摘要
目的：脊髓损伤是一种常见病、多发病,可以造成损伤平面以下不可逆的感觉、运动及括约肌功能障碍,临床治疗效果不理想,一直是医学领域的热点和难点。本实验应用对脊髓损伤具有保护作用的红花黄素腹腔注射联合MSCs移植治疗大鼠脊髓损伤模型,探讨红花黄素腹腔注射和MSCs移植对损伤脊髓修复作用的机制,为提高脊髓损伤临床治疗效果寻找一条可行的途径。
方法：贴壁分离法分离纯化大鼠MSCs,观察原代及传代MSCs的形态特点、生长特性,探讨其作为种子细胞的可行性并用流式细胞分析技术鉴定MSCs表面蛋白表达。携带GFP基因的腺病毒感染MSCs,荧光显微镜观察MSCs转染情况并计算转染效率,观察GFP-MSCs形态特点、生长特性,研究GFP标记对MSCs生物学性状的影响。55只成年S-D大鼠,雌雄各半,根据处理方法的不同,随机分为5组：A组(红花黄素腹腔注射+MSCs移植组)、B组(生理盐水腹腔注射+MSCs移植组)、C组(红花黄素腹腔注射+PBS组)和D组(生理盐水腹腔注射+PBS组),以上每组各10只；E组为补充组,15只。改良Allen法T7平面制作脊髓损伤模型。造模成功后,A组和B组损伤局部即时直接移植标记后的MSCs (GFP-MSCs) 6×106/60μl,C组和D组注射同等体积PBS。A组和C组术后按40mg/kg的剂量腹腔注射红花黄素,B组和D组注射同等剂量的生理盐水,每日1次,共用2w。实验过程中,各组的死亡动物后从E组中随机选取补充,重新开始计算模型动物的喂养处理时间,以保证各组实验动物数目的相同。术后1w、2w、3w和4w时采用BBB评分法进行后肢功能评分后心脏灌注取材,制作冰冻切片,荧光显微镜观察移植的MSCs; HE染色观察组织形态学变化；免疫组化检测GFAP、NeuN和NGF的表达。
结果：
1、贴壁分离法分离纯化的贴壁细胞形态、生长特性与MSCs一致,流式细胞分析技术检测细胞表面蛋白的表达：CD45阳性率14.47%,CD44阳性率89.26%,CD29阳性率93.27%提示获得的细胞是MSCs;
2、携带GFP基因的腺病毒感染MSCs后,荧光显微镜观察证实MSCs的GFP标记成功,腺病毒的转染效率为(93%±2%),转染后MSCs的形态学、生长特性无明显变化；
3、荧光显微镜下,GFP-MSCs移植后各个时间点1w、2w、3w和4w时的冰冻组织切片均可观察到GFP-MSCs发出的明亮的绿色荧光,证明移植的MSCs可以在大鼠损伤的脊髓中存活；
4、术后2w-4W,红花黄素腹腔注射或MSCs移植的单独应用均可提高大鼠BBB评分的得分,二者联用作用更加明显,差异有显著性(P<0.05),术后1w时,各组间评分的差异性不显著(P>0.05)；
5、术后1w、2w、3w和4w各个时间点冰冻组织切片HE染色显示红花黄素腹腔注射或MSCs移植的单独应用均可改善神经组织病变情况,减少空腔的形成,其改善程度和BBB评分得分情况一致,二者联用病变作用更加明显；
6、免疫组化检测显示各组GFAP、NeuN和NGF表达均随着术后时间的延长而增加,相同时间点同一指标A组、B组和C组的表达水平明显高于D组,差异有显著性(P<0.05),其中A组的表达水平又高于B组和C组,差异有显著性(P<0.05),B组的表达水平高于C组,差异有显著性(P<0.05)。
结论：
1、贴壁分离法是一种较好的MSCs分离方法,所获贴壁细胞生长迅速,活力良好,流式细胞分析技术证实为MSCs,可用作组织工程的种子细胞；
2、应用携带GFP基因的腺病毒感染MSCs,GFP转染效率高,对MSCs的生物学特性没有明显影响,标记后的MSCs能够在大鼠的脊髓中成活、显示,GFP是一种较好的MSCs标记方法；
3、红花黄素腹腔注射或MSCs移植单独应用能够提高NGF的分泌,促使干细胞向神经细胞和神经胶质细胞方向分化,有利于受损神经组织的修复,二者联用具有协同作用。
Objective:Spinal cord injury (SCI) is very frequent in our society, and often results in loss of voluntary motor and sensory function below the lesion site, and causes the dysfunction of urination and defecation, which decreases the life quality seriously. The treatment of SCI remains to be a puzzling and challenging issue in medical science. There are some reports that safflower yellow can promote the neural function to resume, has close relationship with repairing of the nervous system. In this experience, we study the recovery effects of the combination safflower yellow by intraperitoneal injection and transplantation MSCs on spinal cord injury in Rats, discuss the mechanisms of safflower yellow by intraperitoneal injection and transplantation MSCs on the SCI model, and examine whether the combination of these protocols is more effective than a single protocol.
Methods:MSCs were collected from S-D rat bone marrow with adherence separation method, expanded in vitro. The morphology was observed and CD44, CD29 and CD45 of P3 MSCs were detected with flow cytometry analysis. GFP genes were transfected into MSCs with adenovirus vector (Adv-GFP), GFP positive MSCs (GFP-MSCs) were observed with fluorescent microscopy, the morphology and the growth curve were also observed in order to study biocharacteristics. By the striking of the spinal cord at the level of T7 with Allen's device, acute spinal cord injury was induced in anesthetized rats. Fifty-five SCI model rats were randomly divided into 5 groups:group A (safflower yellow +MSCs), B (saline+MSCs), C (safflower yellow+PBS), D (saline +PBS) and E (supplementary group). There were 10 rats in every group except group E which has 15 rats. The labeled MSCs (GFP-MSCs) in a dose of 6×106/60μl were injected into SCI space in group A and B, the identical volume of PBS in group C and D. A dose of saff lower yellow (40mg/kg) was administered by intraperitoneal injection at post-lesion from 1d tol4d in group A and C and the identical volume of saline in group B and D. At 1w、2w、3w 4w after SCI, Neurological functional changes was evaluated by another man who was proficient in the regulations with open field Basso, Beattie, Bresnahan (BBB)locomotors rating scale. At the same time of post-operation, cardiac perfusion by Para-formaldehyde was performed; the injury spinal cord was made into frozen section. histomorphology change was observed by HE staining, the survival of MSCs by fluorescence microscopy and the expressions of GFAP、NeuN and NGF through immunohistochemical staining technique and image analysis. During the experiment, if any rat died, another one would be selected from group E instead of it and calculate feeding time from beginning, which in order to make sure the same number in each group.
Results:(1) It was believed that the harvested cells was identical to MSCs, because the morphology and biocharacteri-stics were similar to MSCs'and flow cytometry analysis showed the expression of CD44, CD45 and CD29 were 89.26%,14.47%and 93.27%.
(2) After GFP genes were transfected into MSCs, Nearly (93±2)%of cells expressed GFP genes, which be confirmed Adv-GFP can efficient transfect MSCs and make GFP mass expression in MSCs. The morphology and biocharacteristics of GFP-MSCs were found no difference.
(3) GFP positive MSCs were observed on frozen section by fluorescent microscopy at 1w、2w、3w 4w after GFP-MSCs transplantation, which be conformed MSCs can survive in the injured spinal cord of rats.
(4) From the second week to the fourth week postoperatively, the BBB score of in the group B and C were higher than those in the group D, those in the group B and C was compared with those in the group D, there was significant difference (P<0.05), the BBB score of in the group A was higher than those in the group B, C and D, there was significant difference (P<0.05), while there was no obvious difference among in every group (P>0.05) at the first week postoperatively.
(5) The application of either safflower yellow intra-peritoneal injection or MSCs transplantation can improve histomorphology of nerve tissue at every time point postoperatively. Improvement effects of the combination safflower yellow by intraperitoneal injection and transplantation MSCs was significant better than either safflower yellow intraperitoneal injection or MSCs transplantation. The degree of improvement was parallel to the BBB score.
(6) Immunohistochemistry staining results showed that the expression of GFAP、NeuN and NGF was consistently increased in all of groups. At identical time point the expression of GFAP、NeuN and NGF was higher in the group A, B and C than those in the group D, there was significant difference (P<0.05); Those in the group A than those in the group B and those group C, and those in the group B than those in the group C, there was significant difference (P<0.05).
Conclusions:(1) Adherence separation method might be an ideal method for isolation and purified MSCs from bone marrow, because the harvested adherence cells proliferated fast, and flow cytometry analysis showed the cells were identical to MSCs. all those results shows MSCs are suitable for seeding cells in tissue engineering.
(2) MSCs which be transfected GFP gene carry bright green fluorescence, which be observed in injured spinal cord of rats, and have no impact on biological function of MSCs, so Adv-GFP transfected MSCs is an ideal method for GFP labeled of MSCs, with its characteristics of high accuracy, high ratio of the labeled and stable expression.
(3) The application of either safflower yellow intra-peritoneal injection or MSCs transplantation can increase expression of NGF, enhance ratio of the differentiation into neurons and neuroglial cells, and facilitate recovery of injured nerve tissue on SCI model in rats. The combination safflower yellow by intraperitoneal injection and transplantation MSCs may result in better effect. It provides the better theoretical basis for more extensive study application.
引文
[1]李建军,周红俊,洪毅,等.2002年北京市脊髓损伤发病率调查[J].中国康复理论与实践,2004,10(7)：412-413
[2]Wyndaele M, Wyndaele J J. Incidence, prevalence and epidemiology of spinal cord injury:What learns a worldwide literature survey [J].Spinal Cord,2006,44(9):523-529
[3]Carlson GD, Gordon C. Current developments in spinal cord injure research [J]. Spine J,2002,2(2):116-128
[4]周天健.21世纪与我国脊髓损伤及康复事业[J].伤残医学杂志,2005,13(3)：54-58
[5]Hagg T, Oudega M. Degenerative and spontaneous regenerative processes after spinal cord injury [J].J Neurotrauma,2006,23(3-4):264-280
[6]刘涛,邵增务,李健,等.脊髓损伤传统治疗及细胞移植基因治疗的进展[J].中国组织工程研究与临床康复,2007,11(28)：5598-5601
[7]Nandoe Tewarie RD, HurtadoA, Levi AD, et al. Bone marrow stromal cells for repair of the spinal cord:towards clinical application[J].Cell Transplant 2006,15 (7):563-577
[8]Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cellsfor the repair of central nervous system injury [J]. Bone Marrow Transplant.2007,40(7):609-619
[9]林建华,雷盛民,康德智,等.静脉注射骨髓间质干细胞对脊髓损伤修复作用的实验研究[J].中华骨科杂志,2005,25(9)：556.
[10]Lu P, Jone LL, Tuszynski MH. Axon regeneration through scars and into sites of chronic spinal cord injury [J].Exp Neurol 2007,203(1):8-21
[11]施永彦,喻爱喜,张功礼,等.神经生长因子基因转染间充质干细胞移植对损伤脊髓保护的实验研究[J].中华显微外科杂 志,2008,31(3)：199-201
[12]赵丽娟,杜遵义,李恋.关于红花的研究进展[J].中国民族医药杂志,2007,13(3)：75-77
[13]祝美珍,胡国恒,肖建.羟基红花黄色素A的应用研究进展[J].中西医结合心脑血管病杂志,2007,5(11)：1108-1110
[14]金鸣,李金荣,吴伟.羟基红花黄色素A抗氧化作用的研究[J].中草药,2004,35(6)：665-666
[15]隋吉生,吴小涛,徐小彬.红花注射液对脊髓损伤早期自由基影响的实验研究[J].中国骨伤,2007,20(2)：94-96
[16]练克俭,林山,翟文亮,等.红花注射液对脊髓缺血再灌注损伤后体感诱发电位的影响[J].中国中医骨伤科杂志,2007,15(10)：1-3
[17]梁辉,范金英,李爱华,等.羟基红花黄色素A对大鼠局灶性脑缺血再灌注NMDAR1蛋白表达的影响[J].中华老年心脑血管病杂志,2004,6(3)：19
[18]莫尚武,陈恒留,刘宇.用45Ca研究红花对大鼠脑主动脉Ca2+内流的影响[J].中草药,26(10)：532-534
[1]Friedenstdein AJ, Gorskaja UF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs [J].Exp Hematol,1976; 4 (5):267-274
[2]Owen M. Marrow stromal stem cells [J]. J Cell Sci Suppl, 1988; 10:63-76
[3]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 Cellular Physiology,1998,176:57-66
[4]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues [J].Seienee.1997:276:71-74
[5]Bianeo P, Riminueei M, Gronihos S, et al. Bone marrow stromal stem cells:nature, biology, and potential applications [J]. Stem Cells,2001; 19:180-192
[6]Deans RJ, Moseley AB. Mesenchymal stem cells:biology and potential clinical uses [J]. Exp. Hematol,2000; 28 (8):875-884
[7]郭立迭,王捷,夏冰.改良法培养骨髓基质干细胞向成骨细胞分化的实验研究[J].实用医学杂志,2006,22(2)：131
[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
[9]王超,徐蕴,宋文刚,等.大鼠骨髓间充质干细胞分离培养方法的建立及其表型分析[J].细胞与分子免疫学杂志,2007,23(5)：466-468
[10]司徒镇强,吴军正.细胞培养[M].第一版,西安：世界图书出版社.2003,181-182；179
[11]Dressler MR, Butler DL, Boivin GP. Effects of age on the repair ability of mesenchymal stem cells in rabbit tendon. J Orthop Res,2005,23 (2):287-293
[12]Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells:in vitro cultivation and trans-plantation in diffusion chambers [J]. Cell Tissue Kinet,1987, 20 (3):263-272
[13]Jiang YH, Jahagirda BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature,2002,418 (4):41-49
[14]Guo Z, Yang J, Liu X, et al. Biological features of mesenchymal stem cells from human bone marrow [J].Chin Med J(Engl),2001,114(9):950-953
[15]张承韶,杨利民,张其亮,等.大鼠骨髓间充质干细胞的分离培养及生物学特性[J].山东医药,2007,47(5)：26-27
[16]Lisignoli G, Remiddi G, Cattini L, et al. An elevated number of differentiated osteoblast colonies can be obtained from rat bone marrow stromal cells using a gradient isolation procedure [J]. Connect Tissue Res,2001,42(1):49-58
[17]Zohar R, Sodek J, Mcculloch CA. Characterization of stormal progenitor cells enriched by f low cytometry[J]. Blood, 1997,90(9):3471-3481
[18]Encina NR, Billotte WG, Hofmann MC. Immunomagnetic isolation of osteoprogenitors from human bone marrow stromal [J].Lab Invest,1999,79 (4):449-457
[19]刘品端,王伟,梅晰凡.大鼠骨髓间充质干细胞的分离与培养-贴壁分离和消化控制相结合可否为简便高效的技术方法[J].中国临床康复,2005,9(18)：38-40
[20]Mareschi K, Ferrero I, Rustichelli D, et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow [J]. J Cell Biochem,2006,97 (4):744-754
[21]Zhang H, Fazel S, Tian H, et al. increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy [J]. Am J Physiol Heart Circ Physiol.2005,289 (5):H2089-2096
[23]Strube P, Mehta M, Baerenwaldt A, et al. Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity [J]. Bone, 2009,45(6):1065-1072
[24]Martinez C, Hofmann T J, Marino R, et al. Human bone marrow mesenchymal stromal cells express the neural ganglio-side GD2:a novel surface marker for the identification of MSCs[J]. Blood,2007,109 (4):1743-1751
[25]王广军,王爱萍,郑修启,等.骨髓间充质干细胞与基因治疗[J].泰山医学院学报,2006,26(2)：179-181
[26]Morris MC. Fluorescent biosensors of intracellular targets from genetically encoded reporters to modular poly-peptide probes [J]. Cell Biochem Biophys,2010,56(1):19-37
[27]Wiedenmann J, Oswald F, Nienhaus GU. Fluorescent proteins for live cell imaging:opportunities, limitations, and challenges [J]. IUBMB Life,2009,61 (11):1029-1042
[28]Shiotani A, Saito K, Araki K, et al. Gene therapy for laryngeal paralysis [J]. Ann Otol Rhinol Laryngol 2007, 116(2):115-122
[29]Yoo YM, Lee CJ, Lee U, et al. Neuroprotection of adenoviral-vector-mediated GDNF expression against kainic-acid-induced excitotoxicity in the rat hippocampus [J]. Exp Neurol 2006,200(2):407-417
[30]Vasita R, Shanmugam I K, Katt DS. Improved biomater-ials for tissue engineering applications:surface modification of polymers [J]. Curr Top Med Chem,2008,8(4):341-353
[1]朱玉海,冯世庆,王雪.大鼠脐带华通胶来源MSCs修复脊髓损伤的实验研究[J].中国修复重建外科杂志,2009,23(12)：1491-1496
[2]Jung DI, Ha J, Kang BT, et al. A comparison of autologous and allogenic bone marrow-derived mesenchymal stem cell transplantation in canine spinal cord injury [J]. J Neurol Sci. 2009,285 (1-2):67-77
[3]Wright KT, Masri WE, Osman A, et al. The cell culture expansion of bone marrow stromal cells from humans with spinal cord injury:implications for future cell transplantation therapy [J]. Spinal Cord.2008,46 (12):811-817
[4]施永彦,喻爱喜,张功礼,等.神经生长因子基因转染间充质干细胞移植对损伤脊髓保护的实验研究[J].中华显微外科杂志,2008,31(3)：199-201
[5]Urdzikova L, Jendelova P, Glogarova K, et al. Trans-plantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats [J]. J Neurotrauma.2006, 23(9):1379-1391
[6]金鸣,李金荣,吴伟.羟基红花黄色素A抗氧化作用的研究[J].中草药,2004,35(6)：665-666
[7]隋吉生,吴小涛,徐小彬.红花注射液对脊髓损伤早期自由基影响的实验研究[J].中国骨伤,2007,20(2)：94-96
[8]练克俭,林山,翟文亮,等.红花注射液对脊髓缺血再灌注损伤后体感诱发电位的影响[J].中国中医骨伤科杂志,2007,15(10)：1-3
[9]梁辉,范金英,李爱华,等.羟基红花黄色素A对大鼠局灶性脑缺血再灌注NMDAR1蛋白表达的影响[J].中华老年心脑血管病杂志,2004,6(3)：19
[10]莫尚武,陈恒留,刘宇.用45Ca研究红花对大鼠脑主动脉Ca2+内流的影响[J].中草药,26(10)：532-534
[11]Basso DM, Beattie M, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats [J].J Neurotrauma,1995,12:1-21
[12]Pearse DD, Lo TP Jr, Cho KS, et al. Histopathological and behavioral characterization of a novel cervical spinal cord displacement contusion injury in the rat [J]. J Neurotrauma, 2005,22 (6):680-702
[13]Fiford RJ, Bilston LE, Waite P, et al. A vertebral dislocation model of spinal cord injury in rats [J]. J Neuro-trauma,2004,21 (4):451-458
[14]Basso DM, Beattie MS, Bresnahan JC. Graded histology-cal and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transaction [J]. Exp Neurol, 1996,139 (2):244-256
[15]Saruhashi Y, Young W, Sugimori M, et al. Evidence for serotonin sensitivity of adult rat spinal axons:studies using randomized double pulse stimulation [J]. Neuroscience.1997, 80 (2):559-566
[16]郭维淮主编.平乐正骨[M].北京：中国中医药出版社,1996：474
[17]杜立建,冯胜格,周冀杭.中医药对脊髓损伤的研究[J].河北中医,2009,31(9)：1413-1416
[18]党杰,任学通,刘保健,等.中医药治疗脊髓损伤实验研究进展[J].中医药导报,2007,13(10)：85-87
[19]李文超,付光明,何振辉,等.中医药治疗脊髓损伤研究进展[J].中医正骨,2002,14(11)：55-57
[20]杨松涛,胡晓梅.合理应用中药提高脊髓的可塑性[J].四川中医,2000,18(6)：9-10
[21]李根林,尹君.中医药治疗外伤性截瘫研究进展[J].河南中医,2008,28(9)：102-105
[22]王善金,夏英鹏,张学利.脊髓损伤的中药治疗研究进展[J].中医正骨,2008,20(2)：71-72
[23]刘汝专.活血化瘀中药治疗脊髓损伤的研究概况[J].云南中医中药杂志,2007,28(4)：43-45
[24]杨晓君,吴桂荣.红花的现代研究[J].农垦医学,2004,26(4)：301-304
[25]赵丽娟,杜遵义,李恋.关于红花的研究进展[J].中国民族医药杂志,2007,13(3)：75-77
[26]刘扬,林晓丹.红花注射液的临床应用[J].人民军医,2006,49(1)：39-41
[27]张承丽.中药穴位注射治疗糖尿病周围神经病变患者的效果观察[J].护理学杂志综合版,2009,24(11)：44-45
[28]王雅丽刘慧琴.红花注射液降脂作用的临床观察[J].中国现代药物应用,2009,3(19)：136
[29]HiramatsuM, Takahashi T, KomatsuM, et al. Antioxidant and neuroprotective activities of Mogami-benibana (safflower, Carthamus tinctorius Linne) [J]. Neurochem Res.2009,34(4): 795-805
[30]Tian J, Li G, Liu Z, et al. Hydroxysafflor yellow A inhibits rat brain mitochondrial permeability transition pores by a free radical scavenging action [J]. Pharmacology,2008, 82 (2):121-126
[31]罗嘉,方治平,周黎明,等.红花注射液对大鼠局灶性脑缺血后梗死体积和神经元凋亡相关蛋白bcl—2, caspase—3表达的影响[J].中国中药杂志,2004,29(10)：977-980
[32]Zhu HB, Zhang L, Wang ZH, et al. Therapeutic effects of hydroxyl-safflor yellow A on focal cerebral ischemic injury in rats and its primary mechanisms [J]. J Asian Nat Prod Res, 2005,7 (4):607-613
[33]朱海波,王振华,田京伟,等.羟基红花黄色素A对实验性脑缺血的保护作用[J].药学学报,2005,40(12)：1144-1146
[34]陈亭亭,杜玉娟,刘晓雷,等.羟基红花黄色素A对脑缺血大鼠皮层炎症信号转导途径相关因子的抑制作用[J].药学学报,2008,43(6)：570-575
[35]Wang C, Zhang D, Li G, Liu J, Tian J, Fu F, Liu K. Neuroprotective effects of safflor yellow B on brain ischemic injury [J]. Exp Brain Res.2007,177 (4):533-539
[36]Wislet-Gendebien S, Hans G, Leprince P, et al. Plasticity of cultured mesenchymal stem cells:switch from nestin-positive to excitable neuron-like phenotype [J]. Stem Cells,2005,23(3):392-402
[37]Yoon YS, Wecker A, Heyd L, et al. Clonally expanded novel multipotent stem cells from human bone marrow regenerate myocardium after myocardial infarction [J]. J Clin Invest,2005, 115(2):326-338
[38]晏杰,刘玲蓉,张其清.诱导骨髓间充质干细胞向软骨分化的体外研究[J].中国修复重建外科杂志,2006,20(11)：1114-1118
[39]韩冰,付小兵,孙晓庆.骨髓基质细胞基因表达的研究进展[J].中国修复重建外科杂志,2005,19(2)：153-158
[40]董世武,应大军,朱楚洪,等.核心结合因子α1基因修饰骨髓间充质干细胞对骨缺损的修复研究[J].中国修复重建外科杂志,2006,20(5)：555-559
[41]Dennis JE, Charbord P. Origin and differentiation of human and murine stroma [J]. Stem Cells,2002,20(3):205-214
[42]David JA, Fred HG, Irying LW. Can stem cells cross the lineage boundaries [J] Nature Medicine,2001,7(4):393-395
[43]Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells:isolation, in vitro expansion and characterization [J]. Handb Exp Pharmacol.2006, (174):249-282
[44]Satake K, Lou J, Lenke LG. Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue [J]. Spine,2004,29 (18):1971-1979
[45]Ankeny DP, McTigue DM, Jakeman LB. Bone marrow trans-plants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats [J]. Exp Neurol, 2004,190(1):17-31
[46]Lu P, Jones LL, Tuszynski MH. BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury [J]. Exp Neurol,2005,191(2):344-360
[47]Huang F, Wang J, Chen A. Effects of co-grafts mesen-chymal stem cells and nerve growth factor suspension in the repair of spinal cord injury [J]. J Huazhong Univ Sci Technolog Med Sci.2006,26 (2):206-210
[48]Urdzikovd L, Jendelova P, Glogarova K, et al. Trans-plantation of bone marrow stem cells as well as mobilization by granulocyte-colony stimulating factor promotes recovery after spinal cord injury in rats [J]. J Neurotrauma.2006, 23(9):1379-1391
[49]Zhang HT, Luo J, Sui LS, et al. Effects of differentiat-ed versus undifferentiated adipose tissue-derived stromal cell grafts on functional recovery after spinal cord contusion [J]. Cell Mol Neurobiol.2009,29 (8):1283-1292
[50]Lee JH, Chang HS, Kang EH, et al. Percutaneous trans-plantation of human umbilical cord blood-derived multipotent stem cells in a canine model of spinal cord injury [J]. J Neurosurg Spine,2009,11 (6):749-757
[51]施永彦,喻爱喜,张功礼,等.神经生长因子基因转染间充质干细胞移植对损伤脊髓保护的实验研究[J].中华显微外科杂志,2008,31(3)：199-201
[52]Pekny M, Wilhelmsson U, Bogestal YR, et al. The role of astrocytes and complement system in neural plasticity [J]. Int Rev Neurobiol,2007,82:95-111
[53]Zhu H, Dahlstrom A. Glial fibrillary acidic protein-expressing cells in the neurogenic regions in normal and injur-ed adult brains [J]. J Neurosci Res.2007,85(12):2783-2792
[54]Nawashiro H, Messing A, Azzma N, et al. Mice lacking GAFP are hyper-sensitive to traumatic cerebrospinal injury [J].Neuro RePort,1998,9:1691-1696
[55]Menet V, VPrieto M, Privat A, et al. Axonal Plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes [J].Proc Natl Acad Sci USA,2003,100:8999-9004
[56]Menet V, Gimenez Y, Riboatt M, et al. Inactivation of the glial fibrillary acidic protein gene, but not that of vimentin, improves neuronal survival and neuriet growth by modifying adhesion molecule expression [J].J Neurosci,2001, 21:6147-6158
[57]Wolf HK, Buslei R, Schmidt-Kastner R, et,al. NeuN:a useful neuronal marker for diagnostic histopathology [J]. J Histochem Cytochem,1996,44 (10):1167-1171
[58]Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates [J]. Development,1992,116(1): 201-211
[59]Weyer A, Schilling K. Developmental and cell type-specific expression of the neuronal marker NeuN in the murine cerebellum [J]. J Neurosci Res.2003,73(3):400-409
[60]Englund C, Alvord EC Jr, Folkerth RD, et, al. NeuN expression correlates with reduced mitotic index of neoplastic cells in central neurocytomas [J]. Neuropathol Appl Neurobiol, 2005,31 (4):429-438
[61]贾小力,陈少强.生长因子和细胞外基质与脊髓损伤.医学综述,2007,13(18)：1368-1370
[62]Esper RM, Loeb JA. Rapid axoglial signaling mediated by neuregulin and neurotrophic factors [J].J Neurosei,2004, 24(27):6218-6227
[63]Brown A, Ricci MJ, Weaver LC. NGF message and Protein distribution in the injured rat spinal cord [J].ExP Neurol, 2004,188(1):115-127
[64]Hari A, Djohar B, Skutella T, et al. Neurotrophins and extracellular matrix molecules modulate sensory axon out-growth [J].Int J Neurosci,2004,22 (2):113-117
[65]高明勇,郑启新,郭晓东,等.急性脊髓损伤后胶质细胞源性神经营养因子的表达变化.中华创伤杂志,2004,20(9)：559-560
[66]刘锦波,唐天驷,杨惠林,等.大鼠脊髓损伤后神经营养素及受体表达的变化.中国矫形外科杂志,2004,12(18)：1404-1406
[67]宋海涛,贾连顺,陈坚,等.大鼠脊髓损伤后GDNF mRNA表达变化及意义.颈腰痛杂志,2001,22(2)：107-110
[68]Linker R, Gold R, Luhder F. Function of neurotrophic factors beyond the nervous system:inflammation and autoimmune demyelination [J]. Crit Rev Immunol,2009,29(1):43-68
[69]Skaper SD. The biology of neurotrophins, signalling pathways, and functional peptide mimetics of neurotrophins and their receptors [J]. CNS Neurol Disord Drug Targets.2008, 7(1):46-62
[70]MiceraA, LambiaseA, Stampachiacchiere B, et al. Nerve growth factor and tissue repair remodeling:trkA(NGFR) and p75(NTR), two receptors one fate [J]. Cytokine Growth Factor Rev,2007,18 (3-4):245-256
[71]Lykissas MG, Batistatou AK, Charalabopoulos KA, et al. The role of neurotrophins in axonal growth, guidance, and regeneration [J]. Curr Neurovasc Res,2007,4(2):143-151
[72]Tanaka S, Koike T. Activation of protein kinase C delays apoptosis of nerve growth factor-deprived rat sympathetic neurons through a Ca2+-influx dependent mechanism [J]. Neurosci Lett,2001,313(1-2):9-12
[73]Cao X, Tang C, Luo Y. Effect of nerve growth factor on neuronal apoptosis after spinal cord injury in rats [J]. Chin J Traumatol,2002,5(3):131-135
[74]Tang C, Cao X, Wang D. Effects of nerve growth factor on neuronal nitric oxide production after spinal cord injury in rats [J]. Chin J Traumatol,2002,5 (3):165-168
[75]Seki S, Sasaki K, Igawa Y, et al. Suppression of detrusor-sphincter dyssynergia by immunoneutralization of nerve growth factor in lumbosacral spinal cord in spinal cord injured rats [J]. J Urol,2004,171 (1):478-482
[76]Qin DX, Zou XL, Luo W, et al. Expression of some neurotrophins in the spinal motoneurons after cord hemisection in adult rats [J]. Neurosci Lett,2006,27,410(3):222-227