本文關(guān)鍵詞：低頻弱磁誘導(dǎo)移植神經(jīng)干細(xì)胞上調(diào)β-Ⅲtubulin mRNA表達(dá)修復(fù)腦損傷大鼠爬行功能的研究　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 低頻弱磁 腦損傷 神經(jīng)干細(xì)胞 細(xì)胞移植 分化

【摘要】：目的:通過體內(nèi)體外實驗設(shè)計觀察低頻弱磁(low frequency magnetic flux)在骨髓源神經(jīng)干細(xì)胞移植對腦損傷大鼠(rats with brain injury)爬行功能改善的作用。觀察到相關(guān)神經(jīng)元標(biāo)志物以及神經(jīng)遞質(zhì)的表達(dá)改變,為腦損傷的臨床治療提供基礎(chǔ)研究依據(jù)。方法:建立腦損傷大鼠模型,將64只SD大鼠隨機(jī)分成四組,每組16只大鼠,分別為對照組(C組)、BM-NPCs組(B組)、磁場組(L組)和磁場+BM-NPCs組(LB組)。取3周齡SD大鼠骨髓來源的骨髓間充質(zhì)干細(xì)胞,體外培養(yǎng)骨髓源神經(jīng)干細(xì)胞(Bone Marrow Stroma Cells-derived Neural Progenitor Cells,BM-NPCs),在成功造模第7d,移植體外CD-Dil標(biāo)記的BM-NPCs。其中,L組和LB組分別進(jìn)行低頻弱磁場(50Hz、5mT)干預(yù),60min/d,連續(xù)干預(yù)60d。在實驗過程中,分別在移植1d、3d、7d、30d和60 d對各組大鼠進(jìn)行水平爬樓梯評分、Wayne clark行為學(xué)評分,并于移植1d、7d和30d取材進(jìn)行HE染色,觀察各組腦組織病理表現(xiàn);在外傷區(qū),免疫熒光檢測移植細(xì)胞的神經(jīng)細(xì)胞標(biāo)志物NeuN、GFAP、β-Ⅲtubulin表達(dá)。在體外實驗中,使用50Hz、5mT弱磁場對BM-NPCs進(jìn)行處理,時間為60min/天,連續(xù)作用15天,同時設(shè)置對照組。細(xì)胞免疫熒光技術(shù)檢測Q-PCR分析BM-NPCs表達(dá)Nestin(神經(jīng)干細(xì)胞標(biāo)志物)、PSA-NCAM、β-Ⅲtubulin(神經(jīng)元標(biāo)志物)、ACHE、5-HT、GABA(神經(jīng)遞質(zhì))等情況。結(jié)果:BM-NPCs移植后第1d,經(jīng)Wayne clark評分以及水平爬樓梯評分后,各組結(jié)果差異無統(tǒng)計學(xué)意義(P0.05);移植后第3d、7d、30d、60d,各組Wayne clark評分、水平爬樓梯評分結(jié)果顯示,組間比較差異有統(tǒng)計學(xué)意義(P0.05),C組與其他三組比較有明顯差異,不同時間點也存在差異,不同處理因素隨時間增加而行為學(xué)評分的變化趨勢不同,其中LB組運動功能評分改善更為明顯。HE染色結(jié)果顯示,造模后第7d,各組大鼠腦損傷灶周圍組織血管受壓變形、組織腫脹壞死形成。BM-NPCs移植后第7d,C組和L組腦損傷灶周圍組織水腫明顯減輕,有較大的囊性空洞,周圍炎癥細(xì)胞浸潤,神經(jīng)細(xì)胞減少明顯;B組和LB組腦損傷灶周圍組織水腫較輕,出現(xiàn)范圍局限的囊性空洞,可見膠質(zhì)細(xì)胞。移植后第30d,除對照組外,其他三組囊性空洞面積均較少,B組水腫組織消失,LB組腦損傷灶周圍組織細(xì)胞排類整齊,炎癥細(xì)胞基本消失。移植后第60d,與對照組相比,各實驗組腦損傷組織結(jié)構(gòu)大部分修復(fù)。留取標(biāo)本進(jìn)行免疫熒光技術(shù)檢測顯示,BM-NPCs移植后第30d,B組和LB組大鼠腦損傷組織周圍出現(xiàn)Dil標(biāo)記的NeuN和β-Ⅲtubulin陽性細(xì)胞;移植后第60d,Dil標(biāo)記的NeuN和β-Ⅲtubulin陽性細(xì)胞與腦損傷周圍組織融合生長。BM-NPCs移植后第30d和第60d,C組大鼠腦損傷組織周圍可見大量GFAP陽性細(xì)胞,而L組和LB組較C組少。在體外實驗過程中,體外培養(yǎng)的BM-NPCs形成細(xì)胞球并經(jīng)弱磁場干預(yù)后,可觀察到神經(jīng)元樣細(xì)胞出現(xiàn)。免疫熒光技術(shù)檢測發(fā)現(xiàn)體外培養(yǎng)的BM-NPCs Nestin呈陽性表達(dá);對照組和磁場組神經(jīng)元樣細(xì)胞Tuj-1和GFAP均呈陽性表達(dá)。與對照組相比,Q-PCR結(jié)果顯示磁場組Nestin、PSA-NCAM、Ach、GABA、5-HT基因表達(dá)水平與體外誘導(dǎo)前有顯著性差異(P0.01),弱磁場組β-ⅢtubulinmRNA表達(dá)水平顯著高于對照組(P0.05)。結(jié)論:低頻弱磁能改善腦損傷大鼠患側(cè)爬行功能,也能促進(jìn)骨髓源神經(jīng)干細(xì)胞移植腦損傷大鼠模型的修復(fù),該過程可能與低頻弱磁場上調(diào)β-Ⅲtubulin mRNA表達(dá)水平促進(jìn)骨髓源神經(jīng)干細(xì)胞向神經(jīng)細(xì)胞分化有關(guān)。
[Abstract]:Objective: To observe the in vivo and in vitro experimental design of low frequency weak magnetic field (low frequency magnetic flux) in bone marrow derived neural stem cells on rat brain injury (rats with brain injury) function to improve the crawling effect. To observe related neuronal markers and neurotransmitter expression changes, and provide basic evidence for the clinical treatment of brain injury methods: the rat model of brain injury was established, 64 SD rats were randomly divided into four groups, 16 rats in each group, including control group (C group), BM-NPCs group (group B), the magnetic field and the magnetic field group (group L) +BM-NPCs group (group LB). Take 3 weeks SD rat bone marrow derived mesenchymal stem cells, neural stem cells derived from bone marrow in vitro (Bone Marrow Stroma Cells-derived Neural Progenitor Cells, BM-NPCs), the successful models of 7D, CD-Dil labeled BM-NPCs. in vitro transplantation, L group and LB group were low frequency magnetic fields (50Hz, 5 MT) 60min/d, 60d. continuous intervention, intervention in the experimental process, respectively in 3D, 7d, transplantation of 1D, 30d and D of rats in each group were 60 level stair climbing score, Wayne score, Clark behavior, and to transplant 1D, 7d and 30d were analysed by HE staining of brain tissue was observed with pathological manifestation; in the area of trauma, neural cell marker NeuN, cell immunofluorescence detection of GFAP expression of beta III tubulin. Using 50Hz in vitro, 5mT weak magnetic field treatment for BM-NPCs, time is 60min/ days, for 15 days, the control group at the same time. The detection of Q-PCR by immunofluorescence analysis of the expression of BM-NPCs Nestin (neural stem cell marker), PSA-NCAM, beta III tubulin (neuron marker), ACHE, 5-HT, GABA (neurotransmitter) etc. RESULTS: after transplantation, BM-NPCs 1D, Wayne Clark score and the level of the stair climbing score, no significant difference between the results of each group (P0.05); After transplantation, 3D, 7d, 30d, 60d, Wayne were Clark score, the level of stair climbing scores showed that there were significant differences between the groups (P0.05), C group compared with the other three groups have obvious differences, there are differences in different time, different factors and behavioral trends with different scores time increases, which group LB movement function score improved more significantly.HE staining showed that after modeling 7d, vascular compression tissue surrounding the brain injury rats focal deformation, formation of.BM-NPCs and 7d after transplantation of tissue swelling and necrosis, C group and L group brain injury perihematomal edema was reduced, cystic large voids, inflammatory cell infiltration, nerve cells decreased significantly; B group and LB group of brain injury foci surrounding tissue edema, cystic confined cavity, visible glial cells. After transplantation, 30d, except the control group, the other three groups of cystic cavity surface Products are less, edema in group B disappeared, LB group of brain injury foci surrounding tissue cells row class neatly, inflammatory cells disappeared. After transplantation, 60d, compared with the control group, the experimental group of brain tissue damage repair. Display structure of most specimens for immunofluorescence detection of BM-NPCs after transplantation, 30d, Dil mark NeuN and beta III tubulin positive cells appeared around the B group and LB group rat brain tissue injury after transplantation; 60d, Dil labeled NeuN and beta III tubulin positive cells surrounding tissue and brain injury fusion growth after transplantation of.BM-NPCs 30d and 60d C around the rats brain injury tissue a large number of GFAP positive cells, L group and LB group than in C group. In vitro experiment, cultured BM-NPCs cells and the formation of the ball by the weak magnetic field intervention can be observed in neuron like cells. Immunofluorescence assay showed that BM-NPCs cultured in vitro Ne The expression of stin was positive; the control group and the magnetic field group neuron like cells were positive for Tuj-1 and GFAP. Compared with the control group, Q-PCR results show that the magnetic field group of Nestin, PSA-NCAM, Ach, GABA, 5-HT gene expression level in vitro and there was significant difference before induction (P0.01), weak magnetic field group beta III tubulinmRNA expression significantly higher than the control group (P0.05). Conclusion: low frequency weak magnetic energy to improve the brain damage ipsilateral crawling function in rats, also can promote the repair of bone marrow derived neural stem cell transplantation for brain injury model in rats, which may be associated with the low frequency weak magnetic field by beta III tubulin mRNA expression of bone marrow derived neural stem cells into nerve cell differentiation.

【學(xué)位授予單位】：南方醫(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

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