视网膜神经节细胞诱向(营养)因子的生物效应的体外研究

IN VITRO STUDY OF THE BIOLOGICAL EFFECT OF RETINAL GANGLION NEURONOTROPHIC FACTOR

新生大鼠RGCs预先用明胶海绵吸粒蓝(GB)经顶盖逆行标记,2天后分离视网膜细胞进行培养,用于检测Te的生物效应。以Thyl.1特异性抗体纯化RGCs,建立纯化的RGCs培养系统,检测由Te纯化的RGNTF的生物效应。结果发现含Te培养48小时的视网膜细胞,GB标记的细胞有大(直径18～50μm)、中(10～18μm)和小(<10μm)三型,分别占31.5%、28.1%和40.4%。GB标记细胞存活数量在Te组比无Te组高3.3倍,且约50%表达神经突起。在纯化的RGCs培养中,含RGNTF培养48小时的RGCs,生长非常活跃,胞体均>18μm,有>95%的RGCs表达神经突起。无任何因子的对照组,RGCs培养48小时后全部死亡。四唑盐MTT定量结果显示RGNTF组RGCs存活的OD值比对照组高5.3倍。这进一步表明RGNTF对RGCs在体外存活、生长及神经突起表达具有特异性促进的效应。

Retinal ganglion cells (RGCs) from neonatal rats were retrograde-prelabelabeled with granular blue (GB) by applying foam gel soaked with GB solution next to the tectal area. After 2 days, retinal cells were isolated and cultured for studying the biological effect of tectal extract (Te). In order to establish a purified retinal ganglion cell culture system, retinal ganglion cells were purified using the specific anti-rat Thy 1.1 antibody. Effect of retinal ganglion neuronotrophic factor(RGNTF) from tecta on purified RGCs was studied. It was found that the GB-labeled RGCs could be classified into three sizes : Large (with diameters varied from 18-50 um), medium (with diameters varied from lC-18um) and small (with diameters<10 um=.The average percentages of the three sizes were 31.5, 28.1 and 40.0%, respectively in 4S h cultures with Te. The number of surviving RGCs in cultures with Te was 3.3 times higher than those cultures without Te and nearly 50% of these suprving RGCs expressed neurites. These RGCs survived and grew actively with an average diameter >18um, and>95% of them exnressed neurites after 43 h culture with nurified RGNTF. In cultures without any trophic factor, all of the RGCs degenerated and died after 48 h culture. The. sizes of all these RGCs were less than 10 um. MTT assay showed that the optical density nf RGCs survival in the culture with RGNTF is 5.3 times higher than those cultures without RGNTF. These results indicated further that the RGNTF played a specific role on the survival, growth and neurite xpression of RGCs in vitro.