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

Efficient isolation of bovine keratocytes utilizing two step enzymatic digestion

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。应用成本较低的Ⅰ型胶原酶，通过改良的两步酶消化法可能达到高效、快速、低成本分离牛角膜原代基质细胞的目的。目的评价设计的Ⅰ型胶原酶两步酶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0．5g／L及1．0g／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的变化；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F—actin的分布。结果牛角膜经两步酶消化法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）×10。个基质细胞，细胞存活率（91．693±3．551）％，贴壁率（81．195±1．214）％。原代培养的牛角膜基质细胞贴壁呈树突样，铺伸至星状，融合时树突连接呈网状，其F—aetin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。

Background Efficient and lowcost way to isolate keratocytes is helpful for research on cornea. Either relatively expensive or inefficient is the shortage of those means now applied, while raising the keratocytes through passage will change the phenotype of them quickly. Our aim is to approach the way getting keratocytes effectively utilizing modified two step enzymatic digestion by type Ⅰ collagenase. Objective To evaluate the effect of isolating the bovine keratocytes utilizing two step enzymatic digestion and observe the morphological changes of the keratocytes during cultivation in vitro. Methods Keratocytes were isolated from bovine corneas using 0.5 mg/mL and 1 mg/mL type I collagenase digestion. The harvesting rate and viability rate of the primary keratocytes were evaluated. During the primary cultivation in vitro, the morphological changes of the keratocytes and their F-action distribution were observed. Results （1） The extracellular matrix of the bovine corneas were almost dissolved by the two step enzymatic digestion, followed the keratocytes completely isolated from the solid matrix. The amount of the harvested keratocytes from each cornea was （2. 11±0. 15 ）×10^6 on average while the viability rate was （91.69± 3.73 ） % and the inoculation rate was （ 81.20± 1.25 ） %. （2） The primary keratocytes attached and spreaded out with dendritic and stellate morphology. After 3 days cultured, the branches of the keratocytes were contacting and formed networks. The F-actin detected by phalloidin binding showed a limited cortical localization. Conclusion （ 1 ） The method of two step enzymatic digestion can make the extracellular matrix of bovine cornea stroma completely degraded with the advantages in high efficiency of harvesting keratocytes and high cell viability and relatively simple manipulation. （2） The primary bovine keratocytes have dendritic morphology and with limited F-action distribute in cellular cortex.