本文關(guān)鍵詞： Nestin NeuN GFAP 凋亡 人胚胎 脊髓　出處：《昆明醫(yī)學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： [目的]探討人胚胎脊髓發(fā)育過(guò)程中神經(jīng)干細(xì)胞、神經(jīng)元、星形膠質(zhì)細(xì)胞的發(fā)育變化和規(guī)律以及細(xì)胞凋亡的變化。 [方法]采用免疫組織化學(xué)方法檢測(cè)神經(jīng)干細(xì)胞、神經(jīng)元、星形膠質(zhì)細(xì)胞的標(biāo)記蛋白Nestin,NeuN,GFAP在3w～8m人胚胎脊髓中的表達(dá)及變化;應(yīng)用TUNEL法檢測(cè)3w～8m人胚胎脊髓中的原位凋亡細(xì)胞。 [結(jié)果]1.Nestin染色結(jié)果:Nestin在整個(gè)人胚胎脊髓發(fā)育過(guò)程中均可見(jiàn)到,3w即可在神經(jīng)管見(jiàn)到陽(yáng)性細(xì)胞;5w～6w,陽(yáng)性細(xì)胞在神經(jīng)上皮背側(cè)較腹側(cè)多,套層、邊緣層中陽(yáng)性細(xì)胞開始出現(xiàn)并增多,脊神經(jīng)節(jié)內(nèi)可見(jiàn)陽(yáng)性反應(yīng)物;7w～8w,套層可見(jiàn)大量突起較長(zhǎng)的陽(yáng)性細(xì)胞(P＜0.05);10w～11w以后神經(jīng)上皮、灰白質(zhì)內(nèi)陽(yáng)性細(xì)胞密度和強(qiáng)度均逐漸減少,中央管、白質(zhì)陽(yáng)性細(xì)胞于3m、5m后趨于穩(wěn)定,灰質(zhì)內(nèi)可見(jiàn)少量有短突起的陽(yáng)性細(xì)胞。 2.NeuN染色結(jié)果:NeuN陽(yáng)性細(xì)胞在5w的套層中始見(jiàn),數(shù)量少且染色淺,邊緣層未見(jiàn)NeuN陽(yáng)性細(xì)胞;6w～7w,陽(yáng)性細(xì)胞明顯增多,尤以基板為甚,脊神經(jīng)節(jié)內(nèi)也可見(jiàn)到陽(yáng)性細(xì)胞;8w～9w,套層內(nèi)陽(yáng)性細(xì)胞進(jìn)一步增多(P＜0.05);10w,腹角內(nèi)陽(yáng)性細(xì)胞有所減少,后根處始見(jiàn)少量陽(yáng)性細(xì)胞;11w～3m,灰質(zhì)內(nèi)陽(yáng)性細(xì)胞數(shù)明顯降低(P＜0.05),腹角見(jiàn)體積較大、胞核深染的運(yùn)動(dòng)神經(jīng)元;4m～6m,灰質(zhì)內(nèi)的陽(yáng)性細(xì)胞數(shù)繼續(xù)減少(P＜0.05),后根處陽(yáng)性細(xì)胞減少(P＜0.05);7m～8m,灰質(zhì)內(nèi)陽(yáng)性細(xì)胞數(shù)量未有明顯變化。 3.GFAP染色結(jié)果:GFAP陽(yáng)性細(xì)胞始見(jiàn)于8w的腹側(cè)神經(jīng)管上皮;9w,神經(jīng)管上皮陽(yáng)性纖維密度有所增加(P＜0.05);10w～11w,灰、白質(zhì)內(nèi)陽(yáng)性纖維均明顯增多(P＜0.05),中央管11w明顯減少,后正中隔見(jiàn)到密集的陽(yáng)性纖維;3m,中央管陽(yáng)性纖維進(jìn)一步減少(P＜0.05),其后至8m未有明顯變化。灰質(zhì)內(nèi)偶見(jiàn)胞體、突起呈陽(yáng)性的星形膠質(zhì)細(xì)胞,背角內(nèi)陽(yáng)性纖維數(shù)量增多(P＜0.05);4m,腹角內(nèi)陽(yáng)性纖維數(shù)量明顯增多(P＜0.05),白質(zhì)內(nèi)纖維明顯較灰質(zhì)纖維密集,背角內(nèi)陽(yáng)性纖維減少(P＜0.01);5m之后陽(yáng)性細(xì)胞繼續(xù)減少并持續(xù)到8m。 4.TUNEL凋亡細(xì)胞檢測(cè)結(jié)果:3w～4w即可在神經(jīng)上皮見(jiàn)到大量陽(yáng)性細(xì)胞;5w,基板出現(xiàn)陽(yáng)性細(xì)胞;6w～7w,神經(jīng)上皮陽(yáng)性細(xì)胞減少(P＜0.05),基板內(nèi)的陽(yáng)性細(xì)胞較5w明顯增多(P＜0.05),脊神經(jīng)節(jié)也可見(jiàn)陽(yáng)性細(xì)胞,邊緣層未見(jiàn);8w,神經(jīng)上皮陽(yáng)性細(xì)胞進(jìn)一步減少(P＜0.05),套層中可見(jiàn)胞核深染的陽(yáng)性細(xì)胞,背根節(jié)內(nèi)也可見(jiàn)到陽(yáng)性細(xì)胞;9w,邊緣層開始出現(xiàn)少量陽(yáng)性細(xì)胞;10w～11w,神經(jīng)上皮陽(yáng)性細(xì)胞繼續(xù)減少(P＜0.05),腹角、背角內(nèi)均可見(jiàn)大量陽(yáng)性細(xì)胞,白質(zhì)內(nèi)陽(yáng)性細(xì)胞較9w有所增加(P＜0.05);3m～6m,灰質(zhì)內(nèi)陽(yáng)性細(xì)胞密度降低(P＜0.05),腹角見(jiàn)到陽(yáng)性運(yùn)動(dòng)神經(jīng)元,白質(zhì)內(nèi)可見(jiàn)大量小陽(yáng)性細(xì)胞;7m～8m,神經(jīng)上皮仍有少量陽(yáng)性細(xì)胞,灰、白質(zhì)內(nèi)均可見(jiàn)到體積較小的陽(yáng)性細(xì)胞,數(shù)量較6m明顯減少(P＜0.05)。 [結(jié)論]1.Nestin陽(yáng)性細(xì)胞在神經(jīng)上皮貫穿整個(gè)發(fā)育過(guò)程,5w基板、翼板形成后即可見(jiàn)到陽(yáng)性物,而邊緣層6w出現(xiàn),中央管、基板、翼板、邊緣層內(nèi)陽(yáng)性細(xì)胞達(dá)高峰的時(shí)間分別是6w、7w、8w、9w,而其出現(xiàn)明顯減少的時(shí)間分別是8w、10w、9w、10w,隨著脊髓的發(fā)育成熟,灰、白質(zhì)內(nèi)陽(yáng)性物逐漸減少,而中央管陽(yáng)性細(xì)胞數(shù)至3m后較為穩(wěn)定。 2.NeuN陽(yáng)性細(xì)胞僅在基板(腹角)、翼板(背角)及后根可見(jiàn),套層內(nèi)出現(xiàn)的時(shí)間是5w～6w,于9w達(dá)高峰,背角于3m又出現(xiàn)一次小高峰,隨后逐漸減少,白質(zhì)內(nèi)陽(yáng)性細(xì)胞10w出現(xiàn),持續(xù)到6m后消失。 3.GFAP出現(xiàn)時(shí)間最晚,中央管、腹角、白質(zhì)均8w出現(xiàn),高峰出現(xiàn)時(shí)間為10w、4m、4m,而背角于10w始見(jiàn),高峰出現(xiàn)時(shí)間為3m,隨后灰、白質(zhì)陽(yáng)性細(xì)胞均逐漸減少,而中央管于3m后趨于穩(wěn)定。 4.脊髓神經(jīng)上皮凋亡細(xì)胞在整個(gè)發(fā)育過(guò)程中均可見(jiàn)到,5w達(dá)高峰,基板、翼板內(nèi)凋亡細(xì)胞出現(xiàn)時(shí)間分別為5w、6w,高峰出現(xiàn)時(shí)間為6w、11w,邊緣層于9w始見(jiàn)凋亡細(xì)胞,5m出現(xiàn)高峰,隨后逐漸減少。
[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.

【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329.2

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