脂质体转染影响糖尿病大鼠视网膜下神经干细胞移植的实验研究
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摘要
目的观察阳离子脂质体法转染增强型绿色荧光蛋白的效果,探讨NSCs作为基因靶细胞和EGFP示踪的可行性;探讨阳离子脂质体转染对糖尿病大鼠视网膜下移植NSCs的影响;探讨神经干细胞糖尿病大鼠视网膜下移植后发生炎性反应的可能性。
方法用链尿佐菌素(streptozotocin,STZ)腹腔注射制作大鼠糖尿病视网膜病变(diabetic retinopathy,DR)模型?于模型建立后4周、8周及12周处死大鼠,取眼球做石蜡切片及视网膜铺片?用免疫组化法检测TLR4(toll receiptor 4)、转化因子NF-кB及VEGF在视网膜的表达?用胰酶消化视网膜并行PAS视网膜血管染色,观察管视网膜血管形态变化?从孕12 d的Wistar大鼠胚胎脑泡培养神经干细胞,无血清培养,免疫组织化学技术鉴定。将报告基因pEGFP-N1经Lipofectamine介导转染神经干细胞(EGFP组)观察EGFP表达,以同期培养未转染的细胞作为对照组,测定细胞活力、生长曲线和转染效率。在一组实验(A实验)中,将报告基因pEGFP-N1经Lipofectamine介导转染神经干细胞(EGFP组),以同期培养未转染的细胞作为对照组,将两组神经干细胞分别行糖尿病大鼠视网膜下移植,rt-PCR、明胶酶谱法检测TOLL受体4(TLR4)、核因子NF-κBmRNA、蛋白质的表达。试验分3组:NSCs移植组(A组),DMEM填充组(B组)和非手术对照组(C组)。在另一组实验(B实验)中,大鼠糖尿病成模后,A组视网膜下移植大鼠胚胎神经干细胞,B组视网膜下填充DMEM,C组不做手术处理。western-blot检测核因子NF-κB的蛋白表达。
结果糖尿病大鼠成模后视网膜VEGF的表达持续上调,而视网膜血管自4周出现周细胞的病理改变,8周有微血管瘤形成,12周可见无细胞血管?体外培养出大鼠胚胎神经干细胞。荧光显微镜观察到被转染的神经干细胞长期表达绿色荧光蛋白,两组细胞具有相似的形态学变化和生长曲线,流式细胞仪结果显示转染率最高可达到35.2%。在A实验中,两组细胞分别行糖尿病大鼠视网膜下移植,TLR4,NF-κB mRNA,蛋白质的表达无显著性差异。在B实验中,糖尿病大鼠视网膜下移植大鼠胚胎神经干细胞后,A组大鼠NF-κB持续表达阳性,B组仅术后1周NF-κB阳性表达,C组NF-κB表达为阴性。各组NF-κB表达有显著性差异。
结论链尿佐菌素腹腔注射制作大鼠糖尿病视网膜病变模型成功?成模4周已有病理性改变。大鼠胚胎神经干细胞能够在体外适宜的条件下长期培养;转染EGFP对胚胎神经干细胞的体外增殖无明显的影响;阳离子脂质体Lipofectamine介导转染神经干细胞效率较高,报告基因表达时间长,不影响糖尿病大鼠神经干细胞视网膜下移植,为做标记细胞移植研究及糖尿病视网膜病变的神经干细胞治疗研究奠定基础。糖尿病大鼠NSCs视网膜下移植后NF-κB持续表达阳性,提示术后有严重的炎性反应,或NSCs诱发了免疫反应。
Objective To observe the expression of enhanced green fluorescent protein(EGFP) in NSCs, explore the feasibility of NSCs being gene target cells and EGFP being the tracer. To investigate the effects of Lipofectamine on NSCs transplanted into the subretinal space of rats with streptozotocin-induced diabetes. To learn the inflammation or the immunization after NSCs transplantation.
Methods Diabetic rats were induced by injection of streptozotocin(STZ) intraperitondally. Four week、eight weeks and twelve weeks after the model being builded, the eyeballs were removed for making the retinal vascular network,PAS stained,HE stained and immunohistochemically stained.The NSCs were isolate from Wistar rat embryo brains, then cultured in serum free medium, and identified by immunocytochemisty. The NSCs were either transfected with pEGFP-N1 by Lipofectamine (as EGFP group) or uninfected (as control). The expression of EGFP in NSCs was detected by fluoresecent microscopy. Compare with the control, the cellular viability, the growth curves of the labeled cells were respectively analyzed. Transfection efficiencies were evaluated by flow cytometry. IN one experiment(A experiment), the NSCs were either transplanted into the subretinal space of rats. Toll-like receptor 4(TLR4) were measured by RT-PCR; the protein expression of TLR4 and activation of NF-κB were detected by Western-blot.IN another experiment (B xkperiment), The diabetic rats were randomly divided into three groups, NSCs group(A group)、DMEM group(B group) and contract group(C group).To transplant NSCs into the subretinal space of rats of A group,DMEM into the subretinal space of rats of B group,no special deal with C group. Activation of NF-κB in retina were detected by Western blot.
Results After the model being builded,the expression of VEGF are obviously up-regulated;there were patho-transforms in retinal vascular pericytes after four weeks;the formation of small capillary microaneuryms was occasionally recorded after eight weeks;the acellular capillaries occurred after twelve weeks.The NSCs were successfully cultured; The transfected NSCs expressed EGFP for a long-term. Similar morphological development and growth curves were found in 2 groups. And flow cytometry revealed that the highest transfection rate was up to 35.2%. IN A experiment,the expression of TLR4 and NF-κB had no differentiation between two groups.IN B experiment, compared with C group,western blotting showed up-regulation of NF-κB in A group and B group.
Conclusions Diabetic rats model induced by injection of streptozotocin(STZ) was sucessful. The NSCs originated from Wistar rat embryo brains can be cultured in vitro under appropriate condition, and transfection of EGFP shows no significant effect on the proliferation of NSCs. Furthermore, transfection of NSCs mediated by Lipofectamine is effective and the reported gene has a long-term expression. That me thod can be further applied for the transplantation study of labeled cells. Lipofecta- mine has no effects on the NSCs subretinal transplantation in rats with diabetes. That method can be further applied for the transplantation study of NSCs. In diabetic rats ,the protein of NF-κB has a long-term expression after the NSCs subretinal transplantia- tion. This suggests that NSCs transplantiation induced the serious inflammation or the immunization.
方法用链尿佐菌素(streptozotocin,STZ)腹腔注射制作大鼠糖尿病视网膜病变(diabetic retinopathy,DR)模型?于模型建立后4周、8周及12周处死大鼠,取眼球做石蜡切片及视网膜铺片?用免疫组化法检测TLR4(toll receiptor 4)、转化因子NF-кB及VEGF在视网膜的表达?用胰酶消化视网膜并行PAS视网膜血管染色,观察管视网膜血管形态变化?从孕12 d的Wistar大鼠胚胎脑泡培养神经干细胞,无血清培养,免疫组织化学技术鉴定。将报告基因pEGFP-N1经Lipofectamine介导转染神经干细胞(EGFP组)观察EGFP表达,以同期培养未转染的细胞作为对照组,测定细胞活力、生长曲线和转染效率。在一组实验(A实验)中,将报告基因pEGFP-N1经Lipofectamine介导转染神经干细胞(EGFP组),以同期培养未转染的细胞作为对照组,将两组神经干细胞分别行糖尿病大鼠视网膜下移植,rt-PCR、明胶酶谱法检测TOLL受体4(TLR4)、核因子NF-κBmRNA、蛋白质的表达。试验分3组:NSCs移植组(A组),DMEM填充组(B组)和非手术对照组(C组)。在另一组实验(B实验)中,大鼠糖尿病成模后,A组视网膜下移植大鼠胚胎神经干细胞,B组视网膜下填充DMEM,C组不做手术处理。western-blot检测核因子NF-κB的蛋白表达。
结果糖尿病大鼠成模后视网膜VEGF的表达持续上调,而视网膜血管自4周出现周细胞的病理改变,8周有微血管瘤形成,12周可见无细胞血管?体外培养出大鼠胚胎神经干细胞。荧光显微镜观察到被转染的神经干细胞长期表达绿色荧光蛋白,两组细胞具有相似的形态学变化和生长曲线,流式细胞仪结果显示转染率最高可达到35.2%。在A实验中,两组细胞分别行糖尿病大鼠视网膜下移植,TLR4,NF-κB mRNA,蛋白质的表达无显著性差异。在B实验中,糖尿病大鼠视网膜下移植大鼠胚胎神经干细胞后,A组大鼠NF-κB持续表达阳性,B组仅术后1周NF-κB阳性表达,C组NF-κB表达为阴性。各组NF-κB表达有显著性差异。
结论链尿佐菌素腹腔注射制作大鼠糖尿病视网膜病变模型成功?成模4周已有病理性改变。大鼠胚胎神经干细胞能够在体外适宜的条件下长期培养;转染EGFP对胚胎神经干细胞的体外增殖无明显的影响;阳离子脂质体Lipofectamine介导转染神经干细胞效率较高,报告基因表达时间长,不影响糖尿病大鼠神经干细胞视网膜下移植,为做标记细胞移植研究及糖尿病视网膜病变的神经干细胞治疗研究奠定基础。糖尿病大鼠NSCs视网膜下移植后NF-κB持续表达阳性,提示术后有严重的炎性反应,或NSCs诱发了免疫反应。
Objective To observe the expression of enhanced green fluorescent protein(EGFP) in NSCs, explore the feasibility of NSCs being gene target cells and EGFP being the tracer. To investigate the effects of Lipofectamine on NSCs transplanted into the subretinal space of rats with streptozotocin-induced diabetes. To learn the inflammation or the immunization after NSCs transplantation.
Methods Diabetic rats were induced by injection of streptozotocin(STZ) intraperitondally. Four week、eight weeks and twelve weeks after the model being builded, the eyeballs were removed for making the retinal vascular network,PAS stained,HE stained and immunohistochemically stained.The NSCs were isolate from Wistar rat embryo brains, then cultured in serum free medium, and identified by immunocytochemisty. The NSCs were either transfected with pEGFP-N1 by Lipofectamine (as EGFP group) or uninfected (as control). The expression of EGFP in NSCs was detected by fluoresecent microscopy. Compare with the control, the cellular viability, the growth curves of the labeled cells were respectively analyzed. Transfection efficiencies were evaluated by flow cytometry. IN one experiment(A experiment), the NSCs were either transplanted into the subretinal space of rats. Toll-like receptor 4(TLR4) were measured by RT-PCR; the protein expression of TLR4 and activation of NF-κB were detected by Western-blot.IN another experiment (B xkperiment), The diabetic rats were randomly divided into three groups, NSCs group(A group)、DMEM group(B group) and contract group(C group).To transplant NSCs into the subretinal space of rats of A group,DMEM into the subretinal space of rats of B group,no special deal with C group. Activation of NF-κB in retina were detected by Western blot.
Results After the model being builded,the expression of VEGF are obviously up-regulated;there were patho-transforms in retinal vascular pericytes after four weeks;the formation of small capillary microaneuryms was occasionally recorded after eight weeks;the acellular capillaries occurred after twelve weeks.The NSCs were successfully cultured; The transfected NSCs expressed EGFP for a long-term. Similar morphological development and growth curves were found in 2 groups. And flow cytometry revealed that the highest transfection rate was up to 35.2%. IN A experiment,the expression of TLR4 and NF-κB had no differentiation between two groups.IN B experiment, compared with C group,western blotting showed up-regulation of NF-κB in A group and B group.
Conclusions Diabetic rats model induced by injection of streptozotocin(STZ) was sucessful. The NSCs originated from Wistar rat embryo brains can be cultured in vitro under appropriate condition, and transfection of EGFP shows no significant effect on the proliferation of NSCs. Furthermore, transfection of NSCs mediated by Lipofectamine is effective and the reported gene has a long-term expression. That me thod can be further applied for the transplantation study of labeled cells. Lipofecta- mine has no effects on the NSCs subretinal transplantation in rats with diabetes. That method can be further applied for the transplantation study of NSCs. In diabetic rats ,the protein of NF-κB has a long-term expression after the NSCs subretinal transplantia- tion. This suggests that NSCs transplantiation induced the serious inflammation or the immunization.
引文
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[1] Taylor R M, Mccarthy N, Zhao G, et al. Engraftment, migration and differentiation of genetically modified neural stem cells in the twitcher model of progressive demyelinatlon. EXP Neurol,2002,175(2):434
[2] Baeurele PA. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta,1991,1072:63-80
[3] Marumo T,Schini-Kerth VB,Busse R. Vascular Endothelual Growth Factor Activates Nuclear Factor B and Induces Monocyte Chemoattractant Protein-1 in Bovine Retinal Endothelial Cells[J].Diabetes,1999,48(5):1131-1137
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[2]Stemple D L,Anderson D J. Isolation of a stem cell forneurons and glia from the mammalian neural crest. Cell,1992,71(6):973~985
[3]Reynolds B A,Weiss S.Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J].Science,1992,255(5052):1707-1710.
[4]Mckay R. Stem cells in the central nervous system. Science,1997,276(5309):66
[5]Gage HF. Mammalian neural stem cells[J]. Science,2000,287(5457):1433-1438.
[6]Philips MF, Maattiasson G, Wieloch T, et al. Neuroprotective and behavioral efficacy of nerve growth factor-transfected hippocampal progenitor cell transplants after experimental traumatic brain injury[J]. J Neu2rosury,2001,94(5):765-774.
[7]Kim JH, Auerbach JM, VelascoI, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of parkinson’s disease[J]. Nature,2002,418(4):50-56.
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[13]Johekk, hazel TG, Muller T, et al. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system[J]. Genes Dev,1996,10(24):3129~3140
[14]Ghosh A, Greenberg ME. Distinct roles for bFGF and NT3 in the regulation of cortical neurogenesis[J]. Neuron,1995,15(1):89~103
[15]Bonni A,Sun Y, Nadal-Vicens M, et al. Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway[J]. Science,1997,278(5337):477~483
[16]Williams BP, Park JK, Alberta JA, et al. APDGF regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells[J]. Neuron,1997, 18 (4):553~562
[17]Kokuzawa J, Yoshimura S, Kitajima H, et al. Hepatocyte growth factor promot or proliferation and neuronal differetiation of neural stem cells from mouse embryos[J]. Mol Cell Neuro sci.2003,24(1):190~197
[18]Marie-Claude A, Bruce AC, Gerald ME, et al. N-CAM binding inhibits the proliferation of hippocampal progenitor cells and promotes their diffentiation to a neural phenotype[J]. J Neuro sci,2000,20(10):3631~3640
[19]Akita J, Takahashi M, Hojo M, et al. Neuronal differentiation of adult rat hippocampus derived neural stem cells transplanted into embryonic rat explanted retinas with retinoicacid pretreatment[J] .Brain Res,2002,954(2):2862293.
[20]Dong X, Pulido JS, Sugaya K. Differentiation of human neural stem cells into retinal cells[J]. Neuro report,2003,14(1):1432146.
[21]Groszer M, Erickson R, Scripture-Adams DD, et al. Negative regulation of neural stem: progenitor cell proliferation by the pten tumor suppressor gene in vivo[J]. Science,2001, 294(5549):2186-2189.
[22]Zhong W, Jiang MM, Schonemann MD, et al. Mouse numb is an essential gene involved in cortical neurogenesis[J]. Proc Natl Acad Sci USA,2000,97(12):6844-6849.
[23]张艳君,范祥,胡利民.不同治则中药单体对体外培养神经干细胞分化的影响[J].天津中医药,2004,21(2):156~157
[24]陈东,孟晓婷,刘佳梅,等.鹿茸多肽对胎大鼠脑神经干细胞体外诱导分化的实验研究[J].解剖学报,2004,35(3):240~243
[25]黄镇,金国华,张新化,等.银杏内酯B对成年大鼠神经干细胞向神经元分化的促进作用[J].解剖学报,2003,34(4):367~371
[26]孙晋浩,扬琳,高英茂.补阳还五汤对神经干细胞生长分化的影响[J].山东大学学报,2002,40(5):406~408
[27]Amar AP, Zlokovic BV, Apuzzo MLJ. Endovascular restorative neuro-surgery: A novel concept for molecular and cellular therapy of the nervous system[J]. Neurosurgery,2003,52(2):402-413.
[28]Modo M, Rezaie P, Heuschling P, et al. Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response[J]. Brain Research,2002,958(8):70-82.
[29]Temple S. The development of neural stem cells[J]. Nature,2001,414(1):112-117.
[30]冯定庆,陈晓蓉.神经干细胞的基础研究进展及应用前景[J].解剖学研究,2004,26(1):66 [31]Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. J Neuro sci Res,2000, 61 (4): 364-368.
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[33]Martinez-Serrano A, Bjorklund A. Ex vivo nerve growth factor gene transfer to the basal forebrain in presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and cognitive impairment during aging[J]. Neurobiology, 1998, 95(4):1858-1863.
[34]Cao Q, Benton RL, Whittemore SR. Stem cell repair of central nervous system injury [J]. J Neuro science,2002,68(5):501-510.
[35]Yang H, Mujtaba T, Venkatraman G, et al. Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation[J]. Proc Natl Acad Sci USA,2000,97(24):13366-13371.
[36]Shihabuddin LS, Homer PJ, Ray J, et al. Adult spinal cord stem cells generateneurons after transplantation in the adult dentate gyrus[J]. J Neuro sci,2000,20(23):8727-8735.
[37]Suhonen JO, Peterson DA, Ray J, et al. Differentiation of adult hippocampus derived progenitors into olfactory neurons in vivo[J]. Nature,1996,383(6601):624-627.
[38]Chacko DM, Das AV, Zhao X, et al. Transplantation of ocular stem cells: the role of injury in incorporation and differentiation of grafted cells in the retina[J]. VisionRes, 2003, 43(8):9372946.
[39]Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in the neocortex of adult mice[J]. Nature,2000,405(6789):9512955.
[40]Cao W, Wen R, Li F, et al. Mechanical injury increases bFGF and CNTFmRNA expression in the mouse retina[J]. Exp Eye Res,1997,65(2):2412248.
[41]Gouras P, Du J, Kjeldbye H, et al. Long-term photoreceptor transplants in dystrophic and normal mouse retina[J]. IOVS,1994,35(8):31452-3153.
[42]Chacko DM, Rogers JA, Turner JE, et al. Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat[J]. Biochem Biophys Res Commun,2000,268(3):842-846.
[43]Hoffelen SJV, Young MJ, Shatos MA, et al. Incorporation of murine brain progenitor cells into the developing mammalian retina[J]. IOVS,2003,44(1):4262434.
[44] Hughes SM,Moussavi-Harami F,Sauter SL,et al. Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol Ther,2002,5:16-24.
[45]Varga CM, Hong K, Lauffenburger DA. Quantitative analysis of synthetic gene delivery vector design properties. Mol Ther,2001,4:438-446.
[46]Zellmer S, Gaunitz F, Salvetter J,et al. Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes. Histochem Cell Biol,2001,115:41-47 .
[47]Akerud P, Canals JM, Sunder EY, et al. Neuroprotection through deliver of glial cellline derived neurotrophic factor by neural stem cell in a mouse model of parkinson’s disease[J]. J Neuro sci,2001,21(20):8108-8118.
[48]Dunn LF, Black PM. The neurosurgeon as local oncologist: cellular and molecular neurosurgery in malignant glioma therapy[J]. Neurosurgery,2003,52(6):1411-1424.
[49]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial glioms[J]. Proc Natl Acad Sci USA,2000,97(23):12846-12851.
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1. Reynolds B A,Weiss S.Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J].Science,1992,255(5052):1707-1710.
2. Mckay R. Stem cells in the central nervous system. Science,1997,276(5309):66
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6. Taylor R M, Mccarthy N, Zhao G, et al. Engraftment, migration and differentiation of genetically modified neural stem cells in the twitcher model of progressive demyelinatlon. EXP Neurol,2002,175(2):434
7. Akerud P, Canals JM, Sunder EY, et al. Neuroprotection through deliver of glial cell line derived neurotrophic factor by neural stem cell in a mouse model of parkinson’s disease[J]. J Neuro sci,2001,21(20):8108-8118.
8. Dunn LF, Black PM. The neurosurgeon as local oncologist: cellular and molecular neurosurgery in malignant glioma therapy[J]. Neurosurgery,2003,52(6):1411-1424.
9.李瑛琦,陆文超,于治国.三叶青的化学成分研究[J].中草药,2003,34(11): 982-983.
10. Zhang G, Gurtu V, Kain SR. An enhanced green fluorecent protein allows sensitive detection of gene transfer in mammalian cells [J]. Biochem Biophys Res Commun, 1996, 227(3): 707-711.
11. Hughes SM, Moussavi-Harami F, Sauter SL, et al. Viral-mediated gene transfer to mouse primary neural progenitor cells. [J]Mol Ther, 2002, 5: 16-24.
12. Wu P, Ye Y, Svendsen CN. Transduction of human neural progenitor cells using recombinant adeno-associtaed viral vector. [J] Gene Ther, 2002, 9: 245-255.
13. Modo M, Rezaie P, Heuschling P, et al. Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response [J]. Brain Research, 2002, 958(8): 70-82.
1.谢平,刘庆淮,戈应宾。糖尿病大鼠早期视网膜功能的损害及其发生机制[J].中国临床康复,2005,2(14):144-146。
2. Taylor R M, Mccarthy N, Zhao G, et al. Engraftment, migration and differentiation of genetically modified neural stem cells in the twitcher model of progressive demyelinatlon. EXPNeurol,2002,175(2):434
3. Brannen CL,Sugaya SO.In vitro differentiation of multipotent human neural progenitor sinserum2 free medium[J].Neuro report,2000,11(5).
4. Meltzer H,Hatton JD,Sang UH. Cell type2 specific development of ro2 dent central nervous system progenitor cells in culture[J].J Neuro Surg, 1998,88(1):93-98.
5. Dan D.Lasic. Novel application of liposomes.TIBTECH,1998,16:307-321.
6. Akira S,Takeda K,Kaisho T. Nat,Immunol,2001,2(8):675-680.
7. Barton GM, Medzhitov R. Toll-like receptor signaling pathways. Science 2003; 300:1524–5.
8. Ohashi K,Burkart V,Flohe S,et al. Cutting edge heat shock protein60 is a putative endogenous ligand of the toll-like receptor-4 complex[J]. J Immunol, 2000, 164: 558 -561.
9. Okamura Y,Watari M,Jerud ES,et al.The extra domain A of Fibronectin activates Toll-like receptor4[J]. J Biol Chem,2001,276:10229-10233.
10. Termeer C,Benedix F,Sleeman J,et al. Oligosaccharides of hyaluronan activate dendritic cells via toll-like receptor4[J]. J Exp Med,2002,195:99-111.
11. Cohen SB, Gaskins C, Nasoff MS. Generation of a monoclonal antibody agonist to toll-like receptor 4.Hybridoma(Larchmt). 2005 Feb;24(1):27-35.
[1] Taylor R M, Mccarthy N, Zhao G, et al. Engraftment, migration and differentiation of genetically modified neural stem cells in the twitcher model of progressive demyelinatlon. EXP Neurol,2002,175(2):434
[2] Baeurele PA. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta,1991,1072:63-80
[3] Marumo T,Schini-Kerth VB,Busse R. Vascular Endothelual Growth Factor Activates Nuclear Factor B and Induces Monocyte Chemoattractant Protein-1 in Bovine Retinal Endothelial Cells[J].Diabetes,1999,48(5):1131-1137
[1]Morrison SJ,Shah NM, Anderson DJ.Stem cells in the central nervous system . Science, 1997, 276(5309):66~79
[2]Stemple D L,Anderson D J. Isolation of a stem cell forneurons and glia from the mammalian neural crest. Cell,1992,71(6):973~985
[3]Reynolds B A,Weiss S.Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J].Science,1992,255(5052):1707-1710.
[4]Mckay R. Stem cells in the central nervous system. Science,1997,276(5309):66
[5]Gage HF. Mammalian neural stem cells[J]. Science,2000,287(5457):1433-1438.
[6]Philips MF, Maattiasson G, Wieloch T, et al. Neuroprotective and behavioral efficacy of nerve growth factor-transfected hippocampal progenitor cell transplants after experimental traumatic brain injury[J]. J Neu2rosury,2001,94(5):765-774.
[7]Kim JH, Auerbach JM, VelascoI, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of parkinson’s disease[J]. Nature,2002,418(4):50-56.
[8]Galli R, Gritti A, Bonfanti L, et al. Neural stem cells: an overview[J]. CirculationResearch,2003,4(4): 598-608.
[9]Vescovi AL, Synder EY. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo[J]. Brain Pathology,1999,9(3):569-598.
[10]Brannen CL, Sugaya SO. In vitro differentiation of multipotent human neural progenitors in serum-free medium[J]. Neuro report,2000,11(5):
[11]Meltzer H, Hatton JD, Sang UH. Cell type2 specific development ofro2dent central nervous system progenitor cells in culture[J].JNeuroSurg,1998,88(1):93-98.
[12]Parmentier M L,Woods D,Greig S,et al.Rapsynoid/partner of inscuteable controls asymmetric division of Larval neuroblasts in Drosophila[J].JNeurosci,2000,20(84):1-5.
[13]Johekk, hazel TG, Muller T, et al. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system[J]. Genes Dev,1996,10(24):3129~3140
[14]Ghosh A, Greenberg ME. Distinct roles for bFGF and NT3 in the regulation of cortical neurogenesis[J]. Neuron,1995,15(1):89~103
[15]Bonni A,Sun Y, Nadal-Vicens M, et al. Regulation of gliogenesis in the central nervous system by the JAK-STAT signaling pathway[J]. Science,1997,278(5337):477~483
[16]Williams BP, Park JK, Alberta JA, et al. APDGF regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells[J]. Neuron,1997, 18 (4):553~562
[17]Kokuzawa J, Yoshimura S, Kitajima H, et al. Hepatocyte growth factor promot or proliferation and neuronal differetiation of neural stem cells from mouse embryos[J]. Mol Cell Neuro sci.2003,24(1):190~197
[18]Marie-Claude A, Bruce AC, Gerald ME, et al. N-CAM binding inhibits the proliferation of hippocampal progenitor cells and promotes their diffentiation to a neural phenotype[J]. J Neuro sci,2000,20(10):3631~3640
[19]Akita J, Takahashi M, Hojo M, et al. Neuronal differentiation of adult rat hippocampus derived neural stem cells transplanted into embryonic rat explanted retinas with retinoicacid pretreatment[J] .Brain Res,2002,954(2):2862293.
[20]Dong X, Pulido JS, Sugaya K. Differentiation of human neural stem cells into retinal cells[J]. Neuro report,2003,14(1):1432146.
[21]Groszer M, Erickson R, Scripture-Adams DD, et al. Negative regulation of neural stem: progenitor cell proliferation by the pten tumor suppressor gene in vivo[J]. Science,2001, 294(5549):2186-2189.
[22]Zhong W, Jiang MM, Schonemann MD, et al. Mouse numb is an essential gene involved in cortical neurogenesis[J]. Proc Natl Acad Sci USA,2000,97(12):6844-6849.
[23]张艳君,范祥,胡利民.不同治则中药单体对体外培养神经干细胞分化的影响[J].天津中医药,2004,21(2):156~157
[24]陈东,孟晓婷,刘佳梅,等.鹿茸多肽对胎大鼠脑神经干细胞体外诱导分化的实验研究[J].解剖学报,2004,35(3):240~243
[25]黄镇,金国华,张新化,等.银杏内酯B对成年大鼠神经干细胞向神经元分化的促进作用[J].解剖学报,2003,34(4):367~371
[26]孙晋浩,扬琳,高英茂.补阳还五汤对神经干细胞生长分化的影响[J].山东大学学报,2002,40(5):406~408
[27]Amar AP, Zlokovic BV, Apuzzo MLJ. Endovascular restorative neuro-surgery: A novel concept for molecular and cellular therapy of the nervous system[J]. Neurosurgery,2003,52(2):402-413.
[28]Modo M, Rezaie P, Heuschling P, et al. Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response[J]. Brain Research,2002,958(8):70-82.
[29]Temple S. The development of neural stem cells[J]. Nature,2001,414(1):112-117.
[30]冯定庆,陈晓蓉.神经干细胞的基础研究进展及应用前景[J].解剖学研究,2004,26(1):66 [31]Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. J Neuro sci Res,2000, 61 (4): 364-368.
[32] Taylor R M, Mccarthy N, Zhao G, et al. Engraftment, migration and differentiation of genetically modified neural stem cells in the twitcher model of progressive demyelinatlon. EXP Neurol,2002,175(2):434
[33]Martinez-Serrano A, Bjorklund A. Ex vivo nerve growth factor gene transfer to the basal forebrain in presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and cognitive impairment during aging[J]. Neurobiology, 1998, 95(4):1858-1863.
[34]Cao Q, Benton RL, Whittemore SR. Stem cell repair of central nervous system injury [J]. J Neuro science,2002,68(5):501-510.
[35]Yang H, Mujtaba T, Venkatraman G, et al. Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation[J]. Proc Natl Acad Sci USA,2000,97(24):13366-13371.
[36]Shihabuddin LS, Homer PJ, Ray J, et al. Adult spinal cord stem cells generateneurons after transplantation in the adult dentate gyrus[J]. J Neuro sci,2000,20(23):8727-8735.
[37]Suhonen JO, Peterson DA, Ray J, et al. Differentiation of adult hippocampus derived progenitors into olfactory neurons in vivo[J]. Nature,1996,383(6601):624-627.
[38]Chacko DM, Das AV, Zhao X, et al. Transplantation of ocular stem cells: the role of injury in incorporation and differentiation of grafted cells in the retina[J]. VisionRes, 2003, 43(8):9372946.
[39]Magavi SS, Leavitt BR, Macklis JD. Induction of neurogenesis in the neocortex of adult mice[J]. Nature,2000,405(6789):9512955.
[40]Cao W, Wen R, Li F, et al. Mechanical injury increases bFGF and CNTFmRNA expression in the mouse retina[J]. Exp Eye Res,1997,65(2):2412248.
[41]Gouras P, Du J, Kjeldbye H, et al. Long-term photoreceptor transplants in dystrophic and normal mouse retina[J]. IOVS,1994,35(8):31452-3153.
[42]Chacko DM, Rogers JA, Turner JE, et al. Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat[J]. Biochem Biophys Res Commun,2000,268(3):842-846.
[43]Hoffelen SJV, Young MJ, Shatos MA, et al. Incorporation of murine brain progenitor cells into the developing mammalian retina[J]. IOVS,2003,44(1):4262434.
[44] Hughes SM,Moussavi-Harami F,Sauter SL,et al. Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol Ther,2002,5:16-24.
[45]Varga CM, Hong K, Lauffenburger DA. Quantitative analysis of synthetic gene delivery vector design properties. Mol Ther,2001,4:438-446.
[46]Zellmer S, Gaunitz F, Salvetter J,et al. Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes. Histochem Cell Biol,2001,115:41-47 .
[47]Akerud P, Canals JM, Sunder EY, et al. Neuroprotection through deliver of glial cellline derived neurotrophic factor by neural stem cell in a mouse model of parkinson’s disease[J]. J Neuro sci,2001,21(20):8108-8118.
[48]Dunn LF, Black PM. The neurosurgeon as local oncologist: cellular and molecular neurosurgery in malignant glioma therapy[J]. Neurosurgery,2003,52(6):1411-1424.
[49]Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial glioms[J]. Proc Natl Acad Sci USA,2000,97(23):12846-12851.
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