【作者】 李长青；

【导师】 戚晓昆；

【作者基本信息】 第二军医大学 ， 神经病学， 2011， 硕士

【摘要】 研究背景和目的:间充质干细胞(Mesenchymal Stem cells, MSCs)具有干细胞的基本特征,即长期的自我更新、向多种组织或细胞分化的能力,是干细胞研究的热点之一。MSCs存在于体内多种组织,包括骨髓、脂肪组织和外周血。MSCs相对于传统的神经干、胚胎干细胞及造血干细胞等有很多优点,如易于获得、更易培养和扩增、自体移植而少有排斥、体外培养较少产生基因变异。目前,骨髓是MSCs的重要来源。但随着研究的不断深入,发现其来源相对贫乏。本实验研究的是一种新的MSCs,即人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, HUMSCs)。通过从脐带中成功提取出HUMSCs,并研究其生物特性,为后期HUMSCs的定向分化奠定基础。研究方法:无菌条件下收集剖宫产新生儿脐带,无菌盐水将脐带清洗后,用组织剪将脐带组织剪碎成2~4cm长度大小,清洗脐带血,人工剥离脐带片段上的动静脉血管,剪碎脐带,加入胶原酶Ⅱ消化,细胞筛过滤,收集滤液,离心,用DMEM/F12完全培养液重悬分离所得细胞,接种细胞培养瓶中。待细胞达到融合状态时,进行消化传代和扩增培养。在倒置显微镜下观察形态学的变化,通过生长曲线的描绘观察其增殖能力,流式细胞仪对其免疫表型进行检测。结果:组织消化第2天,可见少量形态各异的细胞贴壁,细胞形态较小,未见明显的细胞核,大部分未消化的组织块悬浮于培养液表面。随着时间延长,贴壁的细胞越来越多。4天后,可见部分贴壁细胞呈扁平状或长梭形,并有两个突起,约8天后,贴壁细胞明显增多,可见典型的长梭形成纤维样细胞,并呈漩涡状生长,至14天左右时达到80%融合。经过传代培养,可得到逐渐纯化的HUMSCs,同时,细胞形态无明显变化,性质稳定,至少能传代20代以上。对原代及第3、7、14代HUMSCs的增殖能力检测,通过生长曲线描绘,发现第3、7、14代HUMSCs生长曲线有共同的特性,14代以内的细胞生长曲线无明显差别,增殖能力大致相似。同时分别对第3、7、10代HUMSCs进行流式细胞学检测。结果显示这三代细胞免疫表型均无明显差异,CD29、CD73、CD105均呈阳性表达,阳性率分别为99.2%、99.5%、98.9%,而CD34、CD45及HLA-DR均表达率分别为0.1%、0.5%、0.2%。结论:脐带是分娩后的弃物,来源丰富,取材方便,不存在伦理道德问题。通过酶消化法可以从脐带中获得HUMSCs,该细胞是一种成纤维样细胞,具有较强的增殖能力,免疫表型检测显示其表达整合素受体、基质细胞及间充质干细胞标志,而造血干细胞标志及MHC-Ⅱ类抗原均呈极低表达。说明HUMSCs属于间充质干细胞,而且免疫原性低。因此,HUMSCs作为一种新型间充质干细胞,具有干细胞的特性,是组织工程研究中理想的种子细胞来源。研究背景和目的:MSCs具有多向分化的能力,已经证实其可以分化为成骨细胞、软骨细胞、脂肪细胞、心肌细胞及内皮细胞等。同样,HUMSCs具有一定的可塑性,在不同条件下HUMSCs亦能被诱导分化成神经细胞,分化后的细胞能表达特殊的神经干细胞、神经元或胶质细胞相关蛋白。但在不同的诱导条件下,诱导效率不同,虽然均能向神经细胞方向分化,但神经元表达率相对较高,而神经胶质细胞表达率较低。因此,本研究通过在体外特殊诱导环境下,使HUMSCs向神经上皮样细胞分化,特别是向少突胶质样细胞分化,为后期神经系统疾病的修复与治疗提供理想的细胞源,比如中枢神经系统脱髓鞘疾病的治疗。研究方法:HUMSCs在体外DMEM/F12、N2添加剂、胰岛素、bFGF及EGF等诱导液作用下24小时,换分化液包括DMEM/F12、N2添加剂及IGF-I,观察分化后细胞形态变化,并通过免疫荧光方法检测分化后的神经上皮样细胞特异性标记如神经干细胞标志物:巢蛋白Nestin,神经元特异性标志物:微管蛋白β-TubulinⅢ,星形胶质细胞标志物:神经胶质原纤维酸性蛋白GFAP,少突胶质细胞早期祖细胞标记物:A2B5,少突胶质细胞晚期祖细胞标记物:O4,成熟少突胶质细胞特异性标志物:碱性髓鞘蛋白MBP及少突胶质细胞转录因子Olig2。结果:在IGF-1等多种细胞生长因子联合作用下HUMSCs能向神经上皮样细胞分化,分化后的细胞形态有明显的变化,同时还能不同程度的表达Nestin、β-TubulinⅢ、GFAP、MBP、A2B5、O4及Olig2,并随着诱导分化时间的不同,分化后细胞所表达的荧光强度有所变化,混合细胞所含的成分亦不相同。分化后第8天为神经元及成熟少突胶质细胞分化的明显界线,8天前神经元的成分相对较多,而8天后成熟少突胶质样细胞增多。结论:HUMSCs在体外IGF-1等多种细胞生长因子诱导下首先向神经前体细胞分化,再逐渐向神经元样细胞、少突胶质样细胞及星形胶质样细胞等神经上皮样细胞分化。少突胶质细胞的成熟经历了从早期祖细胞到晚期祖细胞再到成熟少突胶质细胞的过程。更多还原

【Abstract】 Background and Purpose: The basic characteristics of mesenchymal stem cells (MSCs) was self-renewal and the multiple differentiative potentials, which is a "hot spot" on the study of stem cells. MSCs can be found in various locations in the body, including bone marrow, adipose tissue, and peripheral blood. Compared with tranditional neural stem cells, embryonic stem cells and haematopoietic stem cells, MSCs have more advantages. MSCs was available in body tissue and easy to culture and to proliferate. MSCs had hardly rejection when transplanting and had lesser genovariation when culturing in vitro. At prensent, the main source of MSCs is bone marrow. However, With the development of MSCs, it was found that the source of bone mesenchymal stem cells is rare. This study is designed to explore HUMSCs, which is a new kind of MSCs. The successful isolation of HUMSCs from umbilical card tissue and study on its biological characteristics will lay the foundation of directional diffrentiation of HUMSCs.Methods: The umbilical cords from full term section patients were collected in sterile condition immediately upon delivery and were washed with sterile saline. The umbilical cords was cut into pieces of 2~4cm. Blood clot in umbilical cords were rinsed out and veins and arteries were removed. Tissue fagments were trypsinized by collagenaseⅡand centrifugate. Then pellets were resuspended in DMEM/F12 medium including fetal bovine serum and penicillin in culture flask. Once adherent cells reached approximately 80~90% confluence, they were re-fed and passaged. We obsevred the cells’morphological changes under inverted phase contrast microscope and their photos were taken. Immunofluorescence and flow cytometry were employed to identify the phenotype of those cells, and the ability of proliferation was observed by means of growth curve.Results: Adherent cells could be found after incubation in 24 hours in vitro,which were small and have no evident nucleus with different appearance in morphology, and most undigestive tissue suspended on culture medium. As time went on, adherent cells were constantly increasing. After 4 days, some adherent cells took on bipolar, flat or long-shuttle shape. Eight days later, the increasing adherent cells looked likes fibroblast cells and grew in whirlpool way. Those cells reached 80% confluence in about 14 days. The passaged cells were gradually purified, and maintained their biological characteristic after at least 20 passages in vitro. It could be found from the growth curve of P3 and P7 and P14 of HUMSCs that their ability of proliferation of had no difference. The phenotype analysis showed that the expressions of CD29, CD73 and CD105 were positive in HUMSCs, and their rates were 99.2%, 99.5% and 98.9% respectively. In contrast,They expressed low level of CD34, CD45 and HLA-DR, and the positive rates were 0.1%, 0.5% and 0.2% respectively. Futhermore, the phenotype analysis showed no significant dieffrence between P3,P7and P10 of HUMSCs.Conclusions: The umbilical cords tissue is disposal after delivery, which is rich, convient to obtain and eliminate the ethical or social concerns. HUMSCs could be harvested by collagenase digesting method to umbilical cords, which had a fibroblast-like appearance and strong ability of proliferation. Surface phenotype analysis indicated that HUMSCs are new member of MSCs family and immunogenicity is low. Therefore, HUMSCs might be an ideal and potential source of cells for tissue engineering. Background and Purpose MSCs was capable of multiple differentiation potential and could differentiate into several types of mature cell, including osteogenic cells, cartilage cells, adipocytes, cardiomyocytes and endotheliocyte. HUMSCs also could be induced to differentiate into neural stem cells, neuron and astroglia-like cells because of its flexibility. The induced efficiency of HUMSCs was distinct under different conditions. The positive rate of neuron-associated protein was higher than gliocyte-associated protein after induced. So the present study was designed to induce HUMSCs to differentiate into neuroepithelium-like cells especially oligodendrocyte-like cells in vitro. This research of inducing differentiation would be possible to provide the source of ideal candidate stem cells for transplantation therapy of nervous system diseases, such as demyelinating disease of central nervous system.Methods: HUMSCs were induced with bFGF, EGF plus N2-supplement and insulin in DMEM/F12. After 24 hours, the medium changed for long-term induction by adding DMEM/F12, N2-supplement and IGF-I. Morphologic change of inducing cells was observed everyday and their pictures were taken. In the same time, immunohistochemistry technique was applied to identify the surface maker of differentiated cells, Such as Nestin,β-TubulinⅢ, GFAP, A2B5, O4, MBP and Olig2, which is respectively the maker of neural stem cell, neuron, astrocyte, oligodendrocyte progentor, preoligodendrocyte, mature oligodendrocyte.Results: Combination of IGF-1 medium with several growth factors synergistically promoted HUMSCs to differentiate into neuroepithelium-like cells. The induced cells not only underwent morphologic changes, but also expressed Nestin、β-TubulinⅢ、GFAP、MBP、A2B5、O4 and Olig2. Moreover, Fluorescence intensity of induced cells was changed with induction time because of different constituents. The borderline between neuron and mature oligodendrocyte occurred on the eighth day after induction. That was to say, the proportion of neuron was relative large first 8 days, and the proportion of oligodendrocyte was relative large after 8 days.Conclusions: HUMSCs was differentiated into neural precursor cell at first. then HUMSCs differentiated into neuroepithelium-like cells including neural, oligodendrocyte and astrocyte-like cells under the condition of combination IGF-1 medium with several growth factors. Meanwhile, the mature of oligodendrocyte experienced a progressive process from early progenitor cells period to late progenitor cells period, then gradually maturity.更多还原