【作者】 高燕；

【导师】 李力燕； 王廷华；

【作者基本信息】 昆明医学院 ， 人体解剖学与组织胚胎学， 2008， 硕士

【摘要】 [目的]探讨人胚胎脊髓发育过程中神经干细胞、神经元、星形胶质细胞的发育变化和规律以及细胞凋亡的变化。[方法]采用免疫组织化学方法检测神经干细胞、神经元、星形胶质细胞的标记蛋白Nestin,NeuN,GFAP在3w～8m人胚胎脊髓中的表达及变化;应用TUNEL法检测3w～8m人胚胎脊髓中的原位凋亡细胞。[结果]1.Nestin染色结果:Nestin在整个人胚胎脊髓发育过程中均可见到,3w即可在神经管见到阳性细胞;5w～6w,阳性细胞在神经上皮背侧较腹侧多,套层、边缘层中阳性细胞开始出现并增多,脊神经节内可见阳性反应物;7w～8w,套层可见大量突起较长的阳性细胞(P＜0.05);10w～11w以后神经上皮、灰白质内阳性细胞密度和强度均逐渐减少,中央管、白质阳性细胞于3m、5m后趋于稳定,灰质内可见少量有短突起的阳性细胞。2.NeuN染色结果:NeuN阳性细胞在5w的套层中始见,数量少且染色浅,边缘层未见NeuN阳性细胞;6w～7w,阳性细胞明显增多,尤以基板为甚,脊神经节内也可见到阳性细胞;8w～9w,套层内阳性细胞进一步增多(P＜0.05);10w,腹角内阳性细胞有所减少,后根处始见少量阳性细胞;11w～3m,灰质内阳性细胞数明显降低(P＜0.05),腹角见体积较大、胞核深染的运动神经元;4m～6m,灰质内的阳性细胞数继续减少(P＜0.05),后根处阳性细胞减少(P＜0.05);7m～8m,灰质内阳性细胞数量未有明显变化。3.GFAP染色结果:GFAP阳性细胞始见于8w的腹侧神经管上皮;9w,神经管上皮阳性纤维密度有所增加(P＜0.05);10w～11w,灰、白质内阳性纤维均明显增多(P＜0.05),中央管11w明显减少,后正中隔见到密集的阳性纤维;3m,中央管阳性纤维进一步减少(P＜0.05),其后至8m未有明显变化。灰质内偶见胞体、突起呈阳性的星形胶质细胞,背角内阳性纤维数量增多(P＜0.05);4m,腹角内阳性纤维数量明显增多(P＜0.05),白质内纤维明显较灰质纤维密集,背角内阳性纤维减少(P＜0.01);5m之后阳性细胞继续减少并持续到8m。4.TUNEL凋亡细胞检测结果:3w～4w即可在神经上皮见到大量阳性细胞;5w,基板出现阳性细胞;6w～7w,神经上皮阳性细胞减少(P＜0.05),基板内的阳性细胞较5w明显增多(P＜0.05),脊神经节也可见阳性细胞,边缘层未见;8w,神经上皮阳性细胞进一步减少(P＜0.05),套层中可见胞核深染的阳性细胞,背根节内也可见到阳性细胞;9w,边缘层开始出现少量阳性细胞;10w～11w,神经上皮阳性细胞继续减少(P＜0.05),腹角、背角内均可见大量阳性细胞,白质内阳性细胞较9w有所增加(P＜0.05);3m～6m,灰质内阳性细胞密度降低(P＜0.05),腹角见到阳性运动神经元,白质内可见大量小阳性细胞;7m～8m,神经上皮仍有少量阳性细胞,灰、白质内均可见到体积较小的阳性细胞,数量较6m明显减少(P＜0.05)。[结论]1.Nestin阳性细胞在神经上皮贯穿整个发育过程,5w基板、翼板形成后即可见到阳性物,而边缘层6w出现,中央管、基板、翼板、边缘层内阳性细胞达高峰的时间分别是6w、7w、8w、9w,而其出现明显减少的时间分别是8w、10w、9w、10w,随着脊髓的发育成熟,灰、白质内阳性物逐渐减少,而中央管阳性细胞数至3m后较为稳定。2.NeuN阳性细胞仅在基板(腹角)、翼板(背角)及后根可见,套层内出现的时间是5w～6w,于9w达高峰,背角于3m又出现一次小高峰,随后逐渐减少,白质内阳性细胞10w出现,持续到6m后消失。3.GFAP出现时间最晚,中央管、腹角、白质均8w出现,高峰出现时间为10w、4m、4m,而背角于10w始见,高峰出现时间为3m,随后灰、白质阳性细胞均逐渐减少,而中央管于3m后趋于稳定。4.脊髓神经上皮凋亡细胞在整个发育过程中均可见到,5w达高峰,基板、翼板内凋亡细胞出现时间分别为5w、6w,高峰出现时间为6w、11w,边缘层于9w始见凋亡细胞,5m出现高峰,随后逐渐减少。更多还原

【Abstract】 [Objective]:To explore the expression and changes of Nestin,NeuN and GFAP in the human embryo fetal spinal cord and investigation on apoptosis in the spinal cord.[Methods]:The expressions and changes of Nestin,NeuN and GFAP were studied systematically in human embryonic spinal cord from the 3 weeks to 8 monthes by the methods of immunohistochemical staining.In situ apoptotic cell detection by TUNEL method.[Results]:.1.Nestin:Nestin-positive immunoreaction appeared throughout the whole period of human embryo fetal spinal cord development,Nestin-positive immunoreaction could be seen on the neurepithelium of neural tube at 3w;at 5w～6w, Nestin-positive immunoreaction began to appeared in mantle layer and marginal layer,more.Nestin-positive immunoreaction appeared on dorsal neurepithelium,and positive structure could be seen on dorsal root ganglion(P＜0.05);at 7w～8w,the length of nemaline Nestin-positive immunoreaction increased;after 10w～11w, density and intensity of Nestin-positive immunoreaction on the neurepithelium,write, gray matter decreased;Nestin-positive structure in ventri-mantle layer was more than dorsal mantle layer with Nestin-positive immunoreaction in mantle and marginal layer being less(P＜0.05),there were a few of positive nestin cells with short processes in gray matter.2.NeuN:NeuN-positive cells in mantle layer appeared at 5w with less amount and light staining,there was no NeuN-positive cells appearing in marginal layer;6w～7w, NeuN-positive cells became more and more,especially in basal plate,some NeuN-positive cells showed up in dorsal root ganglion;8w～9w,alar plate widened, number of NeuN-positive cells in mantle layer kept increasing(P＜0.05);10w,number of NeuN-positive cell in ventral horn went down(P＜0.05),a small amount of NeuN-positive cells began to show up in dorsal root of marginal layer,no NeuN-positive cells in the rest of marginal layer and neurepithelium;11w～3m, amount of NeuN-positive cells in gray matter remarkably reduced(P＜0.05),some motoneurons could be seen in ventral horn;4m～6m,NeuN-positive cells in gray matter kept reducing(P＜0.05),NeuN-positive cells decreased in dorsal root of white matter(P＜0.05);7m～8m,number of NeuN-positive cells in gray matter showed no significant difference.3.GFAP:A small amount of GFAP-positive immunoreaction with light staining began to show up in ventri- neurepithelium at 8w;9w,density of GFAP-positive immunoreaction in neural epithelium increase(P＜0.05);10w～11w,GFAP-positive immunoreaction in write、gray matter increased significantly(P＜0.05),GFAP-positive immunoreaction in neurepithelie decreased;3m,positive GFAP structure on neurepithelie of canalis centralis reduced(P＜0.05),there were also GFAP-positive immunoreaction in gray matter and white matter,accidentally we could find positive astrocyte with cell body and processes,more positive GFAP structure appeared in dorsal horn(P＜0.05);4m,number of GFAP-positive immunoreaction in ventral horn increased further(P＜0.05),structure in white matter much more dense than that in gray matter,positive structure in dorsal horn decreased(P＜0.01);5m,GFAP-positive immunoreaction continually decreased to 8m.4.TUNEL:A lot of TUNEL-positve cells could be seen in neural epithelium at 3w～4w;5w,TUNEL-positve cells began to be detected in basal plate;6w～7w, TUNEL-positve cells in neuroepithelial layer decrease(P＜0.05),TUNEL-positve cells in basal plate increase comparing to 5w(P＜0.05),TUNEL-positve cells also showed up in dorsal root ganglion,no TUNEL-positve cells in marginal layer;8w, TUNEL-positve cells in neurepithelium decreased(P＜0.05),TUNEL-positve cells with strong staining showed in mantle layer,TUNEL-positve cells could be seen in dorsal root ganglion,no TUNEL-positve cells in marginal layer;9w,a few TUNEL-positve cells showed up in marginal layer;10w～11w,TUNEL-positve cells in neurepithelium continued to decrease(P＜0.05),a lot of positive cells in ventral horn and dorsal horn could be seen,there were more positive cells in white matter than 9w(P＜0.05);3m～6m,the numble of positive cells in gray matter decreased(P＜0.05), positive motoneuron could be detected in ventral horn,and smaller positive cells could be found in write matter;7m～8m,TUNEL-positve cells in neurepithelium with less amount than before(P＜0.05),smaller positive cells could be detected in gray matter and white matter with less number than in 6m(P＜0.05).[Conclusion]:The time to show up and peak time are different among neural stem cell,neuron,astrocyte and apoptosis cell,Nestin-positive cells in neurepithelium could be detected throughout the development of human embyro spinal cord, nestin-positive cells could be seen at the same time the basal plate and alar plate formed at 5th week,Nestin-positive cells in marginal layer showed up at 6w. Nestin-positive cells in neurepithelium,basal plate,alar plate,marginal.As spinal cord developed,Nestin-positive cells in write,gray matter decreased gradually,the number of Nestin-positive cells in neurepithelium showed no significant difference after 3m.NeuN-positive cells could only be found in basal plate(ventral horn),alar plate(dorsal horn)during the development of human embyro spinal cord, NeuN-positive cells showed up in basal plate at 5w,NeuN-positve cells peaked at 9w, another peaked in dorsal horn at 3m,then began to decrease.The time for GFAP-positive cells to show up was the latest of all,for neurepithelium,basal plate, marginal layer was 8th week,doral horn at 10w,peak time in canalis centralis neurepithelium was earlier than other parts,which was 10w,peak time in ventral horn and white matter was 4m,in dorsal horn was 3m,then GFAP-positive cells in other parts began gradually to reduce.TUNEL-positive cells in neuroepithelium could be found throughout the development of spinal cord and peaked at 5w.TUNEL-positive cells in basal plate and alar plate could be detected as 5w and 6w.The number of TUNEL-positive cells in basal plate and alar plate peaked at 6w and 11w. TUNEL-positive cells in marginal layer showed up at 9w and peaked at 5m,then decreased gradually.更多还原