骨髓间充质干细胞复合温敏性水凝胶三维微环境体外培养体系的构建与评价

发布时间：2018-11-24 12:15

【摘要】：目的：以温敏性水凝胶作为载体,以骨髓间充质干细胞为种子细胞,构建三维微环境培养体系；评价间充质干细胞与温敏性水凝胶三维支架的相容性；探讨间充质干细胞在三维微环境下分裂增殖与分化的细胞生物学特性。为开展相关的组织工程及应用基础研究提供实验依据,建立实验基础。方法：1.优化三维微环境支架材料：以壳聚糖、β-甘油磷酸钠为原料,羟乙基纤维素为交联剂制备温敏性水凝胶。将壳聚糖、β-甘油磷酸钠、羟乙基纤维素按不同体积比例混合,分为六个实验组,评价其成胶时间及硬度。2.建立稳定的骨髓间充质干细胞资源：从新生SD乳鼠骨髓中分离BMSCs,在体积分数10%胎牛血清的低糖DMEM中培养,细胞达到培养瓶瓶底面积70%,进行传代培养至第四代,后续各代BMSCs的传代时间稳定在4～5d。各代细胞检测证实保持多向分化潜能和低分化状。3.选取生长良好的第3代MSCs为种子细胞,温敏性水凝胶为支架,10%FBS的L-DMEM培养2W,并加入心肌组织裂解液诱导5d,构建三维微环境培养体系,倒置相差显微镜下观察细胞的生长情况,并行HE染色、A0染色,扫描电子显微镜下观察,评价三维微环境中细胞、温敏性水凝胶支架之间的相互作用。结果：1.骨髓间允质干细胞接种于水凝胶上24h后细胞仍维持球形,48h内生长较慢,72h球形细胞开始增多,附着于温敏性水凝胶支架的表面上。连续培养2W,取细胞-支架共培养物行苏木素-伊红染色,镜下可见大量圆形细胞沿着支架表面生长,部分细胞可跨越于支架之间；扫描电子显微镜下,可见大量细胞维持圆形、均质性好、分裂能力强,粘附于支架上,部分细胞表面可见以胞吐方式分泌的膜被颗粒。2.以心肌组织裂解液诱导5d,HE染色可见细胞呈长梭形并包被支架材料,扫描电镜下可观测到细胞开始由圆形变为长梭形,细胞之间出现牵拉。结论：通过优化温敏性水凝胶各组分体积比,可构建具有良好孔径率的三维支架；骨髓间充质干细胞与支架有良好的生物相容性,可保持低分化状态和活跃的分裂增殖能力；在特定的三维微环境培养体系中,能够较好的模拟体内组织结构,促进骨髓间充质干细胞的分化。
[Abstract]:Objective: To construct a three-dimensional micro-environment culture system based on the temperature-sensitive hydrogel as a carrier, and to evaluate the compatibility of the mesenchymal stem cells and the temperature-sensitive hydrogel three-dimensional scaffold. To study the cell biological characteristics of the cell proliferation and differentiation of mesenchymal stem cells in three-dimensional micro-environment. To provide the experimental basis for carrying out the relevant research on the tissue engineering and application, and to establish the experimental basis. The method 1. Optimizing the three-dimensional micro-environment support material: preparing the temperature-sensitive water by using the chitosan, the chitosan-sodium glycerophosphate as the raw material and the hydroxyethyl cellulose as a cross-linking agent; mixing the chitosan, the chitosan-sodium glycerophosphate and the hydroxyethyl cellulose in different volume proportions, and dividing into six experimental groups to evaluate the gel time and the hardness of the two experimental groups;. 2. Stable bone marrow mesenchymal stem cell resources were established: BMSCs were isolated from the bone marrow of neonatal SD rats and cultured in low-sugar DMEM containing 10% fetal bovine serum. The cells reached 70% of the bottom area of the flask, and the cells were subcultured to fourth generation. The passage time of subsequent generation of BMSCs was stable at 4 ~ 5d. The detection of each generation cell confirms that the multi-directional differentiation potential and the low differentiation state are maintained. and 3, selecting the third generation MSCs with good growth as the seed cells, the temperature-sensitive hydrogel as the support, the L-DMEM of the 10% FBS for 2W, and adding the myocardial tissue lysis liquid to induce 5d, constructing a three-dimensional micro-environment culture system, observing the growth of the cells under the inverted phase-difference microscope, and parallel HE staining, A0, the interaction between the cells in the three-dimensional micro-environment and the temperature-sensitive hydrogel support is evaluated under the observation of the dyeing and scanning electron microscope, action. Results: 1. The cells of the bone marrow mesenchymal stem cells were inoculated on the hydrogel for 24 h, the cells remained spherical, the growth of the cells was slower in the 48h, the growth of the 72h spherical cells began to increase, and the cells were attached to the warm-sensitive hydrogel branch. The surface of the rack is continuously cultured for 2W, and the cell-support coculture is subjected to the hematoxylin-eosin staining, a large number of circular cells can be seen under the mirror to grow along the surface of the support, and the part of the cells can be crossed between the supports; under the scanning electron microscope, a large number of cells can be seen to be round, and the homogeneity is good; a membrane that is strong in division, adheres to a stent, and can be seen to be secreted in a cellular manner on the surface of a portion of the cell. The cells were stained with myocardial tissue lysing liquid for 5d. The cells of HE staining were found to be in a long-shuttle shape and coated with the stent material. The cells were observed under the scanning electron microscope from the circular to the long-shuttle-shaped, and the cells Conclusion: The three-dimensional scaffold with good aperture ratio can be constructed by optimizing the volume ratio of each component of the temperature-sensitive hydrogel, and the bone marrow mesenchymal stem cells have good biocompatibility with the support, and the low-differentiation state and the activity can be maintained. in a specific three-dimensional micro-environment culture system, the body tissue structure can be well simulated, the bone marrow is promoted,
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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