血清在构建角膜基质细胞体外三维培养模型中的作用

发布时间：2018-06-25 01:49

本文选题：Pellet三维培养模型 + 角膜基质细胞　；参考：《眼科》2017年05期

【摘要】：目的研究血清在构建角膜基质细胞体外三维培养模型中对细胞生长情况及胶原纤维生成的影响,为体外研究角膜基质细胞外基质(ECM)纤维化提供新的培养模型。设计实验研究。研究对象牛角膜基质细胞。方法分离原代牛角膜基质细胞,取第一代细胞作为种子细胞构建Pellet模型,实验分为对照组(DMEM/F12)和血清组(DMEM/F12+10%胎牛血清FBS)。Pellet模型培养3周后,肉眼观察细胞生长状态,同时进行HE染色及Calcein AM/PI染色测定细胞活性,并应用MASSON染色及透射电子显微镜(TEM)观察胶原纤维的形成、走向及排列情况。主要指标细胞生长状态、细胞活性、胶原纤维走向及排列情况。结果Pellet模型培养3周后,对照组未观察到角膜基质细胞抱团生长现象,且细胞易松散,难以进行后续实验;血清组Pellet模型中细胞明显成簇生长,呈粒状,结合牢固。Calcein AM/PI染色及HE染色可见血清组大多数细胞存活且密集生长,细胞结构完整。MASSON染色示血清组较多胶原纤维显色,并于TEM下观察到胶原纤维密集分布于细胞外且走向紊乱。结论含血清的培养环境能成功构建牛角膜基质细胞体外Pellet三维培养模型,且能生成含有胶原纤维的ECM。
[Abstract]:Objective to study the effect of serum on the growth of corneal stromal cells in vitro and the formation of collagen fibers in order to provide a new culture model for the study of corneal stromal cell (ECM) fibrosis in vitro. Design experimental research. Participants Bovine corneal stromal cells were studied. Methods the primary bovine corneal stromal cells were isolated and used as seed cells to construct pellet model. The cells were divided into two groups: control group (DMEM- F12) and serum group (DMEM- F12% fetal bovine serum FBS). Pellet model was cultured for 3 weeks. At the same time, HE staining and Calcein AM / Pi staining were used to determine the cell activity. The formation, orientation and arrangement of collagen fibers were observed by Masson staining and transmission electron microscopy (TEM). Main outcome measures: state of cell growth, cell activity, direction and arrangement of collagen fibers. Results after the pellet model was cultured for 3 weeks, the corneal stromal cells were not observed in the control group, and the cells were easy to loose and difficult to carry out subsequent experiments, while in the serum group pellet model, the cells grew in clusters and were granular. Combined with firm. Calcein AM / Pi staining and HE staining, most of the cells in the serum group survived and grew intensively. The cell structure integrity. MASson staining showed that there were more collagen fibers in the serum group, and it was observed that the collagen fibers distributed intensively outside the cells and in the direction of disorder under TEM. Conclusion the three dimensional pellet culture model of bovine corneal stromal cells can be successfully constructed in the culture environment containing serum, and ECM containing collagen fibers can be produced.
【作者单位】：广西医科大学第一附属医院眼科;广西医科大学第三附属医院眼科;
【基金】：国家自然科学基金(81360144、81060076) 教育部留学回国人员科研启动基金(2010-1561)
【分类号】：R77

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