骨髓间充质干细胞及其经明胶海绵搭载向神经组织移植的初步研究

发布时间：2018-05-30 14:13

本文选题：骨髓间充质干细胞 + 明胶海绵　；参考：《河北医科大学》2009年硕士论文

【摘要】： 第一部分:骨髓间充质干细胞的分离、培养、鉴定及其自身特性的初步研究目的:通过本实验,对骨髓间充质干细胞进行分离、培养、鉴定并对其自身特性加以研究,同时为下一步明胶海绵载体搭载移植实验提供搭载细胞。方法:首先,利用差速贴壁培养法将骨髓间充质干细胞从大鼠的骨髓之中分离、纯化出来。之后,利用流式细胞学方法对骨髓间充质干细胞的表面标记物加以鉴定。在鉴定其为骨髓间充质干细胞后,以C6细胞系做为对照绘制其在不同代数时期的生长曲线,对其致瘤性加以研究。利用免疫荧光学的方法,研究骨髓间充质干细胞在一定条件下自发向神经细胞转化的特性。利用Transwell方法,研究骨髓间充质干细胞可向受损神经组织定向迁移的特性。最后,研究骨髓间充质干细胞简便的冻存与复苏保存方法。结果:从大鼠骨髓腔中冲出的混杂细胞,在倒置相差显微镜下显示为大量单个或成团均匀分布的小圆形细胞。4～8小时之后部分细胞沉降并铺至培养皿底部。24小时后观察可见铺于培养皿底部的大部分细胞变扁并逐渐伸出突起。3天后未贴壁细胞及其它髓腔内物质经换液被大部分去除。此后贴壁细胞不断扩增,至10～14天可见大量细胞汇集成片,形态不一,成团生长。其中可见大扁平形、多角形、星形、梭形等多种细胞形态。经过传代细胞形态和类型逐渐趋向单一,至第5代细胞大小一致,形态呈梭形,已基本得到纯化。利用流式细胞学方法的检测,细胞体系表达表面标志物CD31、CD34、CD45为阴性,表达表面标志物CD71为阳性。对其不同时期的生长曲线与C6细胞系相比较,骨髓间充质干细胞生长曲线有很明显的压低。通过倒置相差显微镜的观察和免疫荧光学的检测,发现骨髓间充质干细胞在一定条件下可自发分化出神经样细胞并表达神经细胞标志物GFAP、NSE。通过Transwell实验,可以观察到在相同的时间内损伤组骨髓间充质干细胞穿过率明显高于对照组骨髓间充质干细胞的穿过率,经统计P0.05有显著差异。经过简便方法冻存的骨髓间充质干细胞在-80℃条件下至少可以存活3个月,在液氮中保存则至少可以存活半年。骨髓间充质干细胞经过复苏后数量会有较大的损失,但只要复苏后存活的骨髓间充质干细胞达到一定的数量和密度,这些细胞仍可迅速扩增并在较短的时间内获得大量、稳定,可以利用的骨髓间充质干细胞。结论:通过最常应用的差速贴壁培养法可以很稳定地培养出骨髓间充质干细胞,第5代细胞已达到纯化要求。通过细胞表面标志物的鉴定可以确定其为骨髓间充质干细胞。10代以内的骨髓间充质干细胞致瘤性极小或者不存在,可以自发分化出神经元和神经胶质细胞。另外,其具有独特的归巢性可向受损的神经组织迁移。骨髓间充质干细胞通过简便的冻存与复苏方法能够在较长时间内稳定的保存,可为后续应用提供储备细胞。第二部分:骨髓间充质干细胞经明胶海绵载体搭载向神经组织移植可行性的初步研究目的:研究骨髓间充质干细胞被明胶海绵载体搭载后其存活率是否受到影响和其自身特性有否改变,并进一步研究骨髓间充质干细胞经过明胶海绵载体搭载后能否与神经细胞共存。方法:首先,观察经明胶海绵搭载后骨髓间充质干细胞形态有否改变并统计经明胶海绵搭载移植后的骨髓间充质干细胞贴壁存活的数量与未经搭载细胞相比是否有差异。然后,以未经搭载第5代骨髓间充质干细胞为对照绘制经过搭载骨髓间充质干细胞的生长曲线,并对其致瘤性加以研究。之后诱导其向神经细胞方向分化,观察经过搭载的骨髓间充质干细胞与未经搭载的细胞在诱导之后形态的不同,并统计二者所分化出的神经元比例是否有差异。最后,建立体外神经细胞培养体系,并通过倒置相差显微镜研究观察经明胶海绵搭载后的骨髓间充质干细胞能否与神经细胞共同培养。结果:倒置相差显微镜观察,明胶海绵组与对照组相比较细胞的形态未有明显变化,多呈长梭形,扁平老化的现象也很少见。无论是在明胶海绵中部或是边缘部均有骨髓间充质干细胞贴壁,而且在明胶海绵中部贴壁的细胞数量更多一些。通过对明胶海绵组和对照组未贴壁细胞的计数比较P0.05,无统计学差异。经搭载后的第5代骨髓间充质干细胞的生长曲线与未经搭载第5代细胞的生长曲线相比较没有观察到明显的变化。骨髓间充质干细胞经诱导6～8小时后可以观察到细胞胞体开始收缩并伸出突起,部分突起相互连接形成网络状结构。经免疫荧光学检测细胞可表达神经胶质细胞标记物GFAP和神经元细胞标记物NSE。通过流式细胞学方法对明胶海绵组与对照组表达NSE阳性率做比较,经统计P0.05,无显著差异。新生大鼠神经细胞种植后12～24小时后大部分细胞可贴壁并伸出突起。随着培养时间的延长神经元胞突主干和分枝明显延长形成稠密的网络,神经胶质细胞数量则不断增长。原代神经细胞可在体外维持培养14～30天以上。经过搭载的骨髓间充质干细胞移植入神经细胞培养体系以后,倒置相差显微镜下观察可见二者能够共同生长、繁殖。结论:骨髓间充质干细胞经过明胶海绵搭载后,细胞的贴壁、存活未受到明显影响。其低致瘤性的增殖特性以及可分化为神经细胞的特性也未有明显改变。经明胶海绵搭载移植后骨髓间充质干细胞可以与神经细胞共存。
[Abstract]:Part one: isolation, culture, identification and characterization of bone marrow mesenchymal stem cells
Objective: through this experiment, the bone marrow mesenchymal stem cells were isolated, cultured, identified and studied on their own characteristics. At the same time, the cells were provided for the next step of Gelfoam carrier carrying transplantation.
Methods: first, the bone marrow mesenchymal stem cells were isolated and purified from the bone marrow of rats by differential adherence culture. Then, the surface markers of bone marrow mesenchymal stem cells were identified by flow cytometry. After identifying the bone marrow mesenchymal stem cells, the C6 cell line was used as the control to draw their different algebra. The growth curve of the period was used to study the tumorigenicity of the bone marrow mesenchymal stem cells. The characteristics of the spontaneous migration of bone marrow mesenchymal stem cells to the nerve cells under certain conditions were studied. The characteristics of the directional migration of bone marrow mesenchymal stem cells to the damaged nerve tissue were studied by Transwell method. Finally, the bone marrow mesenchymal stem cells were studied. A simple method of cryopreservation and resuscitation.
Results: the hybrid cells washed out from the marrow cavity of the rat were displayed on the inverted phase contrast microscope for a large number of small round cells with uniform distribution of a large number of single or mass groups.4 ~ 8 hours after the cell settlement and spread to the bottom of the culture dish for.24 hours, and observed that the large part of the cell spread at the bottom of the culture dish became flat and gradually extended out for.3 days. The adherent cells and other intramedullary substances were removed most of the fluid. After that, the adherent cells were continuously amplified, and a large number of cell lines were visible to 10~14 days, with different morphologies, such as large flattened, polygonal, star and spindle shape. Through the form and type of the passages, the cells gradually tended to be single, to the fifth generation. The cell size is the same, the shape is spindle shaped, and it has been basically purified. By flow cytometry, the expression surface markers CD31, CD34, CD45 are negative, and the expression surface marker CD71 is positive. The growth curve of bone marrow mesenchymal stem cells has obvious pressure compared with the C6 cell line at different times. Low. Through the observation of inverted phase contrast microscope and the detection of immunofluorescence, it was found that bone marrow mesenchymal stem cells can spontaneously differentiate into the neuro cell and express the nerve cell marker GFAP under certain conditions. Through the Transwell experiment, NSE. can observe that the rate of bone marrow mesenchymal stem cells in the injury group is obviously higher in the same time. The cross rate of bone marrow mesenchymal stem cells in the control group was significantly different by statistical P0.05. The bone marrow mesenchymal stem cells stored in the simple method can survive at least 3 months at -80 C, and can survive for at least half a year in liquid nitrogen. The survival of the bone marrow mesenchymal stem cells in the post Soviet Union reaches a certain amount and density, and these cells can still rapidly expand and obtain a large, stable, and available bone marrow mesenchymal stem cells in a relatively short period of time.
Conclusion: bone marrow mesenchymal stem cells can be cultured stably through the most commonly used differential adherent culture method. The fifth generation cells have reached the requirement of purification. Through the identification of cell surface markers, it can be determined that the bone marrow mesenchymal stem cells in the.10 generation of bone marrow mesenchymal stem cells are very small or not, and can be separated spontaneously. Neurons and glial cells, in addition, have a unique homing ability to migrate to damaged nerve tissue. Bone marrow mesenchymal stem cells can be preserved steadily for a long time through a simple cryopreservation and resuscitation method, which can provide reserve cells for subsequent applications.
The second part: preliminary study on the feasibility of bone marrow mesenchymal stem cells transplanted into nerve tissue via gelatin sponge carrier.
Objective: To investigate whether the survival rate of bone marrow mesenchymal stem cells (MSCs) is affected by Gelfoam carrier and their own characteristics, and to further study whether bone marrow mesenchymal stem cells can coexist with nerve cells after carrying Gelfoam carrier.
Methods: first, whether the morphologic changes of bone marrow mesenchymal stem cells (MSCs) were changed after the gelatin sponge was carried and the number of surviving bone marrow mesenchymal stem cells adhered by Gelfoam sponges was compared with that of unloaded cells. Then, the bone marrow mesenchymal stem cells without carrying fifth generations of bone marrow were plotted through the carrying bone. The growth curve of medullary mesenchymal stem cells and its tumorigenicity were studied. Then it was induced to differentiate into the nerve cell direction and observed the difference in the form after the induction of the bone marrow mesenchymal stem cells and the unloaded cells after the induction, and the difference between the two differentiated nerve elements. Finally, the extracorporeal nerve was established. Cell culture system was used to study whether the bone marrow mesenchymal stem cells loaded with gelatin sponge could co cultured with nerve cells by inverted phase contrast microscope.
Results: in the inverted phase contrast microscope, there was no obvious change in the morphology of the cells in the Gelfoam group and the control group, and there was a long spindle shape, and the flat aging phenomenon was rare. The bone marrow mesenchymal stem cells were adhered to the middle or edge of the gelatin sponge, and the number of cells attached to the middle of the gelfoam sponge was more. There was no statistical difference between the number of unadhered cells in the gelatin sponge group and the control group P0.05. There was no significant difference in the growth curve of the fifth generation bone marrow mesenchymal stem cells after carrying on the fifth generation cells. The bone marrow mesenchymal stem cells could be observed after 6~8 hours of induction. The cell bodies began to contract and protruded, and some of the protuberances were connected to form a network structure. By immunofluorescent detection, the expression of glial cell marker GFAP and neuron cell marker NSE. was compared with the control group by flow cytometry, and there was no significant difference between the expression of NSE and the control group by statistical P0.05. Most of the neurons could be adhered to the wall and protruded after 12~24 hours after the nerve cells were planted. With the prolongation of the incubation time, the main stem and branches of the neurons were prolonged to form a dense network, and the number of glial cells increased continuously. The primary nerve cells could be cultured outside the body for more than 14~30 days. After transplantation of bone marrow mesenchymal stem cells into nerve cell culture system, the inverted phase contrast microscope showed that the two could grow together and reproduce.
Conclusion: after the bone marrow mesenchymal stem cells are loaded with gelatin sponge, the cell adhesion and survival have not been significantly affected. The characteristics of its low tumorigenicity and differentiation into neural cells have not changed obviously. The bone marrow mesenchymal stem cells can coexist with the nerve cells after the transplantation of gelfoam.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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