Long Evans大鼠视网膜神经节细胞培养与鉴定

目的建立Long Evans大鼠视网膜神经节细胞(RGCs)体外培养的方法,为RGCs的体外实验研究奠定基础.方法出生后1～3 d的Long Evans大鼠视网膜神经上皮层,胰蛋白酶 透明质酸酶消化制成单细胞悬液,用DMEM培养液进行培养,观察细胞生长规律,图像分析系统分析有突起的RGCs数目、胞体面积和最长的突起长度,Thy1.1单克隆抗体和微管相差蛋白2多克隆抗体荧光双标法鉴定RGCs.结果 Long Evans大鼠RGCs体外培养存活4 d;荧光双标显示RGCs纯度为80%～90%.结论在普通培养基中Long Evans大鼠RGCs体外培养能获成功.胰蛋白酶 透明质酸酶联合消化较单独用胰蛋白酶消化效果好.

Culture and identification of retinal ganglion cells in Long Evans rats

Objective To establish a culture method for in vitro culture of retinal ganglion cells (RGCs) in Long Evans rats. Methods Epithelium of neurosensory retina of Long Evans rats (postnatal 1-3 d) were digested by trypsin and hyaluronidase and cultured in DMEM. The growth of RGCs in vitro was observed under phase contrast microscope. The number of RGCs with processes, cell body area, and length of the longest processes of RGCs were measured by image analysis system. RGCs were identified with fluorescent double labeling method. Results RGCs of Long Evans rats could survive for 4 d in DMEM. The purity of RGCs in the experiment was 80%-90%. Conclusion The cultured RGCs of Long Evans rats can survive in DMEM. Trypsin combined with hyaluronidase is better than trypsin used alone in digesting epithelium of neurosensory retina.