骨髓源神经祖细胞向神经元分化促进鼠脑损伤神经再生

发布时间：2018-04-25 16:56

本文选题：间充质干细胞 + 骨髓源神经祖细胞　；参考：《暨南大学》2017年博士论文

【摘要】：背景和目的干细胞与再生医学研究是当今生命科学最受关注的前沿领域,干细胞移植治疗中枢神经损伤疾病在国内外实验研究方面取得了很大进展。其中,间充质干细胞(mesenchymal stem cells,MSC)是其中研究最多的一种干细胞。其原因之一是,MSCs具有强大的增殖能力和多向分化潜能,在适宜的体内或体外环境下,可分化为神经元、胶质细胞等外胚层细胞发挥神经修复作用。2014年全球有关MSCs临床转化研究多达409项目(www.Clinical Trial.gov),美国批准了40余项其在脑损伤方面的临床试验。2016年中国神经科学学会神经损伤与修复分会脑损伤神经功能损害与修复专家共识,将间充质干细胞视为细胞修复治疗脑损伤有前景的治疗策略。然而,MSCs研究中也存在一些问题,如干细胞扩增能力有限,缺乏MSCs在颅内长期存活和参与神经再生的直接证据。在MSCs研究领域,用得最多的是骨髓来源的间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)。尽管近年来不少的研究支持BM-MSCs能够跨越胚层向神经细胞分化,形成骨髓源性神经细胞,但未检测到大量的、成熟的、具有功能的神经细胞,在体内研究中尤其如此。甚至有研究认为,移植的BM-MSCs不能够分化成功能性神经细胞,起到细胞替代作用,而主要是通过抑制凋亡,调节机体免疫减少炎症反应等作用来促进神经功能恢复。我们前期研究发现,在神经细胞培养环境中,骨髓源性神经细胞形成过程中可能存在骨髓源神经祖细胞(bone marrow derived neural progenitor cells,BM-NPCs)阶段。BM-NPCs可能较BM-MSCs更适合作为细胞移植的种子细胞,发挥细胞替代作用治疗中枢神经损伤疾病。因此,如何更好地诱导BM-MSCs向功能性神经细胞定向分化,获取骨髓源性神经祖细胞,观察骨髓源神经元在颅内长期存活,并能够参与神经再生的有力证据,是其临床转化移植治疗中枢神经损伤疾病亟待解决的问题。本课题聚焦在获取BM-NPCs,探讨BM-NPCs筛选纯化方案,证明功能性骨髓源性神经元能够在颅内长期存活,并参与神经元再生,为骨髓源神祖细胞在中枢神经再生中的应用提供有价值的实验依据。第一部分获取骨髓源神祖细胞目的通过神经干细胞悬浮培养法,获取骨髓源神祖细胞(BM-NPCs),观察分析BM-NPCs细胞学特性。方法采用全骨髓贴壁培养法分离培养大鼠BM-MSCs,观察原代及传代细胞的形态及增殖特点,采用流式细胞术检测表面标志物。将第3代BM-MSCs转移到无血清神经干细胞培养基Neurobal-A medium,加1%N2-supplement、2%B27、2 mmol/L L-谷氨酰胺和20 ng/ml b FGFEGF的悬浮培养瓶诱导培养。48h后可见部分细胞聚集成团悬浮生长,Accutase TM酶消化传代,其中部分细胞具有成球悬浮增殖生长能力,取第三代骨髓来源细胞球备用。通过流式细胞术检测这种骨髓来源细胞球的细胞周期,用细胞免疫荧光和RT-PCR方法等鉴定其多项向分化潜能与神经祖细胞相关基因蛋白表达情况,并进行成脂能力检测。结果课题培养的BM-MSCs符合国际干细胞对BM-MSCs的鉴定标准,经流式细胞鉴定CD34/45/3/4/11b/14/133(-)和CD29/105(+)。通过神经干细胞悬浮培养法多次传代扩增获取的骨髓来源细胞球,经流式细胞周期检测发现,第3代细胞球79.2%的细胞停滞在G0/G1期;细胞免疫荧光鉴定表达SOX2/CD133/Nestin蛋白;半定量RT-PCR检测细胞球m RNA水平结果显示,较强表达c-myc/klf4/sox2,弱表达Sca-1,不表达0ct4的多能干细胞基因特点,具有较强表达Muashil1/CD184/CD133,表达CD56/Nestin/Muashil2/Notch1神经祖细胞基因特点,同时,具有成脂诱导分化能力,诱导脂滴红油O染色阳性。结论采用神经干细胞悬浮培养法,从BM-MSCs中获取的骨髓来源细胞球,表达神经祖细胞基因和蛋白,具有向神经细胞分化倾向等神经祖细胞特点,保留多向分化潜能。第二部分骨髓源神经祖细胞向神经元样细胞诱导分化目的通过直接贴壁分化和与神经元共培养诱导方法,观察分析BM-NPCs向神经细胞诱导分化的能力,检测细胞分化过程中基因水平改变特点,为向功能性神经元诱导分化提供依据。方法将BM-MSCs获得的第3代BM-NPCs置于神经元细胞培养基中贴壁诱导观察15d。通过细胞免疫荧光技术分析诱导细胞神经元标志物Tuj-1/NF200和神经胶质细胞标志物GFAP/S100的表达情况,用半定量RT-PCR与定量q PCR检测m RNA水平,诱导前后神经干细胞标志物(Nestin/NCAM1/CD133)基因,神经细胞基因β-III-tubulin/Neu N/5-HT/ACHE/GABA和CNPase,以及神经营养因子NGF/BDNF/GDNF基因表达情况。另外,将CM-Dil细胞示踪剂标记后的第3代BM-NPCs与原代皮层神经元细胞共培养15d,利用倒置显微镜和细胞免疫荧光法观察检测神经元标志物Tuj-1表达情况。结果BM-NPCs直接贴壁诱导的骨髓源神经细胞,10d后可见神经元样形态细胞,其中一些细胞似胶质细胞形态,互相连成网状生长;继续诱导5d,可见较典型的神经样细胞形态,与正常皮层神经元样细胞形态相似,完全不同于BM-MSCs。这些神经样细胞可表达部分神经细胞表型Tuj-1(+)/NF200(-)和GFAP(+)/S100(+),以及表达β-III-tubulin(+)/Neu N/5-HT(+)/ACHE(+)/GABA(-)和CNPase(++)/NGF(+++)/BDNF(+)/GDNF(+)基因特点。定量基因表达检测发现,BM-NPCs较BM-MSCs高表达NCAM1和CD133神经祖细胞基因,贴壁分化后干细胞基因Nestin、NCAM1、CD133表达明显下降,随着细胞分化成不同的神经样细胞,β-III-tubulin/Neu N/5-HT/ACHE基因表达下调,提示实验给予的直接贴壁诱导分化环境,并不利于向神经元分化,无法形成成熟神经元和表达神经递质,培养时间越长更多的细胞可能向容易增殖存活的胶质细胞分化,伴随CNPase表达升高,NGF营养因子基因水平的显著提高。将CM-Dil细胞示踪剂标记后的BM-NPCs置于更适合神经元生长的环境中,与原代皮层神经元细胞共培养,可见BM-NPCs可分化成较多典型神经元样形态特征、Tuj1荧光蛋白呈阳性表达,与正常神经细胞相互连接成网络状生长。结果提示在更适宜神经元生长环境,BM-NPCs可能具备向成熟功能性神经元分化的能力。结论实验中体外诱导的骨髓源性神经元样细胞与完全成熟神经元相比细胞形态相似,但基因表达上仍存在一定差距,悬浮扩增培养的BM-NPCs较BM-MSCs更具有向神经细胞分化的能力,BM-NPCs在合适的环境中可能有能力向功能性神经元分化,在中枢神经损伤疾病中发挥作用。第三部分骨髓源神经元长期存活参与脑神经再生目的寻找骨髓源神经元在脑损伤局部长期存活,并参与脑损伤神经再生的证据,为BM-NPCs移植治疗中枢神经损伤疾病提供有价值的实验依据。方法建立脑损伤大鼠模型7d后,随机将CD-Dil细胞示踪的BM-NPCs 10ul(100万个细胞)通过微量注射器移植至脑损伤部位大鼠设为细胞组,同样条件下注射培养基的大鼠设为对照组,每组20只。分别在移植后的第1d、3d、7d、30d和60 d进行运动功能Wayne clark评分与grooming评分。同时,移植后第7d、30d、60d和90d取材,进行脑组织病理检测,利用组织免疫荧光法检测CM-Dil标记的BM-NPCs在脑损伤区的存活迁移情况及神经细胞标志物Neu N、GFAP表达情况。结果(1)HE染色显示:造模7d,各组大鼠脑损伤灶周围组织碎裂,血管受压变形,血流量减少,神经细胞肿胀坏死变性。细胞移植7d,对照组损伤灶周围组织水肿,可见囊性空洞,神经细胞的数量明显减少,周围炎症细胞浸润;细胞组水肿较轻,囊性空洞范围局限,可见胶质细胞。移植30d,脑损伤灶周围组织有所恢复,与对照组相比,细胞组囊性空洞较小,且周围细胞排类较整齐,组织水肿和炎症细胞消失。(2)组织免疫荧光结果:移植7d,细胞组脑损伤组织周围可见CM-Dil标记的细胞移植到损伤的区域,但未见Neu N阳性的Dil+细胞。移植30d,脑损伤组织周围可见大量GFAP呈阳性表达的胶质细胞,其中部分Dil+细胞;在海马和脑皮层神经元区域,可见散在Dil+细胞表达Neu N,与正常神经细胞整合在一起。移植90d,细胞组仍然可见CM-Dil标记的Neu N阳性细胞整合在脑组织正常神经细胞中,与损伤区周围组织融合生长。(3)动物行为学评分:移植1d,两组Wayne clark、grooming评分结果比较无显著差异(P0.05);移植3d、7d、30d、60d,各组Wayne clark、grooming评分结果显示组间比较有显著意义(P0.05),细胞移植组功能恢复较好.结论移植BM-NPCs具有促进脑损伤大鼠患侧肢体运动功能恢复的作用,骨髓源性神经细胞可在颅内长期存活的,骨髓源神经元整合到了损伤脑组织参与神经再生。
[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.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R329.2

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