牛角膜基质细胞的两步酶消化法高效分离及体外培养观察

发布时间：2018-04-28 08:43

本文选题：牛 + 角膜基质细胞　；参考：《广西医科大学》2011年硕士论文

【摘要】：背景：高效、低成本分离出生物学功能活性高的角膜基质细胞是开展角膜基础研究的需要。目前的分离方法成本高、分离效率低,而通过培养达到扩增细胞数会导致细胞表型快速改变。本研究旨在应用成本较低的I型胶原酶,通过改良的两步消化法达到高效、快速、低成本分离原代牛角膜基质细胞的目的。目的：评价设计的I型胶原酶两步消化法分离原代牛角膜基质细胞的效果,并观察体外培养原代牛角膜基质细胞的形态学变化。方法：分别用基础培养液配制的0.5 g/L及1.0 g/L I型胶原酶以两步酶消化法顺序消化牛角膜组织,分离角膜基质细胞,以细胞计数板进行计数,检测基质细胞收获效率；锥虫蓝染色法检测收获细胞的存活率；分离的细胞进行原代培养,倒置显微镜下观察细胞形态和生长变化；应用Alexa488标记的鬼笔环肽检测原代培养的牛角膜基质细胞中微丝肌动蛋白F-actin的分布。结果：牛角膜经两步酶消化基质逐步解离和降解,绝大多数细胞得以释放和分离,分离的牛角膜基质细胞呈圆形,透亮且大小均匀。每个角膜收获(2.109±0.142)×106个基质细胞,细胞存活率(91.69士3.55)%,黏附率(81.20±1.21)%。原代培养的牛角膜基质细胞附壁呈树突样,铺伸至星状,融合时树突连接呈网状,其F-actin局限性分布于细胞皮质。结论：两步酶消化法可使牛角膜基质完全消化降解,具有高细胞收获率高细胞存活率和操作简便等特点。原代培养的牛角膜基质细胞呈树突状,F-actin分布于细胞皮质。
[Abstract]:Background: high-efficiency and low-cost isolation of corneal stromal cells with high biological activity is necessary for basic corneal research. The current separation methods have high cost and low separation efficiency, and the cell phenotype will change rapidly when the number of expanded cells is reached through culture. The purpose of this study was to separate primary bovine corneal stromal cells from bovine corneal stromal cells at low cost by using a low cost type I collagenase and a modified two-step digestion method. Aim: to evaluate the effect of the designed two step digestion method for the isolation of primary bovine corneal stromal cells and to observe the morphological changes of primary bovine corneal stromal cells cultured in vitro. Methods: bovine corneal tissue was digested with 0.5 g / L and 1.0 g / L I collagenase prepared from basic culture medium respectively by two-step enzymatic digestion. Corneal stromal cells were isolated and counted by cell counter to detect the harvest efficiency of stromal cells. The survival rate of harvested cells was detected by trypanosome blue staining, the isolated cells were cultured in primary culture, and the morphology and growth of the cells were observed under inverted microscope. The distribution of microfilamentactin (F-actin) in primary cultured bovine corneal stromal cells was detected by Alexa488 labeled ghost pen cyclic peptide. Results: the bovine corneal stromal cells were gradually dissociated and degraded by two steps of enzyme digestion, and most of the cells were released and separated. The isolated bovine corneal stromal cells were circular, transparent and uniform in size. The cell survival rate was 91.69 卤3.55%, and the adhesion rate was 81.20 卤1.21% per corneal harvest of 2.109 卤0.142) 脳 106 stromal cells. The primary cultured bovine corneal stromal cells were dendritic, spreading to stellate, reticular dendritic junctions during fusion, and their F-actin was localized in the cortex. Conclusion: the two-step enzyme digestion method can completely degrade bovine corneal stroma, and has the characteristics of high cell harvesting rate, high cell survival rate and simple operation. Primary cultured bovine corneal stromal cells were distributed as dendritic F-actin in the cortex.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329;S823

【参考文献】