本文選題：創(chuàng)傷性腦損傷　切入點：NT-3殼聚糖支架　出處：《首都醫(yī)科大學》2014年碩士論文

【摘要】：研究背景：創(chuàng)傷性腦損傷后，，導致神經(jīng)元死亡或丟失，造成多種神經(jīng)功能缺陷，其中海馬損傷導致認知功能缺陷嚴重影響了患者的空間學習與記憶功能。傳統(tǒng)的觀點認為中樞神經(jīng)通路是固定的、不能改變的。中樞神經(jīng)死亡后不能再生，由膠質(zhì)細胞替代丟失的神經(jīng)細胞。近年研究表明在缺血損傷等條件下，成年大鼠腦海馬內(nèi)存在的內(nèi)源性神經(jīng)前體細胞可以不斷增殖產(chǎn)生新生神經(jīng)元。這為修復中樞神經(jīng)損傷提供了理論基礎(chǔ)。 研究目的：創(chuàng)傷性腦損傷后移植入NT-3殼聚糖支架材料，研究損傷腦組織再生及損傷區(qū)邊緣瘢痕情況；觀察損傷區(qū)神經(jīng)再生過程以及損傷區(qū)內(nèi)新生神經(jīng)元突觸形成，并參與腦神經(jīng)環(huán)路重建的研究。 實驗方法：本研究采用生物吸除裝置造成大鼠腦海馬CA1及以上皮層機械性損傷做為創(chuàng)傷性腦損傷的實驗?zāi)Ｐ�。實驗動物隨機分為三組：分別是單純損傷組、單純殼聚糖支架組、NT-3殼聚糖支架組。分別于術(shù)后3d、7d、14d、28d和60d，各實驗組取4只動物灌殺取腦，冰凍切片免疫化學組織染色觀察。NF染色觀察損傷區(qū)內(nèi)再生的神經(jīng)元及神經(jīng)纖維的情況；GFAP染色觀察損傷邊緣膠質(zhì)瘢痕情況。Nestin免疫組織化學染色觀察損傷區(qū)內(nèi)神經(jīng)前體細胞增殖情況；βtubulinⅢ免疫組織化學染色觀察損傷區(qū)內(nèi)分化的不成熟神經(jīng)元；MAP2免疫組織化學染色觀察損傷區(qū)內(nèi)分化成熟的神經(jīng)元。術(shù)后30d、60d灌殺動物，利用免疫電鏡技術(shù)和免疫組織化學染色觀察損傷區(qū)內(nèi)再生神經(jīng)元的突觸超微結(jié)構(gòu)。術(shù)后60d，應(yīng)用神經(jīng)示蹤方法將BDA-FITC注射到損傷對側(cè)CA3區(qū)，熒光顯微鏡下觀察示蹤劑走形情況。 研究結(jié)果：免疫組織化學結(jié)果顯示：創(chuàng)傷性腦損傷后60天，各組損傷區(qū)內(nèi)都可見不同數(shù)量神經(jīng)再生，統(tǒng)計數(shù)值后發(fā)現(xiàn)：NT-3殼聚糖支架組同單純殼聚糖組、單純損傷組存在明顯的統(tǒng)計學差異；同時觀察損傷邊緣的膠質(zhì)瘢痕情況發(fā)現(xiàn)NT-3殼聚糖支架組GFAP陽性細胞數(shù)明顯少于單純損傷組和單純殼聚糖支架組。以上結(jié)果說明創(chuàng)傷性腦損傷后，移植NT-3殼聚糖支架可以促進損傷區(qū)內(nèi)神經(jīng)再生，抑制損傷區(qū)邊緣膠質(zhì)瘢痕的形成。進一步研究移植NT-3殼聚糖支架到損傷區(qū)后，觀察神經(jīng)前體細胞增殖分化為成熟神經(jīng)元的過程。研究發(fā)現(xiàn)移植NT-3殼聚糖支架組，術(shù)后3天時nestin陽性細胞被激活，7天時nestin陽性細胞數(shù)達高峰，到14天出現(xiàn)下降趨勢。觀察發(fā)現(xiàn)腦損傷后14天βtubulinⅢ陽性細胞數(shù)增多，28天后陽性細胞數(shù)下降。研究發(fā)現(xiàn)腦損傷后28天MAP2陽性細胞數(shù)逐漸增多，此增長趨勢延續(xù)到腦損傷后60天。統(tǒng)計數(shù)值發(fā)現(xiàn)：NT-3殼聚糖支架組nestin、βtubulinⅢ和MAP2陽性細胞數(shù)同其他對照組存在明顯的統(tǒng)計學差異。移植NT-3殼聚糖支架組損傷后30天，通過免疫電鏡方法觀察損傷區(qū)內(nèi)出現(xiàn)BrdU+MAP2+雙陽性的新生神經(jīng)元，并形成成熟突觸結(jié)構(gòu)。熒光顯微鏡下觀察到BDA+神經(jīng)細胞通過右側(cè)海馬CA3錐體細胞的軸突沿著海馬聯(lián)合到達左側(cè)CA1區(qū)，在損傷區(qū)內(nèi)觀察到BDA+細胞，并且部分BDA+細胞表達MAP2+。 研究結(jié)論：NT-3殼聚糖支架可以促進損傷區(qū)內(nèi)神經(jīng)再生，抑制損傷周邊膠質(zhì)瘢痕的形成。損傷區(qū)內(nèi)神經(jīng)前體細胞能夠增殖分化為成熟神經(jīng)元并形成神經(jīng)突觸，參與腦神經(jīng)網(wǎng)絡(luò)的修復重建。
[Abstract]:Background : After traumatic brain injury , the death or loss of neurons leads to a variety of neurological deficits , in which the damage of the hippocampus leads to a severe impairment of cognitive function . The traditional view suggests that the central nervous pathway is fixed and cannot be changed . In recent years , the endogenous neural progenitor cells present in the hippocampus of adult rats can proliferate and produce new neurons . This is a theoretical basis for repairing the central nervous system injury .

Objective : To study the regeneration of injured brain tissue and the scar of injured area after traumatic brain injury .
Observe the nerve regeneration process in the injured area and the synaptic formation of neonatal neurons in the injured area , and participate in the study of the reconstruction of the brain nerve loop .

The experimental animal model of traumatic brain injury was studied by means of biosorption device . The experimental animals were randomly divided into three groups : simple injury group , chitosan stent group and NT - 3 chitosan stent group . The experimental animals were divided into three groups : simple injury group , simple chitosan stent group and NT - 3 chitosan stent group .
GFAP staining was used to observe the glial scar in the injured area . Nestin immunohistochemistry staining was used to observe the proliferation of neural progenitor cells in the injured area .
Non - mature neurons in the injured area were observed by immunohistochemical staining of 尾 - 鈪⑩參 .
The synaptic ultrastructure of regenerated neurons in the injured area was observed by immunohistochemical staining of MAP2 . After the operation , the synaptic ultrastructure of the regenerated neurons in the injured area was observed by immunoelectron microscopy and immunohistochemical staining . After operation 60 days after operation , the experimental tracer was injected into the injured contralateral CA area under the fluorescence microscope .

The results showed that after traumatic brain injury 60 days after traumatic brain injury , different numbers of nerve regeneration were observed in each group , and it was found that NT - 3 chitosan stent group had obvious statistical difference compared with simple chitosan group and simple injury group .
It was found that the number of GFAP - positive cells in NT - 3 chitosan stent group was significantly lower than that of simple injury group and simple chitosan stent group .

Conclusion : NT - 3 chitosan scaffold can promote nerve regeneration in the injured area and inhibit the formation of glial scar in the injured peripheral area . The neural progenitor cells in the injured area can proliferate into mature neurons and form neuroses , and participate in the repair and reconstruction of cranial nerve network .

【學位授予單位】：首都醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R651.15

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