体外诱导分化的神经元在温敏型壳聚糖中的生长观察

发布时间：2018-07-11 15:37

本文选题：骨髓基质细胞 + 神经元样细胞　；参考：《河北医科大学》2007年硕士论文

【摘要】： 目的:脑损伤后不可逆性的神经元网络破坏和胶质细胞脱失,阻碍了脑组织结构的重建,造成相应神经功能缺损。神经组织工程技术的发展为中枢神经系统损伤的修复提供了新思路、新方法。组织工程的关键是种子细胞和生物材料接合构建三维空间复合体。壳聚糖(C)与β-甘油磷酸钠(GP)按一定比例混合得到一种温敏型水凝胶,具有生物相容性好、无毒性、可降解等特点。当它注射到体内后,在温度、pH值等因素作用下完成溶胶-凝胶转变,使操作简单、创伤小、塑型方便,是良好的支架材料。本实验将体外诱导的神经元样细胞与C/GP进行联合培养,观察细胞的生长状况,以了解C/GP对神经元样细胞生长、增殖、分化的影响及与神经元样细胞的生物相容性,探索以C/GP为载体与神经元样细胞制成组织工程材料的可行性,为体外诱导的神经元的脑内移植寻找较合适的可注射性载体提供实验依据。方法:1骨髓基质细胞的培养:50-60g的SD大鼠,麻醉处死,分离双侧胫骨和腓骨,暴露并冲洗骨髓腔,用贴壁培养法,分离出骨髓基质细胞(marrow stromal cell,MSCs)并连续传代、扩增,获取更纯化的MSCs。 2诱导分化神经元:取第3代MSCs,加入0.2mg/ml的三磷酸胞苷二钠(CTP),每天观察细胞变化,约10天左右可见较多的神经元样细胞。做神经元特异性烯醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)的免疫组化染色鉴定细胞。 3温敏型水凝胶的制备:称取脱乙酰度91%的壳聚糖(CS)200mg溶于9mlHCl溶液中,称取β-甘油磷酸钠(β-GP)560mg溶于1ml去离子水中。消毒后室温下按不同体积比逐滴将β-GP溶液滴入不断搅拌着的CS溶液中,分别测混合液的PH值及370C条件下的凝固时间(以凝胶在容器内倒置90°不流动、不变形为凝胶形成的标准),选择最适混合比。 4神经元在C/GP凝胶中的培养与观察:将培养的神经元样细胞制成密度为2×10~5/ml细胞悬液与C/GP混合,接种于96孔培养板,每孔20μl细胞悬液加C/GP复合物10μl,设为实验组;每孔取20μl细胞悬液加10μl完全培养基,设为细胞对照组;每孔取10μlC/GP复合物加完全培养基20μl,设为C/GP对照组。每日换液1次,并在倒置显微镜下观察神经元三维培养的生长情况。 5 MTT法细胞活性检测及生长曲线的绘制:实验组、细胞对照组分别在24h后每组取5孔细胞、C/GP对照组取1孔(测实验组OD值时调0用)加入MTT液,倒置显微镜下观察细胞形态及分布,酶标仪测吸光度(OD值),连续观察7天绘制生长曲线。对两组的OD值作t检验,进行统计学分析。结果:1分离的MSCS呈圆形,胞体透亮。接种24h后,少量细胞贴壁,72h后大部分细胞贴壁,形态为梭形、圆形、多角形,8-10d细胞达到90%融合,细胞呈长梭形排列呈栅栏状,传代后的MSCS贴壁较快约24h完全贴壁,3-7d基本铺满瓶底。 2加诱导剂后3d细胞突起逐渐伸长呈单极或多极同时胞体变圆,光晕明显,随时间的延长不同细胞突起之间有交叉连接现象,10d左右神经元样细胞数量最多。免疫组化呈NSE、GFAP染色阳性表达。 3可注射水凝胶的物理性状:壳聚糖稀盐酸溶液呈淡黄色透明粘稠液体,pH值为5.65, 56%的甘油磷酸钠水溶液呈无色透明液体,pH值为8.91。壳聚糖与β-甘油磷酸钠的体积比不同,C/GP溶液的PH值及37℃凝固时间亦不同(表1)。其中按3:2体积比混合的C/GP、PH为7.2,置于37℃细胞培养箱中胶凝时间约为10min,较为理想。 4神经元细胞在C/GP凝胶中三维培养:24h后倒置显微镜下观察发现大部分细胞保持球形,定植在凝胶中,随时间的延长变形细胞增多,以贴近培养板底面的细胞变形更为明显,胞体呈圆形、多角形,并伸出较长的突起,随时间的延长突起可交叉连接,视角聚集到凝胶的不同层面可见神经元细胞呈三维分布。 5 MTT法细胞活性检测及生长曲线的绘制: MTT染色后,倒置显微镜下观察可见活细胞质内有棕黑色结晶颗粒沉积,间接显示活细胞数量、形态及分布。通过测定OD值绘制两条生长曲线均为“S”型,经过1-2d的潜伏期后进入对数生长期,第3-5d为对数生长期,一周后细胞数量减少,实验组的OD值较对照组略低。 6统计学分析:两组间差异比较用t检验得出P0.05,两组无明显统计学差异,说明神经元样细胞在C/GP凝胶中生长状态良好。结论:1 MSCS具有强大的增殖能力,在一定条件下能分化为神经元样细胞,诱导的神经元性质稳定,可作为良好的种子细胞。 2 CTP在体外可诱导MSCs分化为神经元样细胞 3壳聚糖盐酸溶液与β-甘油磷酸钠按一定体积比(3:2)室温混合后可以得到PH值为7.2的溶胶状混合物,37℃时约10min可发生溶胶-凝胶转变,变成凝胶状,即有利于必要的注射操作的完成又利于收敛细胞。 4神经元在C/GP凝胶中生长良好,与在完全培养基中生长无明显区别,是较为理想的支架材料。
[Abstract]:Objective: the damage of the irreversible neuron network and the loss of glial cells after brain injury hinders the reconstruction of the brain tissue and causes the corresponding neural function defect. The development of neural tissue engineering provides new ideas and new methods for the repair of central nervous system damage. The key of tissue engineering is the joint of seed cells and biomaterials. A three dimensional space complex is constructed. Chitosan (C) and beta glycerphosphate sodium (GP) are mixed to a certain temperature sensitive hydrogel, which has the characteristics of good biocompatibility, non-toxic and biodegradable. When it is injected into the body, the sol-gel transition is completed under the action of temperature and pH, which makes the operation simple, small wound and convenient molding. Good scaffolding material. In this experiment, the cultured neuron like cells were co cultured with C/GP in vitro to observe the growth of cells in order to understand the effect of C/GP on the growth, proliferation, differentiation and biocompatibility with neuron like cells, and to explore the tissue engineering materials made of C/GP as the carrier and neuron like cells. It is feasible to provide experimental evidence for in vitro induced neuronal transplantation in the brain to find more suitable injectable vectors.
Methods: 1 the culture of bone marrow stromal cells: 50-60g SD rats, anesthesia was executed, bilateral tibia and fibula were separated, and the bone marrow cavity was exposed and washed. The bone marrow stromal cells (marrow stromal cell, MSCs) were separated from the bone marrow stromal cells, and the bone marrow stromal cells (stromal cell, MSCs) were isolated and continuously passaged and amplified to obtain a more purified MSCs..
2 induced differentiation neurons: third generation MSCs, 0.2mg/ml three phosphate cytidine two sodium (CTP), the cell changes were observed every day and more neuron like cells were observed for about 10 days. Neuron specific enolase (NSE) and immunohistochemical staining of glial fibrillary acidic protein (GFAP) were used to identify the cells.
The preparation of 3 thermosensitive hydrogels: the chitosan (CS) 200mg of deacetylation degree was dissolved in 9mlHCl solution, and sodium beta glycerphosphate (beta -GP) 560mg was dissolved in 1ml deionized water. After disinfection, the beta -GP solution was dripped by drop by drop in CS solution at different volume ratio at room temperature, and the pH and 370C conditions of the mixture were determined respectively. The best mixing ratio was chosen as the time (in which the gel was inverted 90 degrees in the container without flowing, not being deformed into gel forming standard).
The cultivation and observation of 4 neurons in C/GP gel: the cultured neuron like cells were made into 2 x 10~5/ml cell suspension and mixed with C/GP, inoculated on 96 hole culture plate, 20 mu L cell suspension per pore plus 10 mu C/GP compound, and set as experimental group; 20 mu L cell suspension and 10 mu L complete medium were taken per pore, and the cell control group was set up with 10 mu lC/ per pore. The GP complex was added to the medium of 20 l for C/GP control. 1 times a day, the growth of three dimensional neurons was observed under inverted microscope.
5 MTT method cell activity detection and growth curve drawing: the experimental group, the cell control group took 5 hole cells in each group after 24h, the C/GP control group took 1 holes (the experimental group O time adjustment 0) added MTT solution, the inverted microscope observed the cell morphology and distribution, the enzyme labeling instrument measured the absorbance (o value), and observed the growth curve for 7 days for 7 days. The OD of the two groups The value was tested by T, and the statistical analysis was carried out.
Results: 1 the separation of MSCS was round and the cell body was bright. After inoculation of 24h, a small number of cells were adhered to the wall, and most of the cells were adhered to the wall after 72h. The morphology of the cells was spindle, round and polygonal, and the 8-10d cells reached 90% fusion. The cells showed a long shuttle form of railing, and the MSCS adherent after the passage was almost 24h completely adhered to the wall, and 3-7d was basically paved with the bottom of the bottle.
After 2 addition of inducer, the protuberances of 3D cells gradually elongated to be unipolar or multipolar at the same time and turn round, and the halo was obvious. There was a cross connection between different cell protrusions with time. The number of neuron like cells around 10d was the most. The immunohistochemical staining was NSE and GFAP staining was positive.
3 the physical properties of the injectable hydrogel: the chitosan dilute hydrochloric acid solution shows a light yellow transparent viscous liquid, the pH value is 5.65, the 56% glycerol phosphate water solution is colorless transparent liquid, the pH value is different from the volume ratio of 8.91. chitosan to the sodium beta glycerol phosphate, the pH value of the C/GP solution and the solidification time of the sodium glycerphosphate are different (Table 1). The 3:2 volume ratio is mixed. C/GP and PH were 7.2, and the gelation time was about 10min at 37 C cell incubator.
The 4 neuron cells were cultured in C/GP gel in three dimensions. After 24h, the cells remained spherical, and the cells were colonized in the gel. The cells were increased with time. The cell deformation was more obvious at the bottom of the culture plate, and the cell body was round and polygonal, and extended the protuberance with the extension of time. The junction of neurons is seen in three dimensions.
5 MTT cell activity detection and the drawing of the growth curve: after MTT staining, the deposition of brown black crystalline particles in the living cytoplasm was observed under the inverted microscope, and the number, morphology and distribution of the living cells were indirectly displayed. By measuring the OD value, the two growth curves were all "S", after the incubation period of 1-2D into the logarithmic growth period, 3-5d For logarithmic growth phase, the number of cells decreased after one week, and the OD values in the experimental group were slightly lower than those in the control group.
6 statistical analysis: the difference between the two groups was compared with the P0.05 obtained by t test. There was no significant difference between the two groups, indicating that the neuron like cells grew well in the C/GP gel.
Conclusion: 1 MSCS has strong proliferative ability and can differentiate into neuron like cells under certain conditions. The induced neuron is stable and can be used as a good seed cell.
2 CTP can induce MSCs to differentiate into neuron like cells in vitro.
3 chitosan hydrochloric acid solution and sodium beta glycerphosphate mixed with a certain volume ratio (3:2) room temperature can get a pH value of 7.2 sol-gel mixture. At 37 C, about 10min can have a sol-gel transition and become gelatinous, which is beneficial to the completion of the necessary injection operation and the convergence of the cells.
4 neurons grew well in C/GP gel, and had no obvious difference from those grown in full medium, which is an ideal scaffold material.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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