CAJ | 학술논문

目的观察分析胚胎脊髓细胞悬液(fetal spinal cord cell suspension,FSCS)联合自体激活雪旺细胞(autologus activated Schwann cells,AASCs)在损伤脊髓移植区中的突触发育过程.方法 42只Wistar成年大鼠结扎单侧隐神经,1周后取出结扎远端神经组织,分离、培养、纯化AASCs.以改良Allen法(10 g ×5 cm)打击脊髓,3天后将孕14天(E14)FSCS 20μl联合AASCs植入损伤空腔,移植后2、4、6、8、10和12周,以光学显微镜、电镜、免疫组织化学观察移植物成活、分化及其与宿主之间关系.结果移植区AASCs生长分化良好,胶质瘢痕少.成神经细胞最先展示了胞质突起,随之出现低电子密度的突触前、后膜,突触前、后膜电子密度逐渐增高形成良好的致密突起.突触小泡数量和种类逐渐增多,突触小泡有圆形清亮小泡、椭圆形小泡、颗粒状小泡和扁平小泡-f型.突触的连接方式由单个的胞体-树突突触,出现多个的胞体-树突和树突-树突突触.同时,移植成神经细胞、成少突胶质细胞、成星形细胞的细胞器日渐完善,细胞功能活跃.血脑屏障也随之出现.移植区可见神经微丝、组织胺、降钙素基因相关肽、胶原纤维酸性蛋白阳性纤维.结论 (1 )AASCs辅助下FSCS在成年大鼠损伤脊髓内可发育为成熟的突触；(2)FSCS与宿主脊髓重建突触方式的信息交换具有潜在可行性.
목적 관찰분석배태척수세포현액(fetal spinal cord cell suspension,FSCS)연합자체격활설왕세포(autologus activated Schwann cells,AASCs)재손상척수이식구중적돌촉발육과정.방법 42지Wistar성년대서결찰단측은신경,1주후취출결찰원단신경조직,분리、배양、순화AASCs.이개량Allen법(10 g ×5 cm)타격척수,3천후장잉14천(E14)FSCS 20μl연합AASCs식입손상공강,이식후2、4、6、8、10화12주,이광학현미경、전경、면역조직화학관찰이식물성활、분화급기여숙주지간관계.결과 이식구AASCs생장분화량호,효질반흔소.성신경세포최선전시료포질돌기,수지출현저전자밀도적돌촉전、후막,돌촉전、후막전자밀도축점증고형성량호적치밀돌기.돌촉소포수량화충류축점증다,돌촉소포유원형청량소포、타원형소포、과립상소포화편평소포-f형.돌촉적련접방식유단개적포체-수돌돌촉,출현다개적포체-수돌화수돌-수돌돌촉.동시,이식성신경세포、성소돌효질세포、성성형세포적세포기일점완선,세포공능활약.혈뇌병장야수지출현.이식구가견신경미사、조직알、강개소기인상관태、효원섬유산성단백양성섬유.결론 (1 )AASCs보조하FSCS재성년대서손상척수내가발육위성숙적돌촉；(2)FSCS여숙주척수중건돌촉방식적신식교환구유잠재가행성.
Objective To observe and analyze the synapses developing process of newly generated connections of autologus activated Schwann cells (AASCs) in combination with fetal spinal cord cell suspension (FSCS) in the surrounding area of the spinal cord injury site.Methods A total of 42 Wistar rats underwent unilateral ligation of the saphenous nerve.The portion of nerve tissues distal to the ligation site were harvested 1 week after operation.AASCs were isolated,cultured and purified.Spinal cord injury model produced in 42 Wistar rats on T7 by modified Allen impact method.Three days after injury,20 μl FSCS with a density of 1×105/μl prepared from pregnant rats (El4) in combination with AASCs were injected into the epicenter of the traumatized cavity.Animals were sacrificed at 2,4,6,8,10,12 weeks post transplantation.Light and electronmicroscopic studies as well as immunohistochemical assay were carried out to evaluate the graft survival,its differentation and integration with the host.Results In the transplantation area,AASCs showed good growth and differentiation,and glial scarring surrounding the lesions was less.The neuroblast stretched out the terminal endings 4 weeks after implantation,followed by the presenting of the pre- and post-synaptic membrane.Eight weeks post transplantation,the dense or developed projections were observed in the pre- and post-synaptic membrane,the high electron dense substance full filled the synaptic cleft.All the spherical cleat vesicles,granular vesicles,elliptical vesicles and flattened-f type vesicles were discovered under the electron microscope.Ten weeks after injury,the axosomatic,dendrosomatic,dendro-dendritic,axoaxonic,and dendro-axonic synapses coexisted.Light microscopy showed that the graft cell grew gradually.Immunohistochemical assay showed that NF,5-HT,CGRP and GFAP positive fibers were in the graft.Synapses,glia fibers and blood brain barrier integrated each other.Conclusion 1) The transplanted FSCS combined with AASCs can develop mature synapses with miscellaneous synaptic vesicles in the acute injured spinal cord.2) Co-existing indicate the possibility of synaptic connection between FSCS and host.