【作者】 梁袁昕；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 目的：研究神经营养因子-3(NT-3)修饰的胚胎脊髓来源的神经干细胞移植对于脊髓损伤的治疗作用。方法：构建NT-3基因真核表达载体pEGFPN1-NT-3后，应用核转染技术将其转入神经干细胞内建立NT-3基因修饰的神经干细胞。比较脊髓损伤后不同时间神经干细胞移植的治疗效果，确定脊髓损伤后进行神经干细胞移植的最佳时间。在NT-3修饰的神经干细胞移植入脊髓损伤大鼠后，通过行为学测试，及对损伤脊髓局部移植细胞存活、分化及基因表达的分析，综合评价NT-3修饰的神经干细胞治疗大鼠脊髓损伤的效果。结果：动物行为学实验研究结果表明：与未修饰的神经干细胞移植相比，NT-3基因修饰的神经干细胞移植更能显著改善脊髓损伤大鼠的脊髓功能。免疫组织化学实验研究结果表明：NT-3修饰能使更多的移植细胞存活，并能促进移植的神经干细胞向少突胶质细胞分化。分子生物学实验研究结果表明：NT-3基因修饰的神经干细胞移植后能更好的表达NT-3和MBP。结论：NT-3修饰的神经干细胞移植治疗脊髓损伤具有良好效果，治疗效果优于未修饰的神经干细胞，具有广阔的应用前景。第一部分载体构建目的：构建将NT-3基因真核表达载体pEGFPN1-NT-3。方法：将NT-3基因编码序列克隆入pMD19-T Simple载体；经鉴定确认后，将NT-3基因CDS克隆至真核表达载体pEGFPN1。结果：重组质粒pEGFPN1-NT-3双酶切鉴定与预期结果相同。结论：NT-3基因真核表达载体pEGFPN1-NT-3构建成功。第二部分细胞培养目的：对胚胎脊髓神经干细胞进行稳定的培养。方法：从孕鼠中分离胚胎14天的脊髓组织，培养胚胎脊髓神经干细胞，传代并进行多能性检测。结果：胚胎脊髓神经干细胞神经球形成，并且Nestin阳性。结论：胚胎脊髓神经干细胞培养成功并保持干细胞多能性。第三部分基因修饰目的：构建EGFP标记NT-3基因修饰的神经干细胞。方法：采用核转染将重组质粒pEGFPN1-NT-3转染至大鼠胚胎脊髓神经干细胞后，采用RT-PCR、Western blot及免疫细胞化学方法对转染后的细胞进行鉴定。结果：RT-PCR和Western blot显示转染后细胞NT-3表达强阳性，免疫细胞化学染色结果显示转染后细胞呈NT-3及EGFP阳性。结论：FGFP标记NT-3基因修饰的神经干细胞构建成功并能表达NT-3和EGFP。鼠右侧半脊髓，术后观察大鼠行为学表现并进行行为学测试。结果：脊髓损伤后大鼠右侧后肢瘫痪，损伤后4周时大鼠右侧后肢BBB评分不足2分、网格测试经常摔倒。结论：胸9水平脊髓半切大鼠动物模型建立成功。第五部分细胞移植(一)目的：确定脊髓损伤后神经干细胞的最佳移植时间。方法：比较脊髓损伤后不同时间神经干细胞移植对于大鼠行为功能恢复的治疗效果，确定治疗效果最佳的神经干细胞移植时间。结果：脊髓损伤后3天、7天及14天进行移植大鼠的BBB测试和网格测试结果明显优于对脊髓损伤大鼠，脊髓损伤后7天时进行细胞移植的治疗效果最佳。结论：进行神经干细胞移植的最佳时间为脊髓损伤后一周左右。第六部分细胞移植(二)目的：研究NT-3修饰的胚胎脊髓来源的神经干细胞移植对于脊髓损伤大鼠行为学恢复的治疗作用。方法：采用行为学测试评价NT-3修饰的神经干细胞移植后脊髓损伤大鼠的行为学恢复，并与未修饰的神经干细胞移植的治疗效果进行比较。结果：神经干细胞移植组和NT-3修饰的神经干细胞移植组大鼠的BBB测试结果和网格测试结果明显优于对照组大鼠，NT-3修饰的神经干细胞移植的治疗效果最佳。结论：NT-3修饰能够促进神经干细胞移植对于脊髓损伤大鼠行为学恢复的治疗作用。第七部分细胞检测目的：研究NT-3修饰的胚胎脊髓来源的神经干细胞移植后细胞的存活、分化及基因表达情况。方法：采用免疫组织化学检测NT-3修饰的神经干细胞移植后细胞的存活及分化情况；采用分子生物学技术检测相关基因的表达；并与未修饰的神经干细胞移植进行比较。结果：免疫细胞化学染色表明NT-3修饰的神经干细胞能更好的在损伤脊髓内的存活，分泌NT-3并向少突胶质细胞分化；分子生物学检测表明NT-3修饰能在转录水平和蛋白水平增强NT-3和MBP的表达。结论：NT-3修饰能够促进神经干细胞移植后移植细胞的存活、分化及相关基因表达。更多还原

【Abstract】 Objective:To investigate therapeutic effects of transplantation of Neurotrophin-3(NT-3) modified fetal spinal cord-derived neural stem cells (NSCs) for spinal cord injury(SCI) repairing.Methods:eukaryon expression vector pEGFPN1-NT-3 recombinant plasmid wasconstructed and transfected into NSCs to establish EGFP-labeled NT-3 gene modifiedNSCs via nucleofection.Therapeutic effects of NSCs transplantation at different times aftcrSCI were compared and most suitable time point for NSCs transplantation was determincd.After NT-3-NSCs transplanted into SCI rats,therapeutic effect of NT-3-NSCstransplantation was evaluated by behavioral tests,and analyses of the survival,differentiation and gene expressions of grafted cells at injured sites.Results:Behavioral tests presented the transplantation of NT-3 gene modified NSCscould make SCI rats get more behavioral recovery than the transplantation of unmodifiedNSCs.Immunohistochemical analyses demonstrated NT-3 modification could improvc thesurvival and differentiation into oligodendrocytes of grafted cells.Molecular biologicalexperiments revealed NT-3 modified NSCs could obtain stronger NT-3 and MBPexpression than unmodified NSCs after transplantation.Conclusion:Transplantation of NT-3 modified NSCs is a potential therapy for SCIrepairing,which yields better effect than transplantation of unmodified NSCs, transplantation of NT-3 modified NSCs has a brilliant future for SCI treatment.PART ONE:Construction of recombinant plasmidObjective:To construct NT-3 gene eukaryon expression vector pEGFPN1-NT-3recombinant plasmid.Methods:The coding sequence of NT-3 was amplified andconnected to pMD19-T Simple Vector;after identification,NT-3 gene and plasmid vectorpEGFPN1 were connected.Results:The result of double enzyme digestion of recombinantplasmids was consistent with expectation.Conclusion:NT-3 gene eukaryon expressionvector pEGFPN 1-NT-3 recombinant plasmid was successfully constructed.PART TWO:Culture of neural stem cellsObjective:To stably culture fetal spinal cord derived neural stem cells.Methods:Spinal cord tissue was obtained from fetal Sprague-Dawley rats at embryonic day 14,neural stem cells were cultured and their multipotentiality was detected.Results:Fetalspinal cord neural stem cells formed neurospheres and were Nestin immunoreactivc.Conclusion:Fetal spinal cord neural stem cells were successfully cultured and their stemcell multipotentiality was maintained.PART THREE:NT-3 gene modificationObjective:To construct EGFP-labeled NT-3 gene modified neural stem cells.Methods:After neural stem cells were transfected with recombinant plasmidpEGFPN1-NT-3 via nucleofection,the cells were identified via RT-PCR,Western blot and immunocytochemical assessments.Results:RT-PCR and Western blot results prcscntedstrong expression of NT-3 in transfected cells;immunocytochemical staining demonstratedNT-3 and EGFP immunoreactions in transfected ceils.Conclusion:EGFP-labeled NT-3gene modified neural stem cells were successfully constructed and could express NT-3 andEGFP.PART FOUR:Establishment of spinal cord hemisection modelsObjective:To establish thoracic 9 right-sided spinal cord hemisection rat models.Methods:Spinal cords of female Sprague--Dawley rats were right-sided hemisected atthoracic 9 level,behavioral observation and tests were administered postoperatively.Results:Right hindlimb paralyzed after operation;hemisected rats were scored were lessthan 2 in BBB test and fell frequently in grid test.Conclusion:Thoracic 9 right-sidedspinal cord hemisection rat models were successfully established.PART FIVE:Neural stem cells transplantationObjective:To determine the most suitable neural stem cells transplantation time afterspinal cord injury.Methods:The therapeutic effects of neural stem cells transplantation atdifferent times after spinal cord injury were compared to determine the best transplantationtime point.Results:The rats received neural stem cells transplantation at 3rd,7th and 14thdays after spinal cord injury presented better results than spinal injured rats,rats obtainedbest recovery when neural stem cells transplanted at 7th day after spinal cord injury.Conclusion:The most suitable neural stem cells transplantation time was around one week after spinal cord injury.PART SIX:NT-3 gene modified neural stem cells transplantationObjective:To study the behavioral recovery effect of NT-3 gene modified fetal spinalcord derived neural stem cells transplantation for spinal cord injury rats.Methods:Thebehavioral tests were applied to evaluate the behavioral recovery in spinal cord injury ratsafter transplantation of NT-3 modified neural stem cells;the therapeutic effects of NT-3modified and non-modified neural stem cells transplantation were compared.Results:Therats received NT-3 modified or non-modified neural stem cells transplantation after spinalcord injury presented better results than controls,rats obtained best recovery when receivedNT-3 modified neural stem cells transplantation.Conclusion:The behavioral recoveryeffect of neural stem cells transplantation could be improved by NT-3 gene modification inspinal cord injury rats.PART SEVEN:Detection of transplanted cellsObjective:To study the survival,differentiation and gene expression of transplantedNT-3 modified fetal spinal cord derived neural stem cells.Methods:The survival anddifferentiation of transplanted cells were detected via immunohistochemistries,relatedgenes expressions were detected via molecular biologies;which were compared withtransplantation of non-modified neural stem cells,Results:Immunohistochemical stainingpresented NT-3 modified neural stem cells got better survival,NT-3 secretion anddifferentiation into oligodendrocytes in injuried spinal cords;molecular biologicalassessment demonstrated NT-3 modification could increase NT-3 and MBP expression attranscription and protein levels.Conclusion:NT-3 gene modification could improve the survival,differentiation and related genes expression of transplanted neural stem cells inspinal cord injury rats.更多还原