GDNF基因修饰的神经干细胞移植对大鼠局灶性脑缺血神经干细胞增殖和星形胶质细胞的影响
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摘要
目的：探讨胶质源性神经营养因子(glial cell line-derived neural factor,GDNF)基因修饰的神经干细胞(neural stem cells,NSCs)移植对大鼠脑缺血后神经干细胞增殖与星形胶质细胞的影响。方法：建立大鼠暂时性大脑中动脉梗塞模型(middle cerebral artery occlusion,MCAO),并将其随机分为：生理盐水移植组(NS)、神经干细胞移植组(NSCs)和GDNF基因修饰的神经干细胞移植组(GDNF/NSCs)。再将各组按照再灌注时间不同分为5个亚组(1W、2W、3W、5W、7W,每个时间点6只大鼠)。再灌注3d采用脑立体定位仪将移植物植入大鼠的右侧脑室。再灌注不同时间点(1W、2W、3W、5W、7W,其中免疫组化和免疫印迹各3只大鼠)处死大鼠,取同侧大脑半球,通过免疫组织化学(immunohistochemistry,IHC)的方法,用nestin抗体观察神经干细胞的增殖,用免疫印迹(Western blotting)的方法,用GFAP抗体观察星形胶质细胞反应。结果：1、各实验组的Nestin阳性细胞主要分布于缺血侧的额顶皮质、纹状体、海马部位。2、Nestin阳性细胞统计学分析表明：GDNF/NSCs、NSCs组Nestin阳性细胞数1w最低,3w达高峰,后逐渐下降。NS组Nestin阳性细胞数1w最高,后逐渐下降。而再灌注1-7w,GDNF/NSCs组、NSCs组Nestin阳性细胞数显著高于NS组(P<0.05),而再灌注2w、3w, GDNF/NSCs组Nestin阳性细胞数显著高于NSCs组(P<0.05)。3、GDNF/NSCs组、NSCs组和NS组GFAP蛋白均在2w达到高峰,后逐渐降低。而再灌注1-7w, GDNF/NSCs组的GFAP蛋白显著高于NS组(P<0.05)；GDNF/NSCs组GFAP蛋白显著高于NSCs组(P<0.05)。再灌注1-5w, NSCs组GFAP蛋白显著高于NS组(P<0.05)。结论：GDNF/NSCs移植治疗大鼠缺血再灌注的神经保护作用,其机制可能与促进神经干细胞的增殖和星形胶质细胞数有关。本实验结果为脑中风的基因治疗与细胞移植的临床应用奠定基础。
Abstract:Objective:To investigate the glial derived neurotrophic factor (glial cell line-derived neural factor, GDNF) gene modified neural stem cells (neural stem cells, NSCs) transplantation in rats with cerebral ischemia of neural stem cells and astrocytes effects. Methods:A rat model of transient middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO), and randomly divided into:saline transplantation group (NS), neural stem cell transplantation group (NSCs) and the GDNF gene modified neural stem cell transplantation group (GDNF/NSCs). Then according to reperfusion time in each group were divided into five sub-groups (1W,2W,3W,5W,7W,6 rats at each time point).3d reperfusion brain stereotactic apparatus used to graft implantation in the right lateral ventricle of rats. Reperfusion at different time points (1W,2W,3W,5W,7W, including immunohistochemistry and Western blot all three rats) rats were killed, taking the ipsilateral hemisphere. By immunohistochemistry (immunohistochemistry, IHC) method, using nestin antibody proliferation of neural stem cells, using immunoblotting (Western blotting) methods, Observed with GFAP antibody reactions of astrocytes. Results:1, the experimental group, Nestin-positive cells were mainly distributed in the ischemic side frontoparietal cortex, striatum, hippocampus.2, Nestin-positive cells in statistical analysis showed that:GDNF/NSCs, NSCs group of Nestin positive cells in the lowest 1w,3w peak, then reduced gradually. NS Group Nestin-positive cells 1w highest, then decreased gradually. And reperfusion 1-7w, GDNF/NSCs group, NSCs group Nestin-positive cells was significantly higher than the NS group (P<0.05), whereas reperfusion 2w,3w, GDNF/NSCs group of Nestin-positive cells was significantly higher than the NSCs group (P<0.05).3, GDNF/NSCs group, NSCs group and NS group were GFAP protein 2w peak, then gradually decreased. And reperfusion 1-7w, GDNF/NSCs group of GFAP protein were significantly higher than the NS group (P<0.05); GDNF/NSCs group of GFAP expression was significantly higher than NSCs group (P<0.05). Reperfusion 1-5w, NSCs group GFAP expression was significantly higher than NS group (P<0.05). Conclusion:GDNF/NSCs transplantation rats with ischemia-reperfusion neuroprotective effect, the mechanism may be related to the promotion of neural stem cell proliferation and the number of the astrocytes. Our results for the stroke gene therapy and cell transplantation in clinical applications.
Abstract:Objective:To investigate the glial derived neurotrophic factor (glial cell line-derived neural factor, GDNF) gene modified neural stem cells (neural stem cells, NSCs) transplantation in rats with cerebral ischemia of neural stem cells and astrocytes effects. Methods:A rat model of transient middle cerebral artery occlusion (middle cerebral artery occlusion, MCAO), and randomly divided into:saline transplantation group (NS), neural stem cell transplantation group (NSCs) and the GDNF gene modified neural stem cell transplantation group (GDNF/NSCs). Then according to reperfusion time in each group were divided into five sub-groups (1W,2W,3W,5W,7W,6 rats at each time point).3d reperfusion brain stereotactic apparatus used to graft implantation in the right lateral ventricle of rats. Reperfusion at different time points (1W,2W,3W,5W,7W, including immunohistochemistry and Western blot all three rats) rats were killed, taking the ipsilateral hemisphere. By immunohistochemistry (immunohistochemistry, IHC) method, using nestin antibody proliferation of neural stem cells, using immunoblotting (Western blotting) methods, Observed with GFAP antibody reactions of astrocytes. Results:1, the experimental group, Nestin-positive cells were mainly distributed in the ischemic side frontoparietal cortex, striatum, hippocampus.2, Nestin-positive cells in statistical analysis showed that:GDNF/NSCs, NSCs group of Nestin positive cells in the lowest 1w,3w peak, then reduced gradually. NS Group Nestin-positive cells 1w highest, then decreased gradually. And reperfusion 1-7w, GDNF/NSCs group, NSCs group Nestin-positive cells was significantly higher than the NS group (P<0.05), whereas reperfusion 2w,3w, GDNF/NSCs group of Nestin-positive cells was significantly higher than the NSCs group (P<0.05).3, GDNF/NSCs group, NSCs group and NS group were GFAP protein 2w peak, then gradually decreased. And reperfusion 1-7w, GDNF/NSCs group of GFAP protein were significantly higher than the NS group (P<0.05); GDNF/NSCs group of GFAP expression was significantly higher than NSCs group (P<0.05). Reperfusion 1-5w, NSCs group GFAP expression was significantly higher than NS group (P<0.05). Conclusion:GDNF/NSCs transplantation rats with ischemia-reperfusion neuroprotective effect, the mechanism may be related to the promotion of neural stem cell proliferation and the number of the astrocytes. Our results for the stroke gene therapy and cell transplantation in clinical applications.
引文
1. Ivanova T,Beyer C.Pre-and postnatal expression of brain-derived neurotrophic factor mRNA/protein and tyrosine protein kinase receptor B mRNA in the mouse hippocampus,[J] Neurosci Lett.2001;307(1):21-4
2. Kitagawa H, Hayashi T, Mitsumoto Y, et al. Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats[J]. Stroke,1998;29(7):1417-1422
3. Kobayashi T, Ahlenius H, Thored P, et al. Intracerebral infusion of glial cell line-derived neurotrophic factor promotes striatal neurogenesis after stroke in adult rats[J]. Stroke,2006;37(9):2361-2367
4. Kitagawa H, Sasa K, Sakai K, et al. Adenovirus-mediated gene transfer of glial cell-derived neurotrophic factor prevents ischemic brain injury after transient middle cerebral artery occlusion in rats[J]. J.Cereb Blood Flow Metab,1999;19(12):1336-1344
5. Nicole O, Ali C, Docagne F, et al. Neuroprotection mediated by glial cell line-derived neurotrophic factor:involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway[J]. The Journal of Neuroscience,2001;21(9):3024-3038
6. Jin G, Omori N, Li F, et al. Protection against ischemic brain damage by GDNF affecting cell survival and death signals[J]. Neurol Res,2003; 25(3):249-253
7. Abe K. Gene therapy and neuroprotection for cerebral infarction[J]. Rinsho Shinkeigaku,2003;43(11):894-896
8.马春明,袁琼兰,杨朝鲜等.胶质细胞源性神经营养因子重组腺病毒载体穿梭质粒的构建及鉴定[J].神经解剖学杂志,2006；22(2)：199-202
9. Chen JL,Yi Li,Wang L,etal. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[J]. Stroke,2001;32(4):1005-11
10.J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译.分子克隆实验指南[M].北京：科学出版社,2002；1583
11.徐忠烨、唐文渊.经尾静脉移植神经干细胞对脑损伤大鼠的保护作用[J].中国神经精神疾病杂志,2006,32(4)：351-353
12. Hiroki Toda, Jun Takahashi, Noboru Iwakami, et al. Grafting neural stem cells improved the impaired spatial recognition in ischemic rats [J]. Neuroscience Letters,2001; 316(2001):9-12
13. Kon Chu, Manho Kim, Kyung-Il Park, et al. Human neural stem cells improve sensorimotor deficits in the adult rat brain with experimental focal ischemia[J]. Brain Research,2004; 1016 (2004): 145-153
14. Park KI, Himes BT, Stieg PE,et al. Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury[J].Exp Neurol,2006;199(1): 179-190
15. AndsbergG,Kokaiaz,BjorkhndA,etal.Ameliorationof schemiainduced neuronal death in the rat striatum by NGF-secreting neural stern cells. Eur[J]Neurosci,1998.10:2026-2036
16. Lin LF, Doherty DH, Lile JD, et al. GDNF:a glial cell line-derived neurotrophic facter for midbrain dopaminergic neurons [J]. Science, 1993,260(5111):1130-1132
17. Yagita Y, Kitagawa K, Sasaki T, et al. Differential expression of Musadhil and nestin in the adult rat hippocampus after ischemia. Neurosci Res,2002,69(6):750-756
18.刘鹏种,陆世择,黄娅林,等.成年鼠缺血性脑损伤诱导nestin的表达.生理学报,2002,54(4)：294-299
19.雷万龙,袁群芳,董志宏,等.大鼠局灶脑缺血诱导巢蛋白的反应模式.中国病理生理杂志,2002,18(6)：639-642
20.Douen AG, Dong L, Vanance S, et al. Regullation of nestin expression after cortical ablation in adult rat brain. Brain Res,2004, 1008(2):139-146
21.Maslov AY, Barone TA, Plunkett RJ, et al. Neural stem cell detection,characterization,and age-related changes in the subventricular zone of mice. J Neurosci,2004,24(7):1726-1733
22. Dempsey RJ,Sailor KA,Bowen KK,et al.Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain:effect of exogenous IGF-1 and GDNF [J] Neurochem,2003,87(3): 586-597
23. Zhang R, Zhang ZQZhang Let al. Proliferation anddifferentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral isehemia[J]. Neuroseience,2001, 1050):33-41
24. Takasawa K, Kitagawa K, Yagita Yet al. Increased prolifetmion of neural progenitor cells but reduced survival of cerebralof cerebral cells in the contralateral hlmcampus after focal cerebral ischemia in rats[J]. J Cereb Blood Flow Metab,2002.22:299-307
25.陈波,高小青,杨朝鲜等.移植GDNF基因修饰的神经干细胞对大鼠脑卒中的神经保护作用[J].解剖学杂志,2009；32(3)：.286-290
26.吴旭,张国华.实验性脑挫伤GFAP. PCNA免疫组织化学研究[J]法医学杂志,1999,15(2)：206
27. HozLlnuI,ChiuFC.NottoWT.Biochemical and immuno-cytochemieal changes in glial fibrillary acidic pmtdn after stab wounds[J].Brain Rearch,1990,524:64
28. Pekny M, Nilsson M. Astrocyte activation and reactive gliosis [J]. Glia,2005,50(4):427-434
1. Mckay R. Stem cells in the central nervous system. Science,1997, 276:66-71.
2. Gage HF. Mammalian neural stem cells. Science,2000,287:1433-1438.
3. Abrahams JM,Gokhan S,Flamm ES,et al. Denovo neurogenesis and acute stroke:are exogenous stem cells really necessary[J] Neurosurgery,2004, 54(1):150-156.
4. Bjornson CR, Rietz RL, Reynolds BA,et al. Turning brain into blood:a hematopoietic fate adopted by adult neural stem cells in vivo[J].Science, 1999;2839(5401):534-537.
5. Deng J, Petersen BE, Steindler DA, et al. Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation[J]. Stem Cells,2006,24(4):1054-1064
6. Doetsch F, Caille I, Lim DA, et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell,1999,11;97: 703-716.
7. Englund U, Bjorklund A, Wictorin K, et al. Grafted neural stem cells develop into Functiongal pyramidal neurons integrate into host cortical circuitry. Proc Natl Acad Sci USA,2002,99:17089-17094
8. Suarez-Rodriguez R, Belkind-Gerson J. Cultured nestin-positive cells from postnatal mouse small bowel differentiate ex Vivo into neurons, glia. and smooth muscle[J].Stem Cells,2004,22(7):1373-138
9. Calhoun JD, Lamben NA, Mitalipova MM, et al. Differentiation of rhesus emhronic stem cells to neural progenitors and neurons[J]. Biochem Biophys Res Commun,2003,306(1):191-197.
10. Marzesco AM, Janich P, Wilsch-Brauninger M, et al.Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD 133) from neural progenitors and other epithelial cells[J]. J Cell Sci.2005,118(13):1849-1858.
11. Lee ST, Park JE, Lee K, et al.Noninvasive method of immortalized neural stem-like cell transplantation in an experimental model of Huntington's disease[J] J NeurosciMethods.2006,152(1-2):250-254.
12. Jin K, Sun Y, Xie L,et al. Comparison of ischemia-directemigration of neural precursor cells after intrastriatal,intraventricular,or intravenous transplantion in the rat[J].Neurobiol Dis,2005,18(2):366-374.
13.王霞,杨于嘉,余小河,等.神经干细胞移植对缺氧缺血新生大鼠感觉运动机能的影响.中国行为医学科学,2005.14：596-598.
14.刘伟国罗呜郑学胜陈向日等, 脑外伤后不同时间移植神经干细胞的疗效对比研究,浙江医学2006.28(1)：33-1109
15.杨东波金力,李永利等神经干细胞移植治疗大鼠局灶性脑缺血：移植部位与移植时间的比较.中国临床康复,2006,10{17}30-2
16. Derek van der Kooy, Samuel Weiss. y stem cels[J]Science, 2000, 287(6):1439-1441.
17. Barker RA, Widner H. Immune problems in central nervous system celtherapy[J]. Neuro Rx,2004,1(4):472-481.18.杨清成,梁长春,李敏霞,等.神经干细胞移植治疗脑卒中后遗症59例[J].中国组织工程研究与临床康复,2007,11(20)：4033-4035.19.张儒有,郑永日,胡韶山,等.神经干细胞移植治疗脑卒中后遗症50例临床效果分析[J].中国临床康复,2006,10(9)：138-139.
2. Kitagawa H, Hayashi T, Mitsumoto Y, et al. Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats[J]. Stroke,1998;29(7):1417-1422
3. Kobayashi T, Ahlenius H, Thored P, et al. Intracerebral infusion of glial cell line-derived neurotrophic factor promotes striatal neurogenesis after stroke in adult rats[J]. Stroke,2006;37(9):2361-2367
4. Kitagawa H, Sasa K, Sakai K, et al. Adenovirus-mediated gene transfer of glial cell-derived neurotrophic factor prevents ischemic brain injury after transient middle cerebral artery occlusion in rats[J]. J.Cereb Blood Flow Metab,1999;19(12):1336-1344
5. Nicole O, Ali C, Docagne F, et al. Neuroprotection mediated by glial cell line-derived neurotrophic factor:involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway[J]. The Journal of Neuroscience,2001;21(9):3024-3038
6. Jin G, Omori N, Li F, et al. Protection against ischemic brain damage by GDNF affecting cell survival and death signals[J]. Neurol Res,2003; 25(3):249-253
7. Abe K. Gene therapy and neuroprotection for cerebral infarction[J]. Rinsho Shinkeigaku,2003;43(11):894-896
8.马春明,袁琼兰,杨朝鲜等.胶质细胞源性神经营养因子重组腺病毒载体穿梭质粒的构建及鉴定[J].神经解剖学杂志,2006；22(2)：199-202
9. Chen JL,Yi Li,Wang L,etal. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats[J]. Stroke,2001;32(4):1005-11
10.J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译.分子克隆实验指南[M].北京：科学出版社,2002；1583
11.徐忠烨、唐文渊.经尾静脉移植神经干细胞对脑损伤大鼠的保护作用[J].中国神经精神疾病杂志,2006,32(4)：351-353
12. Hiroki Toda, Jun Takahashi, Noboru Iwakami, et al. Grafting neural stem cells improved the impaired spatial recognition in ischemic rats [J]. Neuroscience Letters,2001; 316(2001):9-12
13. Kon Chu, Manho Kim, Kyung-Il Park, et al. Human neural stem cells improve sensorimotor deficits in the adult rat brain with experimental focal ischemia[J]. Brain Research,2004; 1016 (2004): 145-153
14. Park KI, Himes BT, Stieg PE,et al. Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury[J].Exp Neurol,2006;199(1): 179-190
15. AndsbergG,Kokaiaz,BjorkhndA,etal.Ameliorationof schemiainduced neuronal death in the rat striatum by NGF-secreting neural stern cells. Eur[J]Neurosci,1998.10:2026-2036
16. Lin LF, Doherty DH, Lile JD, et al. GDNF:a glial cell line-derived neurotrophic facter for midbrain dopaminergic neurons [J]. Science, 1993,260(5111):1130-1132
17. Yagita Y, Kitagawa K, Sasaki T, et al. Differential expression of Musadhil and nestin in the adult rat hippocampus after ischemia. Neurosci Res,2002,69(6):750-756
18.刘鹏种,陆世择,黄娅林,等.成年鼠缺血性脑损伤诱导nestin的表达.生理学报,2002,54(4)：294-299
19.雷万龙,袁群芳,董志宏,等.大鼠局灶脑缺血诱导巢蛋白的反应模式.中国病理生理杂志,2002,18(6)：639-642
20.Douen AG, Dong L, Vanance S, et al. Regullation of nestin expression after cortical ablation in adult rat brain. Brain Res,2004, 1008(2):139-146
21.Maslov AY, Barone TA, Plunkett RJ, et al. Neural stem cell detection,characterization,and age-related changes in the subventricular zone of mice. J Neurosci,2004,24(7):1726-1733
22. Dempsey RJ,Sailor KA,Bowen KK,et al.Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain:effect of exogenous IGF-1 and GDNF [J] Neurochem,2003,87(3): 586-597
23. Zhang R, Zhang ZQZhang Let al. Proliferation anddifferentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral isehemia[J]. Neuroseience,2001, 1050):33-41
24. Takasawa K, Kitagawa K, Yagita Yet al. Increased prolifetmion of neural progenitor cells but reduced survival of cerebralof cerebral cells in the contralateral hlmcampus after focal cerebral ischemia in rats[J]. J Cereb Blood Flow Metab,2002.22:299-307
25.陈波,高小青,杨朝鲜等.移植GDNF基因修饰的神经干细胞对大鼠脑卒中的神经保护作用[J].解剖学杂志,2009；32(3)：.286-290
26.吴旭,张国华.实验性脑挫伤GFAP. PCNA免疫组织化学研究[J]法医学杂志,1999,15(2)：206
27. HozLlnuI,ChiuFC.NottoWT.Biochemical and immuno-cytochemieal changes in glial fibrillary acidic pmtdn after stab wounds[J].Brain Rearch,1990,524:64
28. Pekny M, Nilsson M. Astrocyte activation and reactive gliosis [J]. Glia,2005,50(4):427-434
1. Mckay R. Stem cells in the central nervous system. Science,1997, 276:66-71.
2. Gage HF. Mammalian neural stem cells. Science,2000,287:1433-1438.
3. Abrahams JM,Gokhan S,Flamm ES,et al. Denovo neurogenesis and acute stroke:are exogenous stem cells really necessary[J] Neurosurgery,2004, 54(1):150-156.
4. Bjornson CR, Rietz RL, Reynolds BA,et al. Turning brain into blood:a hematopoietic fate adopted by adult neural stem cells in vivo[J].Science, 1999;2839(5401):534-537.
5. Deng J, Petersen BE, Steindler DA, et al. Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation[J]. Stem Cells,2006,24(4):1054-1064
6. Doetsch F, Caille I, Lim DA, et al. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell,1999,11;97: 703-716.
7. Englund U, Bjorklund A, Wictorin K, et al. Grafted neural stem cells develop into Functiongal pyramidal neurons integrate into host cortical circuitry. Proc Natl Acad Sci USA,2002,99:17089-17094
8. Suarez-Rodriguez R, Belkind-Gerson J. Cultured nestin-positive cells from postnatal mouse small bowel differentiate ex Vivo into neurons, glia. and smooth muscle[J].Stem Cells,2004,22(7):1373-138
9. Calhoun JD, Lamben NA, Mitalipova MM, et al. Differentiation of rhesus emhronic stem cells to neural progenitors and neurons[J]. Biochem Biophys Res Commun,2003,306(1):191-197.
10. Marzesco AM, Janich P, Wilsch-Brauninger M, et al.Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD 133) from neural progenitors and other epithelial cells[J]. J Cell Sci.2005,118(13):1849-1858.
11. Lee ST, Park JE, Lee K, et al.Noninvasive method of immortalized neural stem-like cell transplantation in an experimental model of Huntington's disease[J] J NeurosciMethods.2006,152(1-2):250-254.
12. Jin K, Sun Y, Xie L,et al. Comparison of ischemia-directemigration of neural precursor cells after intrastriatal,intraventricular,or intravenous transplantion in the rat[J].Neurobiol Dis,2005,18(2):366-374.
13.王霞,杨于嘉,余小河,等.神经干细胞移植对缺氧缺血新生大鼠感觉运动机能的影响.中国行为医学科学,2005.14：596-598.
14.刘伟国罗呜郑学胜陈向日等, 脑外伤后不同时间移植神经干细胞的疗效对比研究,浙江医学2006.28(1)：33-1109
15.杨东波金力,李永利等神经干细胞移植治疗大鼠局灶性脑缺血：移植部位与移植时间的比较.中国临床康复,2006,10{17}30-2
16. Derek van der Kooy, Samuel Weiss. y stem cels[J]Science, 2000, 287(6):1439-1441.
17. Barker RA, Widner H. Immune problems in central nervous system celtherapy[J]. Neuro Rx,2004,1(4):472-481.18.杨清成,梁长春,李敏霞,等.神经干细胞移植治疗脑卒中后遗症59例[J].中国组织工程研究与临床康复,2007,11(20)：4033-4035.19.张儒有,郑永日,胡韶山,等.神经干细胞移植治疗脑卒中后遗症50例临床效果分析[J].中国临床康复,2006,10(9)：138-139.