7):2295-2313.
[6]Hirose K,Chan PH.Blockate of glutamate excitotoxicity and its clinical application[J].Neurochem Res,1993,18(14):479-483.
[7]Hara H,Friedlander RM,Gagliardini V,et al.Inhibition of interleukin lbeta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage[J].Proc Natl Acad Sci USA,1997,94(5):2007-2012.
[8]Liu T,Donnell PC,Yong PR,et al.Interleukin-1β mRNA expression in ischemic rat cortex[J].Stroke,1993,24(11):1746-1751.
[9]Barone FC,Arvin B,White RF,et al.Tumor necrosis factor-α:a mediator of focal ischemic brain injury[J].Stroke,1997,28(6):1233-1244.
[10]Kudo T,Tada K,Takeda M,et al.Learning impairment and microtubule-associated protein 2 decrease in gerbils under chronic cerebral hypoperfusion[J].Stroke,1990,21(8):1205-1209.
[11]黄敬,刘学源,梅元武.缺血性痴呆大鼠行为学及突触素改变的研究 [J].中国临床神经科学.2007,15(1):28-32.
[12]Erkinjuntti T,Román G.Emerging therapies for vascular dementia and vascular cognitive impairment[J].Stroke,2004,35(4):1010-1017.
[13]Schabitz WR,Schwab S,Spranger M,et al.Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats[J].J Cereb Blood Flow Metab,1997,17(5):500-506.
[14]Kiprianova I,Freiman TM,Desiderato S,et al.Brain-derived neurotrophic factor prevents neuronal death and glial activation after global ischemia in the rat[J].J Neurosci Res,1999,56(1):21-27.
[15]Ando S,Kobayashi S,Waki H,et al.Animal model of dementia induced by entorhinal synaptic damage and partial restoration of cognitive deficits by BDNF and carnitine[J].J Neurosci Res,2002,70(3):519-527.
[16]高唱,王景周,陈曼娥.骨髓间充质干细胞对血管性痴呆大鼠 BDNF 和NGF 的影响[J].中国临床康复,2003,7(25):3434-3435.
[17]张冰清,王玉良.葛根素对血管性痴呆大鼠海马锥体细胞和 BDNF 表达的影响[J].神经解剖学杂志,2007,23(6):615-620.
[18]Chuang CM,Hsieh CL,Lin HY,et al.Panax notoginseng burk attenuates impairment of learning and memory functions and increases ED1,BDNF and beta-secretase immunoreactive cells in chronic stage ischemia-reperfusion injured rats[J].Am J Chin Med,2008,36(4):685-693.
[19]Brandoli C,Sanna A,De Bernardi MA,et al.Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA neurons receptor function in cerebellar granule cells[J].J Neurosci,1998,18(19):7953-7961.
[20]Lindvall O,Kokaia Z,Bengzon J,et al.Neurotrophins and brain insults[J].TINS,1994,17(11):490-496.
[21]Hempstead BL,Martin-Zanca D,Kaplan DR,et al.High-affinity NGF binding requires coexpression of the trk protooncogene and the low-affinity NGF receptor[J].Nature,1991,350(6320):678-683.
[22]Kim DH,Zhao X.BDNF protects neurons following injury by modulation of caspase activity[J].Neurocrit Care,2005,3(1):71-76.
[23]吴伟,王虔,梁浩.重组腺病毒载体转导的脑源性神经生长因子在大鼠体外培养海马神经元谷氨酸损伤模型中的保护作用[J].中华神经科杂志,2005,38(7):457-459.
[24]Pang PT,Teng HK,Zaitsev E,et al.Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity[J].Science,2004,306(5695):487-491.
[25]Horch HW,Katz LC.BDNF release from single cells elicits local dendritic growth in nearby neurons[J].Nat Neurosci,2002,5(11):1177-1184.
[26]Schabitz WR,Berger C,Kollmar R,et al.Effect of brain-derived neurotrophic factor treatment and forced arm use on functional motor recovery after small cortical ischemia[J].Stroke,2004,35(4):992-997.
[27]Marmigere F,Rage F,Tapia-Arancibia L,et al.Regulation of brain-derived neurotrophic factor transc.ripts by neuronal activation in rat hypothalamic neurons[J].J Neurosci Res,2001,66(3):377-389.
[28]Schabitz WR,Schwab S,Spranger M,et al.Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats[J].J Cereb Blood Flow Metab,1997,17(5):500-506.
[29]Wachs FP,Sebastien CD,Engelhardt M,et al.High efficacy of clonal growth and expansion of adult neural stem cells[J].Lab Invest,2003,83(7):949-962.
[30]宣爱国,龙大宏,杨丹迪,等.BDNF 和神经干细胞联用对老年痴呆鼠基底前脑胆碱能神经元和学习记忆能力的影响[J].神经解剖学杂志,2005,21(4):365-371.
[2]Hakuno D,Fukuda k,Makino S,et al.Bone marrow-derived regenerated cardiomyocytes(CMG cells) express functional adrenergic and muscarinic receptors[J].Circulation,2002,105(3):380-386.
[3]江逊,崔鹏程,陈文弦,等.人骨髓间充质干细胞体外培养及生物特性的观察[J].第四军医大学学报,2003,24(4):351-353.
[4]Koc ON,Day J,Nieder M,et al.Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukody strophy(MLD) and Hurler syndrome(MPS-IH).Bone Marrow Transplant,2002,30(4):215-222.
[5]Le Blanc K,Gotherstrom C,Ringden O,et al.Fetal mesenchymal stem cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta.Transplantation,2005,79(11):1607-1614.
[6]Dennis JE,Carbillet JP,Caplan AI,et al.The STRO-1~+ marrow cell population is multipotential[J].Cells Tissues Organs,2002,170(23):73-82.
[7]Tondreau T,Lagneaux L,Dejeneffe M,et al.Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection:phenotype,proliferation kinetics and differentiation potential.Cytotherapy,2004,6(4):372-379.
[8]张本斯,王凡,邓力,等.大鼠骨髓间充质干细胞的分离纯化与初步鉴定[J].中国组织化学与细胞化学杂志,2003,12(2):161-165.
[9]路艳蒙,付文玉,朴英杰,等.人骨髓间充质干细胞超微结构[J].电子显微学报,2002,21(4):373-376.
[10]Minguell JJ,Erices A,Conget P.Mesenchymal stem cells.Exp Biol Med,2001,226(6):507-520.
[11]Bianco P,Riminucci M,Gronthos S,et al.Bone marrow stromal stem cells:nature,biology and potential applications.Stem Cells,2001,19(3):180-192.
[12]Dominici M,Le BK,Mueller I,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement[J].Cytotherapy,2006,8(4):315-317.
[13]Gratzner HG.Monoclonal antibody to 5-bromo and 5-iodo-deoxyuridine.A new reagent for detection of DNA replication[J].Science,1982,218(4571):474-475.
[14]高东,周中和,王景周.实验性血管性痴呆的模型制作问题[J].国外医学(老年医学分册),2002,23(2):59-61.
[15]Pulsinelli WA,Brierley JB.A new model of bilateral hemispheric ischemia in the unanestheized rat[J].Stroke,1979,10(3):267-272.
[16]刘红梅,高天明.急性脑缺血动物模型实验研究进展[J].第一军医大学学报,1999,19(4):368-370.
[17]王蕊,杨秦飞,唐一鹏,等.大鼠拟“血管性痴呆”模型的改进[J].中国病理生理杂志,2000,16(10):914-916.
[18]Tsuchiya M,Sako K,Yura S,et al.Local cerebral glucose utilisation following acute and chronic bilateral carotid artery ligation in wistar rats:relation to changes in local cerebral blood flow[J].Exp Brain Res,1993,95(1):1-7.
[19]刘汇波,叶翠飞,李斌,等.双侧颈总动脉结扎对大鼠学习记忆功能和海马组织形态学的影响[J].基础医学与临床,1998,18(4):54-58.
[20]Savitz SI,Dinsmore JH,Wechsler LR,et al.Cell therapy for stroke[J].Neuro Rx.2004,1(4):406-414.
[21]孙莉,吴江,王守春,等.慢性前脑缺血大鼠学习记忆功能的研究[J].中风与神经疾病杂志,2002,19(1):20-22.
[22]Barbash IM,Chouraqui P,Baron J,et al.Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium:feasibility,cell migration,and body distribution[J].Circulation,2003,108(7):863-868.
[23]Lee JB,Kuroda S,Shichinohe H,et al.A pre-clinical assessment model of rat autogeneic bone marrow stromal cell transplantation into the central nervous system[J].Brain Res Protoc,2004,14(1):37-44.
[24]Chen J,Li Y,Wang L,et al.Caspase inhibition by Z-VAD increases the survival of grafted bone marrow cells and improves functional outcome after MCAO in rats[J].J Neurol Sci,2002,199(1-2):17-24.
[25]Zhao LR,Duan WM,Reyes M,et al.Human bone marrow stem cells exhibit neural phenotypes and meliorate neurological deficits after gafting into the ischemic brain of rats[J].Exp Neurol,2002,174(1):11-20.
[26]刘伟国,陈向阳,郑学胜,等.不同数量神经干细胞移植治疗颅脑外伤疗效的对比研究[J].全科医学临床与教育.2005,3(4):230-232.
[27]Guillermo ME,Akiva JM,Thomas MC,et al.Adult bone marrow stromal cells in the embryonic brain:engraftrment,migration,differentiation,and long-term survival[J].Neuroscience,2004,24(19):4585-4595.
[28]Gage FH.Mammalian neural stem cells[J].Science,2000,287(5457):1433-1438.
[29]Lowenstein DH,Parent JM.Brain,heal thyself[J].Science,1999,283(5405):1126-1127.
[30]高唱,王景周,陈曼娥.骨髓间充质干细胞对血管性痴呆 BDNF 和NGF 的影响[J].中国临床康复,2003,7(25):3434-3435.
[31]James M,Julie C,Hiao Yan,et al.Distribution of BDNF protein and mRNA in the normal adult rat CNS[J].J Neurosci,1997,17(7):2295-2313.
[32]Castillo DV,Figueroa-Guzman Y,Escobar ML.Brain-derived neurotrophic factor enhances conditioned taste aversion retention[J].Brain Res,2006,1067(1):250-255.
[33]von Bohlen und Halbach O,Krause S,Medina D,et al.Regional-and-age-dependent reduction in trkB receptor expression in the hippocampus is associated with altered spine morphologies[J].Biol Psychiatry,2006,59(9):793-800.
[34]Gingrich JA,Hen R.The broken mouse:The role of development,plasticity and environment in the interpretation of phenotypic changes in knockout mice[J].Curr Opin Neurobiol,2000,10(1):146-152.
[35]李爱丽,杨洪发,王彦红,等.老龄大鼠全脑缺血再灌注部分脑区NGFBDNF的表达[J].中风与神经疾病杂志.1999,16(4):214-216.
[36]Majumdar MK,Thiede MA,Mosca JD,et al.Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs) and stromal cells[J].J Cell Physiol,1998,176(1):57-66.
[37]Suzumura.The cytokines and their functions on neural cells[J].Tanpakushitsu Kakusan Koso,1995,40(6):691-699.
[38]Brandoli C,Sanna A,De Bernardi MA,et al.Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA neurons receptor function in cerebellar granule cells[J].J Neurosci.1998,18(19):7953-7961.
[39]Lindvall O,Kokaia Z,Bengzon J,et al.Neurotrophins and brain insults[J].Trends Neurosci,1994,17(11):490-496.
[40]Hempstead BL,Martin-Zanca D,Kaplan DR,et al.High-affinity NGF binding requires coexpression of the trk protooncogene and the low-affinity NGF receptor[J].Nature,1991,350(6320):678-683.
[41]Kim DH,Zhao XR.BDNF protects neurons following injury by modulation of caspase activity[J].Neurocritical Care,2005,3(1):71-76.
[42]Horch HW,Katz LC.BDNF release from single cells elicits local dendritic growth in nearby neurons[J].Nat Neurosci,2002,5(11):1177-1184.
[43]Schabitz WR,Berger C,Kollmar R,et al.Effect of brain-derived neurotrophic factor treatment and forced arm use on functional motor recovery after small cortical ischemia[J].Stroke,2004,35(4):992-997.
[44]Marmigere F,Rage F,Tapia-Arancibia L,et al.Regulation of brain-derived neurotrophic factor transcripts by neuronal activation in rat hypothalamic neurons[J].J Neurosci Res,2001,66(3):377-389.
[45]Pang PT,Teng HK,Zaitsev E,et al.Cleavage of pro-BDNF by tPA/plasmin is essential for long-term hippocampal plasticity[J].Science,2004,306(5695):487-491.
[1]Endres M,Fan G Hirl L,et al.Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene espression[J].J Cereb B100d Flow Metab,2000,20(1):139-144.
[2]Castillo DV,Figueroa-Guzman Y,Escobar ML.Brain-deriVed neurotrophic factor enhances conditioned taste aversion retention[J].Brain Res,2006,1067(1):250-255.
[3]von Bohlen und Halbach O,Krause S,Medina D,et al.Regional-and-age-dependent reduction in trkB receptor expressiOn in the hippocampus is associated with altered spine morphologies[J].Biol Psychiatry,2006,59(9):793-800.
[4]Hall J,Thomas KL,Everitt BJ.Rapid and selective induction of BDNF expression in the hippocampus during contextual learning[J].Nat Neurosci.2000,3(6):533-535.
[5]James M,Julie C,Hiao Yan,et al.Distribution of BDNF protein and mRNA in the normal adult rat CNS:evidence for anterograde axonal transport[J].J Neurosci,1997,17(7):2295-2313.
[6]Hirose K,Chan PH.Blockate of glutamate excitotoxicity and its clinical application[J].Neurochem Res,1993,18(14):479-483.
[7]Hara H,Friedlander RM,Gagliardini V,et al.Inhibition of interleukin lbeta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage[J].Proc Natl Acad Sci USA,1997,94(5):2007-2012.
[8]Liu T,Donnell PC,Yong PR,et al.Interleukin-1β mRNA expression in ischemic rat cortex[J].Stroke,1993,24(11):1746-1751.
[9]Barone FC,Arvin B,White RF,et al.Tumor necrosis factor-α:a mediator of focal ischemic brain injury[J].Stroke,1997,28(6):1233-1244.
[10]Kudo T,Tada K,Takeda M,et al.Learning impairment and microtubule-associated protein 2 decrease in gerbils under chronic cerebral hypoperfusion[J].Stroke,1990,21(8):1205-1209.
[11]黄敬,刘学源,梅元武.缺血性痴呆大鼠行为学及突触素改变的研究 [J].中国临床神经科学.2007,15(1):28-32.
[12]Erkinjuntti T,Román G.Emerging therapies for vascular dementia and vascular cognitive impairment[J].Stroke,2004,35(4):1010-1017.
[13]Schabitz WR,Schwab S,Spranger M,et al.Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats[J].J Cereb Blood Flow Metab,1997,17(5):500-506.
[14]Kiprianova I,Freiman TM,Desiderato S,et al.Brain-derived neurotrophic factor prevents neuronal death and glial activation after global ischemia in the rat[J].J Neurosci Res,1999,56(1):21-27.
[15]Ando S,Kobayashi S,Waki H,et al.Animal model of dementia induced by entorhinal synaptic damage and partial restoration of cognitive deficits by BDNF and carnitine[J].J Neurosci Res,2002,70(3):519-527.
[16]高唱,王景周,陈曼娥.骨髓间充质干细胞对血管性痴呆大鼠 BDNF 和NGF 的影响[J].中国临床康复,2003,7(25):3434-3435.
[17]张冰清,王玉良.葛根素对血管性痴呆大鼠海马锥体细胞和 BDNF 表达的影响[J].神经解剖学杂志,2007,23(6):615-620.
[18]Chuang CM,Hsieh CL,Lin HY,et al.Panax notoginseng burk attenuates impairment of learning and memory functions and increases ED1,BDNF and beta-secretase immunoreactive cells in chronic stage ischemia-reperfusion injured rats[J].Am J Chin Med,2008,36(4):685-693.
[19]Brandoli C,Sanna A,De Bernardi MA,et al.Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA neurons receptor function in cerebellar granule cells[J].J Neurosci,1998,18(19):7953-7961.
[20]Lindvall O,Kokaia Z,Bengzon J,et al.Neurotrophins and brain insults[J].TINS,1994,17(11):490-496.
[21]Hempstead BL,Martin-Zanca D,Kaplan DR,et al.High-affinity NGF binding requires coexpression of the trk protooncogene and the low-affinity NGF receptor[J].Nature,1991,350(6320):678-683.
[22]Kim DH,Zhao X.BDNF protects neurons following injury by modulation of caspase activity[J].Neurocrit Care,2005,3(1):71-76.
[23]吴伟,王虔,梁浩.重组腺病毒载体转导的脑源性神经生长因子在大鼠体外培养海马神经元谷氨酸损伤模型中的保护作用[J].中华神经科杂志,2005,38(7):457-459.
[24]Pang PT,Teng HK,Zaitsev E,et al.Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity[J].Science,2004,306(5695):487-491.
[25]Horch HW,Katz LC.BDNF release from single cells elicits local dendritic growth in nearby neurons[J].Nat Neurosci,2002,5(11):1177-1184.
[26]Schabitz WR,Berger C,Kollmar R,et al.Effect of brain-derived neurotrophic factor treatment and forced arm use on functional motor recovery after small cortical ischemia[J].Stroke,2004,35(4):992-997.
[27]Marmigere F,Rage F,Tapia-Arancibia L,et al.Regulation of brain-derived neurotrophic factor transc.ripts by neuronal activation in rat hypothalamic neurons[J].J Neurosci Res,2001,66(3):377-389.
[28]Schabitz WR,Schwab S,Spranger M,et al.Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats[J].J Cereb Blood Flow Metab,1997,17(5):500-506.
[29]Wachs FP,Sebastien CD,Engelhardt M,et al.High efficacy of clonal growth and expansion of adult neural stem cells[J].Lab Invest,2003,83(7):949-962.
[30]宣爱国,龙大宏,杨丹迪,等.BDNF 和神经干细胞联用对老年痴呆鼠基底前脑胆碱能神经元和学习记忆能力的影响[J].神经解剖学杂志,2005,21(4):365-371.