神经干细胞移植延缓失神经肌萎过程中突触形成及其功能的实验研究
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摘要
第一部分神经干细胞体外培养扩增，体内移植后延缓失神经肌萎的作用
目的验证胚胎脊髓神经干细胞的提取、体外培养和增殖方法，研究神经干细胞移植到损伤的外周神经后，延缓失神经肌萎的效果。
方法取孕10—12天的SD大鼠，通过机械吹打法提取脊髓神经干细胞、以无血清限定性培养基进行培养，扩增及纯化得到大量增生的神经干细胞，以神经干细胞特异性抗体Nestin进行鉴定，将神经干细胞进行体外分化，通过特异性抗体染色检验其分化为神经终末细胞的能力。传2—3代后，将扩增的神经干细胞球分散成单细胞悬液并调整密度为10~6／ul，备用。制作胫神经切断，小腿三头肌失神经支配模型，以微量注射器移植10~6／ul×5ul干细胞悬液至切断的胫神经远端；设立对照组，仅移植5ul神经干细胞培养液，每组各24只。分别于术后3月，5月和7月取材，每次8只，分别观察移植神经干细胞在体内存活及分化成为神经元的情况；测量三头肌湿重及肌纤维横截面积维持率，验证神经干细胞移植能否延缓失神经支配骨骼肌的萎缩及其程度。
结果利用机械吹打和无血清限定性培养基可以得到大量的脊髓神经干细胞，得到的神经干细胞可以在体外大量扩增并分化为多种神经终末细胞，证明取得的神经干细胞活性良好；神经干细胞移植后3、5、7月取材及免疫组化染色显示，移植段胫神经内有大量存活的神经干细胞及其分化而来的神经元，神经元可以合成大量的突触素，随着时间延长，分化而来的神经元明显增加；随着时间延长，移植和对照组三头肌湿重，肌纤维横截面积均进行性下降，但对照组下降更明显(P〈0．05)。
结论1、机械吹打及无血清限定性培养基适用于脊髓神经干细胞的提取及培养；2、神经干细胞在损伤的外周神经系统中可以长期存活并分化为神经元；3、神经干细胞移植到外周神经系统内可以延缓失神经肌萎的程度。
第二部分移植神经干细胞与靶肌肉形成新突触过程的实验研究
目的将体外培养、扩增得到的神经干细胞移植到胫神经切断模型中，观察神经干细胞能否与失神经支配三头肌重新形成突触，以及新形成突触的功能。
方法采用机械吹打和无血清限定性培养基得到大量活性良好的神经干细胞，建立三头肌失神经支配模型，按第一部分方法分成移植组和对照组，每组30只。于移植术后3，5，7月进行检测，通过三头肌内突触(神经肌肉接头)免疫组化染色，神经电生理传导，示踪剂顺向和逆向传导实验，比较神经干细胞移植后对突触后膜的影响，研究神经干细胞能否与靶肌肉形成新的突触，以及新形成的突触是否具有电传导和物质传递功能。
结果3月时：移植组中突触前膜消失，突触后膜萎缩退变，未能检测到电传导，对照组中，突触前膜消失，突触后膜萎缩退变更加明显，同时数量减少，无电传导能力；5月时：移植组中检测到少量不完整的新生突触前膜，突触后膜趋向正常，能检测到电传导，对照组中突触前膜消失，后膜碎裂明显，无电传导能力；7月时：移植组中发现形态上呈各个发育阶段突触前膜，但数量少于突触后膜，而突触后膜形态基本正常，能检测到明显的电传导现象，同时顺向和逆向示踪均为阳性结果，对照组中，突触前后膜均完全消失，电生理检测和顺向逆向示踪均为阴性结果。
结论1、神经干细胞移植到损伤的外周神经后，可以延缓突触后膜萎缩退变和数量减少的发生；2、移植神经干细胞能够与失神经支配靶肌肉形成新的突触联系；3、新形成的突触具有物质传递和电传导功能。
第三部分宿主再生神经轴突与移植神经干细胞形成突触的实验研究
目的研究神经干细胞移植到外周神经系统后，宿主再生神经轴突与移植神经干细胞及其分化而来细胞之间的关系。
方法取孕10—12天F344大鼠，提取、培养并纯化脊髓神经干细胞；移植3cm长的F344神经干至GFP转基因大鼠的远端胫神经，同时移植10~6／ul×5ul F344神经干细胞悬液至移植段F344神经干中；2周后吻合宿主胫神经近端及移植段F344神经．术后2周取材，并进行免疫荧光染色和共聚焦观察，研究宿主再生神经轴突能否与移植并分化而来的神经元形成突触联系。
结果在干细胞移植后4周(神经吻合后2周)，神经干细胞可存活并分化为神经元；近端再生的神经轴突能与移植并分化而来的神经元形成突触联系。
结论宿主再生神经轴突能与移植分化而来的神经元形成突触联系。
PartⅠThe study of Culture,Increase and Transplant Neural StemCells in Delaying Denervated Muscle Atrophy.
Objective To Verify the Process validity in obtain,culture and increase Neural StemCells.Study the effect of Neural Stem Cells in delaying the denervated muscleatrophy when it was transplanted into peripheral nerveous system.
Methods Spinal Neural Stem Cells (NSCs) were obtained from Pregnant 10-12th dayembryo spinal cords of SD rats mechanically.The NSCs were cultured in no serumculture medium,after 2-3 generations,the neural stem cells were blew into singlecells,suspensioned and transplanted into the distal part of tibial nerve.Cell culturemedium was injected into the distal tibial nerve in contral group.3,5 and 7 monthspost-transplantation,the distal part of the tibial nerve and muscles were harvested andidentified with specific markers,by means of indirect immunofluorescent stainingrespectively.To evaluate the survival and differentiation of neural stem cells inperipheral nerve,The gastrocnemius muscles were excised and immediately weighedand the muscle fibers' area was measured and compaired to see whether thetransplanted NSCs can delay the denervated muscle atrophy.
Results The process of Spinal NSCs obtain and culture can get enough NSCs,andthese NSCs are active enough for transplantation.3,5 and 7 months after NSCstransplanted into tibial nerves,the immunofluorescent staining showed that NSCscould survive and differentiate into neurons.As the time go on,there were moreneurons apaired.What's more,these neurons can synthesis and secrete considerablesynaptophysin.Both of the humid weight and muscle fibers' cross section area oftriceps surae showed that the muscle atrophy was more serious in the control groupthan in the NSCs transplanted group.
Conclusions 1.The process of Spinal NSCs obtain and culture was suitable for theexperiment.2.Embryonic spinal stem cells can differentiate into different neural cells in vivo after transplantation; 3.NSCs' transplantate into PNS can delay thedenervated muscle atrophy.
PartⅡThe Experiment of New Formed Neuromuscular Junctionbetween Transplanted Neurol Stem Cells and Denervated Muscle
Objective Obtain enough neural stem cells and make the same animal models basedon experiment methods verified in PartⅠ,then study the new neuromuscularformation and detect its function.
Methods Based on the verified methods in partⅠ,we obtained enough activityseperated neural stem cells,made the same NSCs transplanted models and controlgroups.3,5,7 months after transplantation,we perform the different detect.Four andtwo weeks before the nerve and gastrocnemius muscle excision,the distal part of thetibial nerve was dissociated respectively to prevent potential nerve regeneration fromother nerves or tissues.Then the gastrocnemius muscle adjacent to the nerve entryplace were exsected and were carried out immunohistochemistry,to detect the changeof the neuromuscular Junction and the new formed Junction.We also usedElectrophysiology and Direct and Retrograde Tracing to detect the function of thenew formed neuromuscular Junction.
Results 3 months after fetal NSCs transplantation,the pre-menbrane dispersed,thepost-menbrane atrophy and cataplasia,no electrophysiology conductive ability; thesimilar results in the control models.5 months later,in 5 of 8 fetal NSCs transplanted rats,the transplant-derived colocalization of premenbrane reappeared and the host-derivedpostmenbrane became more mature than ever.While some of the premenbrane were lesscontinuous and in dot profile,the morphology of these new NMJs were simpler than thenormal NMJs.The CMAP waves were detected and the latent period was 12.5 ms,waveamplitude was 0.9 mv; In the same time,the pre-membrane disappeared and thepost-membrane fell into pieces in control rats,no CMAP can be detected.7 monthsposttransplantation,in 5 of 8 rats,the pre- and post-membrane were detected and moremature than ever,in some specimens,we detected the mature NMJs and thepostmembrane without the pre-menbrane (denervated muscle fiber) simultaneity,suggesting that the muscle reinnervation was incomplete.The latent period of CMAPwas 5 ms and wave amplitude was 1.5 mv.In the control group,we cannot see the shapeof NMJs,nigher pre-menbrane nor the post-menbrane,no CMAP can be detected.Inanterograde and retrograde tracing,the NSC transplant can transfer the true blue chloride while the control group cannot.
Conclusion 1,The transplanted NSCs can protect and delay the atrophy and cataplasia ofthe post-membrane; 2,new neuromuscular Junction can be formed between thetransplanted NSCs and the denervated muscle; 3,new formed NMJs are biologicallyactive.
PartⅢThe Formation of Synapse between Transplanted NSCs andHost's Regenerate Neural Axons
Objective To study the relationship between transplanted NSCs and host's regenerateneural axons after the NSCs transplanted into the impaired PNS.
Methods NSCs were separated from pregnancy 10-12th day spinal cord of F344 ratsmechanically and cultured in no serum culture medium.Transplante 1.Scm F344nerve segment to the distal part of the GFP gene transferred rats' tibial nerve.Meanwhile,inject 10~6/ul×5ul F344 NSCs into the transplanted F344 nerve segment.2 weeks later,suture the proximal tibial nerve stump with the transplanted F344 nervesegment.Another 2 weeks later,the tibial nerve and transplanted nerve segment wereharvested and identified with specific markers,by means of indirectimmunofluorescent staining respectively.To evaluate the survival and differentiationof neural stem cells and to see the relationship between transplanted NSCs and host'sregenerate neural axons.
Results 4 weeks after NSCs transplantation,NSCs survived and differentiated intoneurons.The regenerate axons can form synapse with neurons differentiated from thestem cells.
Conclusions In PNS,the host's regenerate axons can form synapse with the neuronsdifferentiated from the stem cells.
目的验证胚胎脊髓神经干细胞的提取、体外培养和增殖方法，研究神经干细胞移植到损伤的外周神经后，延缓失神经肌萎的效果。
方法取孕10—12天的SD大鼠，通过机械吹打法提取脊髓神经干细胞、以无血清限定性培养基进行培养，扩增及纯化得到大量增生的神经干细胞，以神经干细胞特异性抗体Nestin进行鉴定，将神经干细胞进行体外分化，通过特异性抗体染色检验其分化为神经终末细胞的能力。传2—3代后，将扩增的神经干细胞球分散成单细胞悬液并调整密度为10~6／ul，备用。制作胫神经切断，小腿三头肌失神经支配模型，以微量注射器移植10~6／ul×5ul干细胞悬液至切断的胫神经远端；设立对照组，仅移植5ul神经干细胞培养液，每组各24只。分别于术后3月，5月和7月取材，每次8只，分别观察移植神经干细胞在体内存活及分化成为神经元的情况；测量三头肌湿重及肌纤维横截面积维持率，验证神经干细胞移植能否延缓失神经支配骨骼肌的萎缩及其程度。
结果利用机械吹打和无血清限定性培养基可以得到大量的脊髓神经干细胞，得到的神经干细胞可以在体外大量扩增并分化为多种神经终末细胞，证明取得的神经干细胞活性良好；神经干细胞移植后3、5、7月取材及免疫组化染色显示，移植段胫神经内有大量存活的神经干细胞及其分化而来的神经元，神经元可以合成大量的突触素，随着时间延长，分化而来的神经元明显增加；随着时间延长，移植和对照组三头肌湿重，肌纤维横截面积均进行性下降，但对照组下降更明显(P〈0．05)。
结论1、机械吹打及无血清限定性培养基适用于脊髓神经干细胞的提取及培养；2、神经干细胞在损伤的外周神经系统中可以长期存活并分化为神经元；3、神经干细胞移植到外周神经系统内可以延缓失神经肌萎的程度。
第二部分移植神经干细胞与靶肌肉形成新突触过程的实验研究
目的将体外培养、扩增得到的神经干细胞移植到胫神经切断模型中，观察神经干细胞能否与失神经支配三头肌重新形成突触，以及新形成突触的功能。
方法采用机械吹打和无血清限定性培养基得到大量活性良好的神经干细胞，建立三头肌失神经支配模型，按第一部分方法分成移植组和对照组，每组30只。于移植术后3，5，7月进行检测，通过三头肌内突触(神经肌肉接头)免疫组化染色，神经电生理传导，示踪剂顺向和逆向传导实验，比较神经干细胞移植后对突触后膜的影响，研究神经干细胞能否与靶肌肉形成新的突触，以及新形成的突触是否具有电传导和物质传递功能。
结果3月时：移植组中突触前膜消失，突触后膜萎缩退变，未能检测到电传导，对照组中，突触前膜消失，突触后膜萎缩退变更加明显，同时数量减少，无电传导能力；5月时：移植组中检测到少量不完整的新生突触前膜，突触后膜趋向正常，能检测到电传导，对照组中突触前膜消失，后膜碎裂明显，无电传导能力；7月时：移植组中发现形态上呈各个发育阶段突触前膜，但数量少于突触后膜，而突触后膜形态基本正常，能检测到明显的电传导现象，同时顺向和逆向示踪均为阳性结果，对照组中，突触前后膜均完全消失，电生理检测和顺向逆向示踪均为阴性结果。
结论1、神经干细胞移植到损伤的外周神经后，可以延缓突触后膜萎缩退变和数量减少的发生；2、移植神经干细胞能够与失神经支配靶肌肉形成新的突触联系；3、新形成的突触具有物质传递和电传导功能。
第三部分宿主再生神经轴突与移植神经干细胞形成突触的实验研究
目的研究神经干细胞移植到外周神经系统后，宿主再生神经轴突与移植神经干细胞及其分化而来细胞之间的关系。
方法取孕10—12天F344大鼠，提取、培养并纯化脊髓神经干细胞；移植3cm长的F344神经干至GFP转基因大鼠的远端胫神经，同时移植10~6／ul×5ul F344神经干细胞悬液至移植段F344神经干中；2周后吻合宿主胫神经近端及移植段F344神经．术后2周取材，并进行免疫荧光染色和共聚焦观察，研究宿主再生神经轴突能否与移植并分化而来的神经元形成突触联系。
结果在干细胞移植后4周(神经吻合后2周)，神经干细胞可存活并分化为神经元；近端再生的神经轴突能与移植并分化而来的神经元形成突触联系。
结论宿主再生神经轴突能与移植分化而来的神经元形成突触联系。
PartⅠThe study of Culture,Increase and Transplant Neural StemCells in Delaying Denervated Muscle Atrophy.
Objective To Verify the Process validity in obtain,culture and increase Neural StemCells.Study the effect of Neural Stem Cells in delaying the denervated muscleatrophy when it was transplanted into peripheral nerveous system.
Methods Spinal Neural Stem Cells (NSCs) were obtained from Pregnant 10-12th dayembryo spinal cords of SD rats mechanically.The NSCs were cultured in no serumculture medium,after 2-3 generations,the neural stem cells were blew into singlecells,suspensioned and transplanted into the distal part of tibial nerve.Cell culturemedium was injected into the distal tibial nerve in contral group.3,5 and 7 monthspost-transplantation,the distal part of the tibial nerve and muscles were harvested andidentified with specific markers,by means of indirect immunofluorescent stainingrespectively.To evaluate the survival and differentiation of neural stem cells inperipheral nerve,The gastrocnemius muscles were excised and immediately weighedand the muscle fibers' area was measured and compaired to see whether thetransplanted NSCs can delay the denervated muscle atrophy.
Results The process of Spinal NSCs obtain and culture can get enough NSCs,andthese NSCs are active enough for transplantation.3,5 and 7 months after NSCstransplanted into tibial nerves,the immunofluorescent staining showed that NSCscould survive and differentiate into neurons.As the time go on,there were moreneurons apaired.What's more,these neurons can synthesis and secrete considerablesynaptophysin.Both of the humid weight and muscle fibers' cross section area oftriceps surae showed that the muscle atrophy was more serious in the control groupthan in the NSCs transplanted group.
Conclusions 1.The process of Spinal NSCs obtain and culture was suitable for theexperiment.2.Embryonic spinal stem cells can differentiate into different neural cells in vivo after transplantation; 3.NSCs' transplantate into PNS can delay thedenervated muscle atrophy.
PartⅡThe Experiment of New Formed Neuromuscular Junctionbetween Transplanted Neurol Stem Cells and Denervated Muscle
Objective Obtain enough neural stem cells and make the same animal models basedon experiment methods verified in PartⅠ,then study the new neuromuscularformation and detect its function.
Methods Based on the verified methods in partⅠ,we obtained enough activityseperated neural stem cells,made the same NSCs transplanted models and controlgroups.3,5,7 months after transplantation,we perform the different detect.Four andtwo weeks before the nerve and gastrocnemius muscle excision,the distal part of thetibial nerve was dissociated respectively to prevent potential nerve regeneration fromother nerves or tissues.Then the gastrocnemius muscle adjacent to the nerve entryplace were exsected and were carried out immunohistochemistry,to detect the changeof the neuromuscular Junction and the new formed Junction.We also usedElectrophysiology and Direct and Retrograde Tracing to detect the function of thenew formed neuromuscular Junction.
Results 3 months after fetal NSCs transplantation,the pre-menbrane dispersed,thepost-menbrane atrophy and cataplasia,no electrophysiology conductive ability; thesimilar results in the control models.5 months later,in 5 of 8 fetal NSCs transplanted rats,the transplant-derived colocalization of premenbrane reappeared and the host-derivedpostmenbrane became more mature than ever.While some of the premenbrane were lesscontinuous and in dot profile,the morphology of these new NMJs were simpler than thenormal NMJs.The CMAP waves were detected and the latent period was 12.5 ms,waveamplitude was 0.9 mv; In the same time,the pre-membrane disappeared and thepost-membrane fell into pieces in control rats,no CMAP can be detected.7 monthsposttransplantation,in 5 of 8 rats,the pre- and post-membrane were detected and moremature than ever,in some specimens,we detected the mature NMJs and thepostmembrane without the pre-menbrane (denervated muscle fiber) simultaneity,suggesting that the muscle reinnervation was incomplete.The latent period of CMAPwas 5 ms and wave amplitude was 1.5 mv.In the control group,we cannot see the shapeof NMJs,nigher pre-menbrane nor the post-menbrane,no CMAP can be detected.Inanterograde and retrograde tracing,the NSC transplant can transfer the true blue chloride while the control group cannot.
Conclusion 1,The transplanted NSCs can protect and delay the atrophy and cataplasia ofthe post-membrane; 2,new neuromuscular Junction can be formed between thetransplanted NSCs and the denervated muscle; 3,new formed NMJs are biologicallyactive.
PartⅢThe Formation of Synapse between Transplanted NSCs andHost's Regenerate Neural Axons
Objective To study the relationship between transplanted NSCs and host's regenerateneural axons after the NSCs transplanted into the impaired PNS.
Methods NSCs were separated from pregnancy 10-12th day spinal cord of F344 ratsmechanically and cultured in no serum culture medium.Transplante 1.Scm F344nerve segment to the distal part of the GFP gene transferred rats' tibial nerve.Meanwhile,inject 10~6/ul×5ul F344 NSCs into the transplanted F344 nerve segment.2 weeks later,suture the proximal tibial nerve stump with the transplanted F344 nervesegment.Another 2 weeks later,the tibial nerve and transplanted nerve segment wereharvested and identified with specific markers,by means of indirectimmunofluorescent staining respectively.To evaluate the survival and differentiationof neural stem cells and to see the relationship between transplanted NSCs and host'sregenerate neural axons.
Results 4 weeks after NSCs transplantation,NSCs survived and differentiated intoneurons.The regenerate axons can form synapse with neurons differentiated from thestem cells.
Conclusions In PNS,the host's regenerate axons can form synapse with the neuronsdifferentiated from the stem cells.
引文
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