本文選題：間充質(zhì)干細(xì)胞 + 骨髓源神經(jīng)祖細(xì)胞�。� 參考：《暨南大學(xué)》2017年博士論文

【摘要】：背景和目的干細(xì)胞與再生醫(yī)學(xué)研究是當(dāng)今生命科學(xué)最受關(guān)注的前沿領(lǐng)域,干細(xì)胞移植治療中樞神經(jīng)損傷疾病在國(guó)內(nèi)外實(shí)驗(yàn)研究方面取得了很大進(jìn)展。其中,間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSC)是其中研究最多的一種干細(xì)胞。其原因之一是,MSCs具有強(qiáng)大的增殖能力和多向分化潛能,在適宜的體內(nèi)或體外環(huán)境下,可分化為神經(jīng)元、膠質(zhì)細(xì)胞等外胚層細(xì)胞發(fā)揮神經(jīng)修復(fù)作用。2014年全球有關(guān)MSCs臨床轉(zhuǎn)化研究多達(dá)409項(xiàng)目(www.Clinical Trial.gov),美國(guó)批準(zhǔn)了40余項(xiàng)其在腦損傷方面的臨床試驗(yàn)。2016年中國(guó)神經(jīng)科學(xué)學(xué)會(huì)神經(jīng)損傷與修復(fù)分會(huì)腦損傷神經(jīng)功能損害與修復(fù)專家共識(shí),將間充質(zhì)干細(xì)胞視為細(xì)胞修復(fù)治療腦損傷有前景的治療策略。然而,MSCs研究中也存在一些問題,如干細(xì)胞擴(kuò)增能力有限,缺乏MSCs在顱內(nèi)長(zhǎng)期存活和參與神經(jīng)再生的直接證據(jù)。在MSCs研究領(lǐng)域,用得最多的是骨髓來(lái)源的間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BM-MSCs)。盡管近年來(lái)不少的研究支持BM-MSCs能夠跨越胚層向神經(jīng)細(xì)胞分化,形成骨髓源性神經(jīng)細(xì)胞,但未檢測(cè)到大量的、成熟的、具有功能的神經(jīng)細(xì)胞,在體內(nèi)研究中尤其如此。甚至有研究認(rèn)為,移植的BM-MSCs不能夠分化成功能性神經(jīng)細(xì)胞,起到細(xì)胞替代作用,而主要是通過抑制凋亡,調(diào)節(jié)機(jī)體免疫減少炎癥反應(yīng)等作用來(lái)促進(jìn)神經(jīng)功能恢復(fù)。我們前期研究發(fā)現(xiàn),在神經(jīng)細(xì)胞培養(yǎng)環(huán)境中,骨髓源性神經(jīng)細(xì)胞形成過程中可能存在骨髓源神經(jīng)祖細(xì)胞(bone marrow derived neural progenitor cells,BM-NPCs)階段。BM-NPCs可能較BM-MSCs更適合作為細(xì)胞移植的種子細(xì)胞,發(fā)揮細(xì)胞替代作用治療中樞神經(jīng)損傷疾病。因此,如何更好地誘導(dǎo)BM-MSCs向功能性神經(jīng)細(xì)胞定向分化,獲取骨髓源性神經(jīng)祖細(xì)胞,觀察骨髓源神經(jīng)元在顱內(nèi)長(zhǎng)期存活,并能夠參與神經(jīng)再生的有力證據(jù),是其臨床轉(zhuǎn)化移植治療中樞神經(jīng)損傷疾病亟待解決的問題。本課題聚焦在獲取BM-NPCs,探討B(tài)M-NPCs篩選純化方案,證明功能性骨髓源性神經(jīng)元能夠在顱內(nèi)長(zhǎng)期存活,并參與神經(jīng)元再生,為骨髓源神祖細(xì)胞在中樞神經(jīng)再生中的應(yīng)用提供有價(jià)值的實(shí)驗(yàn)依據(jù)。第一部分獲取骨髓源神祖細(xì)胞目的通過神經(jīng)干細(xì)胞懸浮培養(yǎng)法,獲取骨髓源神祖細(xì)胞(BM-NPCs),觀察分析BM-NPCs細(xì)胞學(xué)特性。方法采用全骨髓貼壁培養(yǎng)法分離培養(yǎng)大鼠BM-MSCs,觀察原代及傳代細(xì)胞的形態(tài)及增殖特點(diǎn),采用流式細(xì)胞術(shù)檢測(cè)表面標(biāo)志物。將第3代BM-MSCs轉(zhuǎn)移到無(wú)血清神經(jīng)干細(xì)胞培養(yǎng)基Neurobal-A medium,加1%N2-supplement、2%B27、2 mmol/L L-谷氨酰胺和20 ng/ml b FGFEGF的懸浮培養(yǎng)瓶誘導(dǎo)培養(yǎng)。48h后可見部分細(xì)胞聚集成團(tuán)懸浮生長(zhǎng),Accutase TM酶消化傳代,其中部分細(xì)胞具有成球懸浮增殖生長(zhǎng)能力,取第三代骨髓來(lái)源細(xì)胞球備用。通過流式細(xì)胞術(shù)檢測(cè)這種骨髓來(lái)源細(xì)胞球的細(xì)胞周期,用細(xì)胞免疫熒光和RT-PCR方法等鑒定其多項(xiàng)向分化潛能與神經(jīng)祖細(xì)胞相關(guān)基因蛋白表達(dá)情況,并進(jìn)行成脂能力檢測(cè)。結(jié)果課題培養(yǎng)的BM-MSCs符合國(guó)際干細(xì)胞對(duì)BM-MSCs的鑒定標(biāo)準(zhǔn),經(jīng)流式細(xì)胞鑒定CD34/45/3/4/11b/14/133(-)和CD29/105(+)。通過神經(jīng)干細(xì)胞懸浮培養(yǎng)法多次傳代擴(kuò)增獲取的骨髓來(lái)源細(xì)胞球,經(jīng)流式細(xì)胞周期檢測(cè)發(fā)現(xiàn),第3代細(xì)胞球79.2%的細(xì)胞停滯在G0/G1期;細(xì)胞免疫熒光鑒定表達(dá)SOX2/CD133/Nestin蛋白;半定量RT-PCR檢測(cè)細(xì)胞球m RNA水平結(jié)果顯示,較強(qiáng)表達(dá)c-myc/klf4/sox2,弱表達(dá)Sca-1,不表達(dá)0ct4的多能干細(xì)胞基因特點(diǎn),具有較強(qiáng)表達(dá)Muashil1/CD184/CD133,表達(dá)CD56/Nestin/Muashil2/Notch1神經(jīng)祖細(xì)胞基因特點(diǎn),同時(shí),具有成脂誘導(dǎo)分化能力,誘導(dǎo)脂滴紅油O染色陽(yáng)性。結(jié)論采用神經(jīng)干細(xì)胞懸浮培養(yǎng)法,從BM-MSCs中獲取的骨髓來(lái)源細(xì)胞球,表達(dá)神經(jīng)祖細(xì)胞基因和蛋白,具有向神經(jīng)細(xì)胞分化傾向等神經(jīng)祖細(xì)胞特點(diǎn),保留多向分化潛能。第二部分骨髓源神經(jīng)祖細(xì)胞向神經(jīng)元樣細(xì)胞誘導(dǎo)分化目的通過直接貼壁分化和與神經(jīng)元共培養(yǎng)誘導(dǎo)方法,觀察分析BM-NPCs向神經(jīng)細(xì)胞誘導(dǎo)分化的能力,檢測(cè)細(xì)胞分化過程中基因水平改變特點(diǎn),為向功能性神經(jīng)元誘導(dǎo)分化提供依據(jù)。方法將BM-MSCs獲得的第3代BM-NPCs置于神經(jīng)元細(xì)胞培養(yǎng)基中貼壁誘導(dǎo)觀察15d。通過細(xì)胞免疫熒光技術(shù)分析誘導(dǎo)細(xì)胞神經(jīng)元標(biāo)志物Tuj-1/NF200和神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物GFAP/S100的表達(dá)情況,用半定量RT-PCR與定量q PCR檢測(cè)m RNA水平,誘導(dǎo)前后神經(jīng)干細(xì)胞標(biāo)志物(Nestin/NCAM1/CD133)基因,神經(jīng)細(xì)胞基因β-III-tubulin/Neu N/5-HT/ACHE/GABA和CNPase,以及神經(jīng)營(yíng)養(yǎng)因子NGF/BDNF/GDNF基因表達(dá)情況。另外,將CM-Dil細(xì)胞示蹤劑標(biāo)記后的第3代BM-NPCs與原代皮層神經(jīng)元細(xì)胞共培養(yǎng)15d,利用倒置顯微鏡和細(xì)胞免疫熒光法觀察檢測(cè)神經(jīng)元標(biāo)志物Tuj-1表達(dá)情況。結(jié)果BM-NPCs直接貼壁誘導(dǎo)的骨髓源神經(jīng)細(xì)胞,10d后可見神經(jīng)元樣形態(tài)細(xì)胞,其中一些細(xì)胞似膠質(zhì)細(xì)胞形態(tài),互相連成網(wǎng)狀生長(zhǎng);繼續(xù)誘導(dǎo)5d,可見較典型的神經(jīng)樣細(xì)胞形態(tài),與正常皮層神經(jīng)元樣細(xì)胞形態(tài)相似,完全不同于BM-MSCs。這些神經(jīng)樣細(xì)胞可表達(dá)部分神經(jīng)細(xì)胞表型Tuj-1(+)/NF200(-)和GFAP(+)/S100(+),以及表達(dá)β-III-tubulin(+)/Neu N/5-HT(+)/ACHE(+)/GABA(-)和CNPase(++)/NGF(+++)/BDNF(+)/GDNF(+)基因特點(diǎn)。定量基因表達(dá)檢測(cè)發(fā)現(xiàn),BM-NPCs較BM-MSCs高表達(dá)NCAM1和CD133神經(jīng)祖細(xì)胞基因,貼壁分化后干細(xì)胞基因Nestin、NCAM1、CD133表達(dá)明顯下降,隨著細(xì)胞分化成不同的神經(jīng)樣細(xì)胞,β-III-tubulin/Neu N/5-HT/ACHE基因表達(dá)下調(diào),提示實(shí)驗(yàn)給予的直接貼壁誘導(dǎo)分化環(huán)境,并不利于向神經(jīng)元分化,無(wú)法形成成熟神經(jīng)元和表達(dá)神經(jīng)遞質(zhì),培養(yǎng)時(shí)間越長(zhǎng)更多的細(xì)胞可能向容易增殖存活的膠質(zhì)細(xì)胞分化,伴隨CNPase表達(dá)升高,NGF營(yíng)養(yǎng)因子基因水平的顯著提高。將CM-Dil細(xì)胞示蹤劑標(biāo)記后的BM-NPCs置于更適合神經(jīng)元生長(zhǎng)的環(huán)境中,與原代皮層神經(jīng)元細(xì)胞共培養(yǎng),可見BM-NPCs可分化成較多典型神經(jīng)元樣形態(tài)特征、Tuj1熒光蛋白呈陽(yáng)性表達(dá),與正常神經(jīng)細(xì)胞相互連接成網(wǎng)絡(luò)狀生長(zhǎng)。結(jié)果提示在更適宜神經(jīng)元生長(zhǎng)環(huán)境,BM-NPCs可能具備向成熟功能性神經(jīng)元分化的能力。結(jié)論實(shí)驗(yàn)中體外誘導(dǎo)的骨髓源性神經(jīng)元樣細(xì)胞與完全成熟神經(jīng)元相比細(xì)胞形態(tài)相似,但基因表達(dá)上仍存在一定差距,懸浮擴(kuò)增培養(yǎng)的BM-NPCs較BM-MSCs更具有向神經(jīng)細(xì)胞分化的能力,BM-NPCs在合適的環(huán)境中可能有能力向功能性神經(jīng)元分化,在中樞神經(jīng)損傷疾病中發(fā)揮作用。第三部分骨髓源神經(jīng)元長(zhǎng)期存活參與腦神經(jīng)再生目的尋找骨髓源神經(jīng)元在腦損傷局部長(zhǎng)期存活,并參與腦損傷神經(jīng)再生的證據(jù),為BM-NPCs移植治療中樞神經(jīng)損傷疾病提供有價(jià)值的實(shí)驗(yàn)依據(jù)。方法建立腦損傷大鼠模型7d后,隨機(jī)將CD-Dil細(xì)胞示蹤的BM-NPCs 10ul(100萬(wàn)個(gè)細(xì)胞)通過微量注射器移植至腦損傷部位大鼠設(shè)為細(xì)胞組,同樣條件下注射培養(yǎng)基的大鼠設(shè)為對(duì)照組,每組20只。分別在移植后的第1d、3d、7d、30d和60 d進(jìn)行運(yùn)動(dòng)功能Wayne clark評(píng)分與grooming評(píng)分。同時(shí),移植后第7d、30d、60d和90d取材,進(jìn)行腦組織病理檢測(cè),利用組織免疫熒光法檢測(cè)CM-Dil標(biāo)記的BM-NPCs在腦損傷區(qū)的存活遷移情況及神經(jīng)細(xì)胞標(biāo)志物Neu N、GFAP表達(dá)情況。結(jié)果(1)HE染色顯示:造模7d,各組大鼠腦損傷灶周圍組織碎裂,血管受壓變形,血流量減少,神經(jīng)細(xì)胞腫脹壞死變性。細(xì)胞移植7d,對(duì)照組損傷灶周圍組織水腫,可見囊性空洞,神經(jīng)細(xì)胞的數(shù)量明顯減少,周圍炎癥細(xì)胞浸潤(rùn);細(xì)胞組水腫較輕,囊性空洞范圍局限,可見膠質(zhì)細(xì)胞。移植30d,腦損傷灶周圍組織有所恢復(fù),與對(duì)照組相比,細(xì)胞組囊性空洞較小,且周圍細(xì)胞排類較整齊,組織水腫和炎癥細(xì)胞消失。(2)組織免疫熒光結(jié)果:移植7d,細(xì)胞組腦損傷組織周圍可見CM-Dil標(biāo)記的細(xì)胞移植到損傷的區(qū)域,但未見Neu N陽(yáng)性的Dil+細(xì)胞。移植30d,腦損傷組織周圍可見大量GFAP呈陽(yáng)性表達(dá)的膠質(zhì)細(xì)胞,其中部分Dil+細(xì)胞;在海馬和腦皮層神經(jīng)元區(qū)域,可見散在Dil+細(xì)胞表達(dá)Neu N,與正常神經(jīng)細(xì)胞整合在一起。移植90d,細(xì)胞組仍然可見CM-Dil標(biāo)記的Neu N陽(yáng)性細(xì)胞整合在腦組織正常神經(jīng)細(xì)胞中,與損傷區(qū)周圍組織融合生長(zhǎng)。(3)動(dòng)物行為學(xué)評(píng)分:移植1d,兩組Wayne clark、grooming評(píng)分結(jié)果比較無(wú)顯著差異(P0.05);移植3d、7d、30d、60d,各組Wayne clark、grooming評(píng)分結(jié)果顯示組間比較有顯著意義(P0.05),細(xì)胞移植組功能恢復(fù)較好.結(jié)論移植BM-NPCs具有促進(jìn)腦損傷大鼠患側(cè)肢體運(yùn)動(dòng)功能恢復(fù)的作用,骨髓源性神經(jīng)細(xì)胞可在顱內(nèi)長(zhǎng)期存活的,骨髓源神經(jīng)元整合到了損傷腦組織參與神經(jīng)再生。
[Abstract]:Background and target stem cells and regenerative medicine are the most important frontiers of life science. The stem cell transplantation for the treatment of central nervous injury has made great progress at home and abroad. Among them, mesenchymal stem cells (MSC) is one of the most studied stem cells. One of the reasons for it is one of the reasons. It is, MSCs has strong proliferation and pluripotent differentiation potential, and can differentiate into neurons in a suitable body or in vitro environment, and glial cells such as ectoderm play a neurologic repair role in the global MSCs clinical transformation study of up to 409 (www.Clinical Trial.gov) for.2014 years. The United States approved more than 40 items in brain damage. Clinical trials,.2016, of the Chinese society of neuroscience and neuroscience in the neurologic injury and repair branch of the Chinese Society for neurologic impairment and repair of brain damage and repair experts consensus, regard mesenchymal stem cells as a promising treatment strategy for the treatment of brain damage by cell repair. However, there are also some problems in the MSCs study, such as the limited capacity of stem cells and the lack of MSCs in the long term The direct evidence of survival and participation in nerve regeneration. In the field of MSCs research, the most used is the bone marrow mesenchymal stem cells (BM-MSCs). Although many studies have supported BM-MSCs in recent years, it has been supported that BM-MSCs can cross the embryo to neural cells and form bone marrow derived neural cells, but not a large number of them have been detected. Mature, functional neurocells, especially in the body, have been studied in the body, and even studies suggest that transplanted BM-MSCs can not differentiate successful energetic neurons, play a role in cell substitution, but mainly by inhibiting apoptosis and regulating immune response to reducing inflammatory response. In the neural cell culture environment, there may be a bone marrow derived neural progenitor cell (bone marrow derived neural progenitor cells, BM-NPCs) during the formation of bone marrow derived neural cells..BM-NPCs may be more suitable as a seed cell for cell transplantation than BM-MSCs. How to induce the directional differentiation of BM-MSCs to functional nerve cells, to obtain bone marrow derived neural progenitor cells, to observe the long-term survival of the bone marrow cells and to participate in the regeneration of the nerve, is an urgent problem to be solved in the clinical transformation and transplantation for the treatment of central nervous injury. This topic focuses on obtaining BM-NPCs. The BM-NPCs screening and purification scheme proves that the functional myeloid neurons can survive in the long term and participate in the regeneration of neurons, and provide valuable experimental basis for the application of bone marrow source God progenitor cells in the regeneration of central nerve. The characteristics of BM-NPCs cytology were observed and analyzed. Methods the whole bone marrow adherent culture method was used to isolate and culture the rat BM-MSCs. The morphology and proliferation characteristics of the original and subcultured cells were observed and the surface markers were detected by flow cytometry. The third generation BM-MSCs was transferred to Neurobal-A medium in the serum-free neural stem cell culture medium. The suspension cultures of 1%N2-supplement, 2%B27,2 mmol/L L- glutamine and 20 ng/ml B FGFEGF were induced and cultured in suspension culture bottle, and some cells were clustered and suspended to grow, and Accutase TM enzyme was digested. Some of the cells had the ability to proliferate and grow in spheroid, and the third generation of bone marrow cells were taken for reserve. The cell cycle of this bone marrow cell was identified by cell immunofluorescence and RT-PCR method, and the expression of multiple differentiation potential and neural progenitor cells related gene protein was identified, and the lipid ability detection was carried out. The result of BM-MSCs was in conformity with the identification standard of international stem cells to BM-MSCs, and CD34/45/3/4/11 was identified by flow cytometry. B/14/133 (-) and CD29/105 (+). The cell ball of bone marrow derived from multiple passages by neural stem cell suspension culture method was detected by flow cytometry. The 79.2% cells of the third generation cell ball stagnated in the G0/G1 stage; the cell immunofluorescence was identified to express the SOX2/CD133/Nestin protein; the semi quantitative RT-PCR was used to detect the level of the cell ball m RNA. It showed strong expression of c-myc/klf4/sox2, weak expression of Sca-1, non expression of 0ct4 gene in pluripotent stem cells, strong expression of Muashil1/CD184/CD133, expression of gene characteristics of CD56/Nestin/Muashil2/Notch1 neural progenitor cells. At the same time, it has the ability to induce adipogenic differentiation and induce the positive staining of lipid droplet O. Conclusion the suspension culture of neural stem cells is used. The bone marrow derived cell balls obtained from BM-MSCs, expressing the gene and protein of the neural progenitor cells, have the characteristics of neural progenitor cells, such as the tendency to differentiate into neural cells, and retain the multidirectional differentiation potential. The second part of the bone marrow derived neural progenitor cells induce differentiation to neuron like cells by direct adherence to wall differentiation and co culture with neurons. Method to observe and analyze the ability of BM-NPCs to induce differentiation to neural cells, detect the change of gene level in the process of cell differentiation, and provide the basis for inducing differentiation to functional neurons. Methods the third generation of BM-NPCs obtained by BM-MSCs was placed in the cell culture medium of the neuron to observe the adhesion of 15d. through cell immunofluorescence technique The expression of cell neuron marker Tuj-1/NF200 and glial cell marker GFAP/S100 was induced. The level of M RNA was detected by semi quantitative RT-PCR and quantitative Q PCR, the neural stem cell marker (Nestin/NCAM1/CD133) gene, the gene beta -III-tubulin/Neu N/5-HT/ACHE/GABA and CNPase, and the neurotrophic factor NGF were induced. In addition, the third generation BM-NPCs labeled by CM-Dil cell tracer and the primary cortical neuron cells were co cultured with 15d, and the expression of Tuj-1 was detected by inverted microscope and cell immunofluorescence. The results showed that BM-NPCs directly adhered to the bone marrow derived nerve cells from the wall, and the neurons were visible after 10d. Like cells, some cells resemble glial cells and grow in reticular formation, and continue to induce 5D, which can be seen as typical neuron like cells, similar to normal cortical neuron like cells, completely different from that of BM-MSCs., which can express partial neurocyte phenotypes Tuj-1 (+) /NF200 (+) and GFAP (+) /S100 (+)). -III-tubulin (+) /Neu N/5-HT (+) /ACHE (+) /GABA (+) and CNPase (+ +) /NGF (+ +) /NGF (+ +) /BDNF (+) /GDNF (+) gene. Quantitative gene expression detection found that BM-NPCs is higher than BM-MSCs NCAM1 and neural progenitor cells gene, after adherent differentiation, the expression of stem cells significantly decreased, with the differentiation of cells into different gods. The expression of beta -III-tubulin/Neu N/5-HT/ACHE gene is downregulated in the sample cells, suggesting that the direct adherence induced differentiation environment is not conducive to the differentiation of neurons, the formation of mature neurons and the expression of neurotransmitters. The longer the culture time, the more cells may differentiate to the glial cells that are easy to proliferate and increase with the expression of CNPase. A significant increase in the gene level of the NGF nutrient factor. The BM-NPCs after the CM-Dil cell tracer was placed in a more suitable environment for neuronal growth and co cultured with the primary cortical neuron cells. It can be seen that BM-NPCs can differentiate into more typical neuron like morphological features. The Tuj1 fluorescent protein is positive and connects with the normal nerve cells. The results suggest that BM-NPCs may have the ability to differentiate into mature functional neurons in a more suitable neuron growth environment. Conclusion the bone marrow derived neuron like cells induced in the experiment are similar to that of fully mature neurons in vitro, but there is still a certain gap between the gene table and the suspension amplification culture. BM-NPCs has the ability to differentiate into neural cells more than BM-MSCs. BM-NPCs may have the ability to differentiate into functional neurons in a suitable environment and play a role in central nervous injury. The long-term survival of the third part of bone marrow neurons in the brain regeneration to find bone marrow derived neurons for long-term survival in brain injury. And participate in the evidence of nerve regeneration of brain injury to provide valuable experimental basis for BM-NPCs transplantation in the treatment of central nervous injury. After the establishment of brain injury rat model 7d, the CD-Dil cells traced BM-NPCs 10ul (1 million cells) were transplanted into the brain injury rats by the micro syringe and the same condition. The rats in the injection medium were set as the control group, with 20 rats in each group. The Wayne Clark score and grooming score were performed at 1D, 3D, 7d, 30d and 60 d after transplantation. Meanwhile, the pathological examination of the brain tissue was carried out in 7d, 30d, 60d and 90d, and the survival of the brain was detected by tissue immunofluorescence. The migration and the expression of Neu N and GFAP. Results (1) HE staining showed that the model was 7d. The tissues around the brain injury were broken, the blood vessels were deformed, the blood flow was reduced, the blood flow was reduced, the cells were swollen and necrotic. The cell transplantation was 7d, and the peripheral tissue was edema in the control group, the number of cystic cavity and the number of nerve cells was obvious. Reduction, infiltration of inflammatory cells around the cell group, the edema of the cell group was light, the scope of cystic cavity was limited, glial cells were visible. The transplantation of 30d, the tissue around the brain injury was recovered. Compared with the control group, the cysts in the cell group were smaller, and the surrounding cells were neatly arranged, tissue edema and inflammatory cells disappeared. (2) immunofluorescence results of tissue transplantation: transplantation of 7D, fine transplantation CM-Dil labeled cells were transplanted to the injured area around the brain injury tissues, but no Neu N positive Dil+ cells were found. A large number of GFAP positive glial cells were found around the tissue of brain injury, including some Dil+ cells. In the hippocampal and cerebral cortex neurons, Neu N was expressed in the Dil+ cells, with the normal God. Cells integrated together. Transplantation of 90d, the cell group still showed that the CM-Dil labeled Neu N positive cells integrated in the normal brain cells and fused with the tissue around the injured area. (3) the animal behavior score: transplantation of 1D, two groups of Wayne Clark, grooming score compared with no significant difference (P0.05); 3D, 7d, 30d, and 3D The results of lark, grooming score showed that there were significant differences between groups (P0.05), and the function of cell transplantation group was better. Conclusion transplantation of BM-NPCs has the effect of promoting the recovery of motor function of the injured side of the brain in rats. Bone marrow derived nerve cells can survive in the brain for a long time, and the bone marrow neurons are integrated into the injured brain tissue to participate in the nerve regeneration. Birth.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R329.2

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