本文關(guān)鍵詞： 創(chuàng)傷性腦損傷 NT3-殼聚糖 神經(jīng)發(fā)生 內(nèi)源性神經(jīng)干細胞 神經(jīng)網(wǎng)絡(luò) 功能恢復(fù) 微環(huán)境　出處：《首都醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：目的:明確NT3-殼聚糖載體能否促進創(chuàng)傷性腦損傷(traumatic brain injury,TBI)后損傷區(qū)內(nèi)新生神經(jīng)元的產(chǎn)生,并探討這些新生神經(jīng)元能否功能性整合入腦環(huán)路以修復(fù)腦功能;觀察TBI及NT3-殼聚糖對內(nèi)源性神經(jīng)干細胞(neural stem cells,NSCs)的激活情況,闡明NT3-殼聚糖對海馬齒狀回(dental gyrus,DG)顆粒細胞下層(subgranular zone,SGZ)和側(cè)腦室室旁區(qū)后部(posterior periventricle,pPV)NSCs的增殖、遷移和神經(jīng)向分化的影響;探討NT3-殼聚糖能否改善損傷區(qū)的微環(huán)境。方法:應(yīng)用自制的腦吸除裝置制備成年大鼠吸除性腦損傷模型,損傷范圍為海馬CA1區(qū)及以上皮質(zhì),術(shù)后立即向損傷區(qū)植入含或不含有神經(jīng)營養(yǎng)因子3(neurotrophin 3,NT3)的殼聚糖載體,實驗分為單純殼聚糖組和NT3-殼聚糖載體組,同時設(shè)假手術(shù)組(只打開骨窗,不接受TBI手術(shù))和損傷對照組(接受TBI手術(shù),但術(shù)后不給予任何治療措施)。HE染色觀察各組腦組織再生情況;大鼠術(shù)后立即腹腔注射BrdU以標(biāo)記增殖的細胞,BrdU/Tuj1和BrdU/NeuN染色檢測術(shù)后不同時間點損傷區(qū)新生神經(jīng)元的再生,同時應(yīng)用vGluT1和GAD67染色觀察損傷區(qū)內(nèi)不同類型神經(jīng)元的再生;BrdU/Tuj1/synapsin1和PSD95免疫熒光標(biāo)記及MAP2免疫電鏡檢測損傷區(qū)內(nèi)新生神經(jīng)元之間的突觸形成;MED64平面微陣列記錄系統(tǒng)檢測損傷區(qū)內(nèi)新生神經(jīng)元之間及新生神經(jīng)元與宿主腦之間的功能性神經(jīng)網(wǎng)絡(luò)形成;BDA神經(jīng)示蹤觀察再生神經(jīng)元在海馬內(nèi)的神經(jīng)環(huán)路重建;morris水迷宮評價大鼠的空間學(xué)習(xí)記憶功能。為了檢測不同腦區(qū)nscs的增殖,大鼠灌流前48小時開始腹腔注射brdu,于術(shù)后不同時間點觀察并計數(shù)海馬dg不同亞區(qū)和ppv區(qū)增殖細胞的數(shù)量;brdu/neun/gfap免疫熒光三標(biāo)記檢測dg增殖細胞的命運;brdu/dcx免疫熒光染色及dil體內(nèi)標(biāo)記觀察sgz和ppv增殖nscs的遷移。cd45和cd11b染色觀察nt3-殼聚糖對tbi后巨噬細胞和小膠質(zhì)細胞的影響,實時定量pcr檢測腦內(nèi)促炎和抗炎因子的表達水平,reca-1染色觀察損傷區(qū)內(nèi)的血管再生。結(jié)果:1.brdu/tuj1和brdu/gfap/neun染色結(jié)果顯示,nt3-殼聚糖能夠促進損傷區(qū)內(nèi)成熟神經(jīng)元的再生,同時抑制膠質(zhì)反應(yīng),并且新生神經(jīng)元包含谷氨酸能神經(jīng)元和gaba能神經(jīng)元。2.brdu/tuj1/synapsin1和psd95免疫熒光染色及map2免疫電鏡結(jié)果顯示,nt3-殼聚糖促進損傷區(qū)內(nèi)新生神經(jīng)元之間的突觸形成。med64進一步證明,nt3-殼聚糖促進新生神經(jīng)元之間及新生神經(jīng)元和宿主腦之間功能性神經(jīng)網(wǎng)絡(luò)的建立。bda神經(jīng)示蹤結(jié)果顯示,新生神經(jīng)元可重建受損的海馬環(huán)路。3.morris水迷宮結(jié)果表明,nt3-殼聚糖可以改善損傷后期大鼠的認知功能,同時恢復(fù)海馬內(nèi)schaffer側(cè)支-ca1突觸的ltp特性。4.brdu/nestin染色結(jié)果顯示,nt3-殼聚糖可激活內(nèi)源性nscs,并促進它們向損傷區(qū)的遷移。5.brdu染色結(jié)果顯示,tbi增加損傷同側(cè)dgbrdu+細胞數(shù),nt3-殼聚糖進一步增加tbi所致的增殖反應(yīng),術(shù)后各時間點損傷同側(cè)nt3-殼聚糖組的增殖細胞均多于單損組。對dg增殖細胞的分布分析結(jié)果顯示,術(shù)后不同時間點nt3-殼聚糖組和單損組brdu+細胞大部分仍位于神經(jīng)發(fā)生區(qū)sgz區(qū),且nt3-殼聚糖組陽性細胞數(shù)多于單損組,其余細胞分布于dg非神經(jīng)發(fā)生區(qū)即分子層和門區(qū)。6.brdu/dcx染色結(jié)果顯示,nt3-殼聚糖增加sgzbrdu+/dcx+細胞數(shù),同時增加BrdU+/Dcx+細胞的比例,說明NT3-殼聚糖不僅增加SGZ新生成神經(jīng)細胞的數(shù)量,而且增加成神經(jīng)細胞在增殖細胞中的比例。BrdU/NeuN/GFAP免疫標(biāo)記顯示,NT3-殼聚糖促進DG增殖細胞的存活,同時增加DG新生顆粒神經(jīng)元的數(shù)量,促進DG神經(jīng)發(fā)生。7.BrdU/Dcx染色結(jié)果表明,NT3-殼聚糖促進SGZ NSCs向損傷區(qū)的遷移。8.BrdU染色結(jié)果顯示,NT3-殼聚糖增加TBI所致的損傷同側(cè)pPV NSCs的增殖,BrdU/Dcx免疫染色和Dil體內(nèi)標(biāo)記實驗顯示,NT3-殼聚糖促進室周區(qū)NSCs向損傷區(qū)的遷移,損傷區(qū)內(nèi)檢測到的Dil+細胞部分表達NSCs標(biāo)記物nestin,部分表達成神經(jīng)細胞標(biāo)記物Dcx,說明室周區(qū)NSCs可能參與了損傷區(qū)新生神經(jīng)元的再生。9.CD45和CD11b染色結(jié)果顯示,NT3-殼聚糖抑制損傷區(qū)內(nèi)巨噬細胞的浸潤和小膠質(zhì)細胞的激活,同時恢復(fù)損傷周圍腦組織不同狀態(tài)小膠質(zhì)細胞的比例。實時定量PCR結(jié)果顯示,NT3-殼聚糖抑制TBI后促炎因子IL-1β和TNF-α的表達,同時增加抗炎因子IL-4和IL-10的水平,提示NT3-殼聚糖的抗炎作用。10.Reca-1熒光染色結(jié)果提示,NT3-殼聚糖促進損傷區(qū)內(nèi)的血管再生,增加損傷區(qū)再生血管的密度和直徑。結(jié)論:NT3-殼聚糖載體通過持續(xù)釋放NT3神經(jīng)營養(yǎng)因子,顯著激活SGZ和pPV內(nèi)源性NSCs,并促進它們向損傷區(qū)的異位遷移。同時該生物活性材料為損傷區(qū)提供了一個抗炎和促血管再生的微環(huán)境,有利于遷移至損傷區(qū)的NSCs分化為成熟的神經(jīng)元,并建立功能性神經(jīng)網(wǎng)絡(luò),進而修復(fù)受損的腦功能。這些結(jié)果為NT3-殼聚糖活性生物材料修復(fù)成年腦損傷的臨床應(yīng)用提供了理論基礎(chǔ)和實驗依據(jù)。
[Abstract]:Objective: to determine the NT3- of chitosan can promote traumatic brain injury (traumatic brain, injury, TBI) after injury of newborn neurons in the region, and to explore these new neurons can integrate into the brain functional loop to repair brain function; to observe the TBI and NT3- of Chitosan on endogenous neural stem cells (neural stem cells, NSCs the activation of NT3-, clarify) chitosan on hippocampal dentate gyrus (dental, gyrus, DG (subgranular) subgranular zone zone, SGZ) and periventricle rear (posterior periventricle, pPV NSCs) proliferation, migration and neural differentiation effect; explore the micro environment of NT3- chitosan can improve the damage zone methods: Using Self-made brain suction device for the preparation of adult rat model of brain injury aspiration, damage range for the CA1 region of the hippocampus and cortex above, immediately after surgery to implant damage zone containing or not containing neurotrophic factor 3 (Neurot Rophin 3, NT3) chitosan carrier, the experiment was divided into chitosan groups and NT3- chitosan group, sham operation group (only open bone window, do not accept the TBI surgery) and injury group (received TBI surgery, but postoperative given no treatment measures) were observed by.HE staining of brain tissue regeneration; rat operation immediately after intraperitoneal injection of BrdU labeled with the proliferation of cells, BrdU/Tuj1 and BrdU/NeuN were detected at different time points after new injury neuron regeneration, while using vGluT1 and GAD67 staining to observe the different types of neurons in the damage zone; formed between BrdU/Tuj1/synapsin1 and PSD95 by immunofluorescence and immunoelectron microscopy MAP2 detection of damage zone of newborn neurons synapse; functional neural network between MED64 plane microarray system measured in the damage zone of new neurons and neurons and host brain formation; BDA Neural tracing observation neural circuits in the hippocampus neuron regeneration and reconstruction; learning and memory function of Morris water maze in rats space. In order to detect NSCs in different brain regions of the proliferation of rats were perfused 48 hours before intraperitoneal injection of BrdU at different time points after operation to observe and count the sea horse DG different number of sub regions and PPV proliferation; brdu/neun/gfap immunofluorescence three markers DG proliferation cell fate; to observe the effect of Chitosan on nt3- TBI macrophages and microglia brdu/dcx immunofluorescence staining and DIL in SGZ and PPV marker to observe the proliferation of NSCs migration of.Cd45 and CD11b staining, the expression of real time quantitative PCR detection of brain proinflammatory and anti-inflammatory the observation of angiogenesis factor, damage zone. Results: 1.brdu/tuj1 reca-1 staining and brdu/gfap/neun staining showed that nt3- chitosan can promote the maturation of neurons and damage zone At the same time, inhibition of glial cells, and neurons contains glutamatergic neurons and GABA neurons.2.brdu/tuj1/synapsin1 and PSD95 immunofluorescence staining and MAP2 immunoelectron microscopy showed that nt3- chitosan to promote in the damage zone of new neurons synapse formation.Med64 further proved that nt3- chitosan to promote new neurons and between neurons and host brain function a neural network.Bda neural tracing showed that new neurons can restore damaged hippocampal loop.3.morris water maze test results show that the cognitive function of nt3- chitosan can improve the late injury in rats, and restore the.4.brdu/nestin characteristics of LTP Schaffer in hippocampus -ca1 collateral synapses staining showed that nt3- chitosan can activate endogenous NSCs, and promote they migrate to the injured area of.5.brdu staining showed that TBI increased the damage of ipsilateral dgbrd The number of u+ cells, nt3- chitosan increased proliferation reaction induced by TBI further, the ipsilateral nt3- chitosan group cells were more than single injury group at different time points after operation. The distribution of damage analysis results on the proliferation of DG cells showed different postoperative time points nt3- chitosan group and the single injury group brdu+ cells are still located in the neurogenesis zone SGZ, nt3- and chitosan group positive cells than single injury group, the remaining cells distributed in the DG region is non neurogenic.6.brdu/dcx molecular layer and hilus staining showed that nt3- chitosan increased the number of sgzbrdu+/dcx+ cells, while BrdU+/ increased the percentage of Dcx+ cells, indicating that NT3- chitosan not only increased the number of SGZ the new generation of nerve cells, and increase the proportion of nerve cells in the proliferation of.BrdU/NeuN/GFAP cells in immune markers showed that NT3- chitosan promotes the survival of DG cells proliferation, and increased DG in newborn granule neuronal number The amount of DG, promote the neurogenesis of.7.BrdU/Dcx staining showed that NT3- chitosan SGZ NSCs to promote the migration of.8.BrdU damage zone staining showed that NT3- chitosan increased TBI damage caused by ipsilateral pPV NSCs proliferation, BrdU/Dcx staining and Dil labeling experiments in vivo, NT3- chitosan to promote the periventricular area of NSCs migration to the damaged area the Dil+ cells in the damage zone detected expression of NSCs marker nestin, expressed as a part of the neuronal marker Dcx, indicating the periventricular area of NSCs may be involved in the damage zone of new neurons to regenerate.9.CD45 and CD11b staining results showed that NT3- chitosan inhibited the activation of macrophage infiltration in the damage zone and microglia at the same time, recovery of injury in different condition of microglia around the brain. The proportion of real-time quantitative PCR showed that the expression of NT3- TBI after chitosan inhibits proinflammatory factor IL-1 alpha beta and TNF-, at the same time The increase of anti-inflammatory factor IL-4 and IL-10 levels, the anti-inflammatory effect of chitosan NT3-.10.Reca-1 fluorescence staining showed that NT3- chitosan promoting vascular regeneration in the damage zone, damage zone increase vascular regeneration density and diameter. Conclusion: NT3- chitosan sustained release NT3 by neurotrophic factors, activate SGZ and pPV endogenous NSCs, and promote them to transfer ectopic injury area. At the same time provides a anti-inflammatory and vascular regeneration microenvironment of the bioactive materials for the damage zone, NSCs is favorable to the differentiation of migrate to the injury area as neurons mature, and the establishment of functional neural network, and then repair the damaged brain function provides a theoretical. These results and experimental basis for clinical application in the repair of NT3- chitosan bioactive material in adult brain injury.

【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R651.15;R318.08

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