人胚胎脊髓发育中Nestin，NeuN，GFAP的表达变化及凋亡细胞的检测

发布时间：2018-02-14 15:11

  本文关键词： Nestin NeuN GFAP 凋亡 人胚胎 脊髓　出处：《昆明医学院》2008年硕士论文　论文类型：学位论文

【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。
[Abstract]:[Objective] to investigate the changes and rules of neural stem cells, neurons and astrocytes in the development of human embryonic spinal cord and the changes of cell apoptosis.
[Methods] immunohistochemical staining was used to detect the expression and change of Nestin, NeuN and GFAP in the 3W to 8m human fetal spinal cord, and the apoptotic cells in the 3W ~ 8m human embryonic spinal cord were detected by TUNEL.
The results of]1.Nestin staining: Nestin in the human embryonic spinal cord development process can be seen, 3W can see the positive cells in the neural tube; 5W ~ 6W positive cells in the dorsal ventral neural epithelium, a layer of positive cells in the edge layer began to appear and increase, spinal ganglia can be seen in the positive reaction; 7W ~ 8W, positive cell layer showed a large number of long protrusions (P < 0.05); 10W ~ 11W neural epithelium, positive cells in the gray matter density and strength were gradually reduced, the central canal, white matter in 3M positive cells, 5m positive cells tended to be stable, a small amount of gray matter in short projections.
Results: 2.NeuN staining was seen in NeuN positive cells in the mantle layer of 5W, small size and light staining, edge layer no NeuN positive cells; 6W ~ 7W, positive cells increased significantly, especially in the substrate even in the spinal ganglia can also see the positive cells; 8W ~ 9W, positive cells within a set of layers step number (P < 0.05); 10W positive cells in the ventral horn, reduced somewhat, after the root began to see a few positive cells; 11W ~ 3M, the number of positive cells in gray matter decreased significantly (P < 0.05), see the larger ventral horn motor neurons, hyperchromatic nucleus; 4m ~ 6m, the number of Yang cells in the gray matter continues to decrease (P < 0.05), the positive cells decreased after root (P < 0.05); 7m ~ 8m, the number of positive cells in the gray matter did not change significantly.
The results of 3.GFAP staining: GFAP positive cells was found in the ventral neural tube 9W, 8W epithelium; neural tube epithelial positive fiber density increased (P < 0.05); 10W ~ 11W, grey, white matter fibers were significantly increased (P < 0.05), the central tube 11W significantly reduced after median septum to see dense 3M positive fibers; the central tube positive fibers further reduced (P < 0.05), then to 8m did not change significantly. The gray matter seen occasionally in the soma, neurite positive astrocytes, the number of positive fibers in the dorsal horn increased (P < 0.05); 4m, the number of positive fibers increased significantly in the ventral horn (P < 0.05), in the white matter fiber than gray matter fiber dense, positive fibers in the dorsal horn decreased (P < 0.01); 5m positive cells continue to reduce and continue to 8m.
The apoptosis of 4.TUNEL cells: 3W ~ 4W to see a large number of positive cells in the neural epithelium; 5W substrate positive cells; 6W ~ 7W, neuroepithelial positive cells decreased (P < 0.05), positive cells within the substrate compared with 5W increased significantly (P < 0.05), spinal ganglia can also see the positive edge of cells. No 8W, neuroepithelial layer; positive cells further decreased (P < 0.05), positive cells in the mantle layer visible hyperchromatic nucleus and dorsal root ganglion in positive cells can be seen; 9W, edge layer began to appear a few positive cells; 10W ~ 11W, reduce the neural epithelium positive cells (P < 0.05 to), the ventral horn and dorsal horn were observed in a large number of positive cells, positive cells in the white matter than 9W increased (P < 0.05); 3M ~ 6m positive cells in the gray matter density decreased (P < 0.05), ventral horn motor neurons were seen in the white matter, showed a large amount of small positive cells; 7m 8m, there is still a small Yang neural epithelium The number of positive cells in sex cells, gray and white matter was smaller than that of 6m (P < 0.05).
[conclusion]1.Nestin positive cells throughout the whole developmental process in the neural epithelium of 5W substrate, wing formation can be seen after positive, while the edge layer 6W, central tube, substrate, wing plate, positive cells reached the peak time in the boundary layer are 6W, 7W, 8W, 9W, and it is significantly reduced the time were 8W, 10W, 9W, 10W, along with the development of spinal cord of mature, grey, white matter were gradually reduced, and the central canal of the number of positive cells after 3M is more stable.
2.NeuN positive cells were seen only in the basal plate (ventral horn), the wing plate (dorsal horn) and the posterior root. The time in the sheath was 5W ~ 6W, reaching the peak at 9W, and a small peak appeared at 3m in the dorsal horn, then gradually decreased, and the positive cells in the white matter appeared 10W and continued to disappear after 6m.
3.GFAP time later, the central canal, ventral horn, white matter 8W, the peak time of 10W, 4m, 4m, and 10W in the dorsal horn at the beginning, the peak time of 3M, then grey, white matter positive cells were gradually reduced, and the central tube after 3M tends to be stable.
4., the apoptotic cells of spinal cord neurons can be seen throughout the development process. 5W reaches the peak. The time of apoptotic cells in the basal plate and wing plate is 5W and 6W respectively. The peak time is 6W, 11W, and the apoptotic cells begin at 9W on the periphery, and the peak of 5m appears, then decreases gradually.

【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329.2

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