BDNF基因转染骨髓间充质干细胞体外诱导分化神经元细胞研究

发布时间：2018-05-01 10:34

本文选题：骨髓间充质干细胞 + 脑源性神经营养因子　；参考：《中南大学》2010年博士论文

【摘要】： 骨髓间充质干细胞(mesenchymal stem cell, MSCs)是存在于骨髓中由中胚层发育的一类非造血干细胞,具有多向分化潜能,不仅能分化为造血细胞,还能分化成多种造血以外的组织细胞,特别是中胚层和神经外胚层来源的组织细胞。MSCs在多个领域,如细胞治疗、组织工程、发育生物学、基因功能研究、药物筛选等领域展示了巨大的应用前景。目的：了解大鼠骨髓间质干细胞体外培养特性,探讨MSCs的体外分离、培养、扩增的规律和特点。建立SD大鼠骨髓间充质干细胞体外培养体系。方法：1.原代培养,取一月龄SD大鼠股骨和胫骨,采用全骨髓培养法,以L-DMEM完全培养基冲出骨髓过滤后直接接种于培养瓶中,3天后首次换液,以后每3天换液一次。至细胞融合达80%以上时,进行传代。2.大鼠骨髓间质干细胞体外扩增的研究,取第1、4、7、10、13代细胞,观察其细胞生长曲线及贴壁率。3.大鼠骨髓间质干细胞表面标志的鉴定,取第4代细胞,采用流式细胞仪检测。结果：1.贴壁筛选法,通过多次换液可以分离、纯化MSCs,该法简单、方便、效果好。2.MSCs在体外培养中有很强的增殖能力,10代以前的细胞形态较一致,增殖能力强,随传代次数的增加,增殖能力减弱,形态发生不规则变化。细胞生长的第1、4、7、10、13代的贴壁率变化规律基本相同,无显著区别。传代后细胞贴壁率随时间延长而增高。3.MSCs的表面标志抗原呈现多样性的特征,MSCs不表达造血细胞表面抗原CD34、CD45,说明MSCs非造血类细胞；而其表达CD90、CD44,说明MSCs的表面标志具有非单一性的特性。它表达了间质细胞、内皮细胞和表皮细胞的表面标志。结论：本实验采用骨髓直接培养,贴壁筛选法来分离培养MSCs,方法简单,效果亦可靠,通过数代的传代扩增就能获得高纯度、高数量的MSCs,以满足组织工程实验研究的需要。目的：1.构建pEGFP-N1-BDNF质粒。2.获得到稳定表达BDNF的MSCs,并转染大鼠骨髓间质干细胞(MSCs),观察其在MSCs内的表达。方法：用RT-PCR法从大鼠骨髓间充质干细胞中扩增出带有XhoⅠ、BamH工酶切位点的BDNF基因片段,将BDNF基因片段克隆到增强型绿色荧光蛋白(EGFP)基因真核表达载体pEGFP-N1上,构建pEGFP-N1-BDNF质粒。通过电转染将pEGFP-N1-BDNF转染入大鼠MSCs。结果：pEGFP-N1-BDNF真核表达载体构建成功,荧光显微镜下观察,pEGFP-N1-BDNF表达载体转染到MSCsl2h后可见BDNF融合蛋白表达。结论：1.成功的构建pEGFP-N1-BDNF质粒。2.pEGFP-N1-BDNF质粒可以转染真核细胞HEK293细胞,证明BDNF-EGFP融合蛋白真核表达质粒可以在真核细胞表达。3.通过电转染和抗性筛选我们获得到了稳定表达BDNF的MSCs。目的：1.采用p-硫基乙醇(p-ME)和碱性成纤维细胞生长因子(bFGF)作为诱导剂,对骨髓间充质干细胞(MSCs)进行体外诱导实验。探讨β-ME和bFGF体外诱导骨髓间充质干细胞分化神经元细胞的机制。2.探讨BDNF基因修饰对MSCs分化神经元细胞的影响。方法：1.MSCs向神经元细胞诱导分化,取P4代的MSCs和BDNF-MSCs,分别以2×104/ml浓度接种于24孔板内,待细胞达到70%-80%融合时,分别加入p-硫基乙醇(p-ME)和碱性成纤维细胞生长因子(bFGF),倒置相差显微镜下连续观察细胞形态变化。2.细胞免疫荧光鉴定,采用细胞免疫荧光方法,对以上两组细胞诱导后5h后检测神经元标志物TuJ1的表达情况,荧光显微镜下观察并拍照记录。3.MSCs诱导为神经元细胞阳性率比较,荧光显微镜下在两组细胞随机取300个细胞,计数TuJ1反应阳性细胞个数,检验各组不同时间点的差异,以及每个时间点各组间的差异。结果：1.第4代MSCs加入诱导剂后可分化为神经元细胞和星形胶质细胞。可见光下观察：前者细胞体积较小数量较少,常有数个短小突起和一个较长的突起,呈典型的双极、多极或锥形,具有较强的折光性；后者细胞体积较大,细胞突起较多且粗,有些形态呈星状,有许多突起呈放射状伸出,平铺在培养皿上。2.细胞免疫荧光结果示细胞诱导后5h,部分细胞TuJ1反应阳性,为神经元细胞。3.两组MSCs诱导为神经元细胞阳性率比较,BDNF-MSCs组分化为神经元细胞的阳性率高于MSCs组,并具有统计学意义。结论：1.在体外MSCs在诱导剂bFGF和β-ME的作用下可分化为神经元样细胞。2.经过BDNF基因修饰,可以显著提高MSCs向神经元细胞分化的阳性率。
[Abstract]:Mesenchymal stem cell (MSCs) is a kind of non hematopoietic stem cells developed from mesoderm in bone marrow. It has multiple differentiation potential. It can not only differentiate into hematopoietic cells, but also differentiate into a variety of hematopoietic tissue cells, especially the tissue cells derived from the mesoembryonic and neuroectoderm cells,.MSCs in a number of collar. Domains, such as cell therapy, tissue engineering, developmental biology, gene function research, drug screening and other fields, have shown great application prospects.
Objective: To investigate the culture characteristics of rat bone marrow mesenchymal stem cells in vitro, and to explore the laws and characteristics of the isolation, culture and amplification of MSCs in vitro. The culture system of bone marrow mesenchymal stem cells (MSCs) in SD rats was established.
Methods: 1. primary culture, the femur and tibia of 1 month old SD rats were taken, and all bone marrow culture was used in full bone marrow culture. The bone marrow filtration was directly inoculated in the culture bottle with the L-DMEM complete medium. After 3 days, the first liquid was changed and then changed every 3 days. When the cell fusion was over 80%, the bone marrow mesenchymal stem cells of.2. rats were amplified in vitro. The cells of the 1,4,7,10,13 generation were taken to observe the cell growth curve and the identification of the surface markers of the bone marrow mesenchymal stem cells in.3. rats. The fourth generation cells were taken and the flow cytometry was used to detect the cells.
Results: the 1. adherent wall screening method can be separated and purified by multiple fluid exchange. The method is simple and convenient, and the effect is good..2.MSCs has strong proliferation ability in the culture in vitro. The cell morphology is more consistent and the proliferating ability is strong before the 10 generation, with the increase of the number of passages, the proliferation ability is weakened, and the morphogenesis is irregular. 1,4,7,10, the cell growth of the cell, is 1,4,7,10, The adherence rate of the 13 generation was basically the same, and there was no significant difference. After the passage, the cell adhesion rate increased with time and increased the surface marker antigen of.3.MSCs. MSCs did not express the hematopoietic cell surface antigen CD34, CD45, indicating that MSCs was not hematopoietic cells; and its surface was CD90, CD44, indicating that the surface markers of MSCs were not single. It expresses the surface markers of interstitial cells, endothelial cells and epidermal cells. Conclusion: this experiment uses bone marrow direct culture and wall screening to isolate and culture MSCs. The method is simple, and the effect is reliable. High purity and high quantity of MSCs can be obtained through generation of passages to meet the needs of tissue engineering experimental research.
Objective: 1. to construct pEGFP-N1-BDNF plasmid.2. and obtain stable MSCs expressing BDNF, and transfect rat bone marrow mesenchymal stem cells (MSCs) to observe its expression in MSCs.
Methods: the BDNF gene fragment with Xho I and BamH was amplified from rat bone marrow mesenchymal stem cells by RT-PCR method, and the BDNF gene fragment was cloned into the eukaryotic expression vector pEGFP-N1 of the enhanced green fluorescent protein (EGFP) gene, and the pEGFP-N1-BDNF plasmid was constructed. PEGFP-N1-BDNF was transfected into MSCs. by transfection of pEGFP-N1-BDNF into rat MSCs..
Results: pEGFP-N1-BDNF eukaryotic expression vector was constructed successfully, under fluorescence microscope, the expression of BDNF fusion protein was found after transfection of pEGFP-N1-BDNF expression vector to MSCsl2h.
Conclusion: 1. the successful construction of pEGFP-N1-BDNF plasmid.2.pEGFP-N1-BDNF plasmid can transfect the eukaryotic HEK293 cells. It is proved that the eukaryotic expression plasmid of BDNF-EGFP fusion protein can be expressed in eukaryotic cells by electric transfection and resistance screening, and we have obtained the MSCs. of the stable expression of BDNF.
Objective: 1. using p- sulfur based ethanol (p-ME) and basic fibroblast growth factor (bFGF) as an inducer, bone marrow mesenchymal stem cells (MSCs) were induced in vitro. The mechanism of beta -ME and bFGF in inducing the differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro.2. to explore the effect of BDNF gene modification on MSCs differentiated neuron cells.
Methods: 1.MSCs induced differentiation to neuron cells, took P4 generation MSCs and BDNF-MSCs, and inoculated into 24 orifice plates at 2 x 104/ml concentration respectively. When the cells reached 70%-80% fusion, p- sulfur based ethanol (p-ME) and basic fibroblast growth factor (bFGF) were added to the cell, and the cell morphological changes of.2. cell immunofluorescence were continuously observed under the inverted phase difference microscope. A cell immunofluorescence method was used to detect the expression of the neuron marker TuJ1 after 5h induction in the above two groups. The positive rate of the neuron cells was observed under the fluorescence microscope and recorded by.3.MSCs. Under the fluorescence microscope, 300 cells were taken randomly in two groups, and the number of TuJ1 positive cells was counted, and the number of the positive cells was counted. The differences between groups at different time points were examined, and the differences among groups at each time point were also examined.
Results: 1. the fourth generation of MSCs can differentiate into neuronal cells and astrocytes after adding the inducer. It is observed under the light of light that the former cells are smaller in volume, often have several short protuberances and a long protuberance, and are typical bipolar, multipolar or cone-shaped, with a strong refraction; the latter is larger in volume and more protruding than in the former. The.2. cell immunofluorescence results showed that 5h was induced by cell immunofluorescence on the Petri dish, and the TuJ1 reaction in some cells was positive. The positive rate of MSCs in the.3. two group of neuron cells was compared with the positive rate of neuron cells, and the positive rate of the BDNF-MSCs group to differentiate into neuron cells was higher than that of the MSCs group. And it has statistical significance.
Conclusion: 1. in vitro, MSCs can differentiate into neuron like cells.2. after BDNF gene modification under the action of inducer bFGF and beta -ME, which can significantly increase the positive rate of MSCs to neuronal cell differentiation.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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