神经短肽NAP对体外培养的兔视网膜神经细胞的作用

Effect of Neuropeptide NAP on Cultured Neuroepithelial Cells from Rabbit Retina

目的以感染神经营养因子4-神经保护短肽融合基因(NT4-NAP)的兔虹膜色素上皮细胞(IPECs)为替代分泌细胞,探讨NAP对兔视网膜神经上皮细胞生长的作用。方法分别培养兔IPECs和视网膜神经上皮细胞;构建NT4-NAP融合基因,同时构建重组腺相关病毒载体rAAV-GFP和rAAV-NAP(含融合基因NT4-NAP),采用斑点杂交法测定病毒滴度。rAAV-GFP感染体外培养的兔IPECs,以荧光表达检测病毒的感染情况。rAAV-NAP感染兔IPECs3d后收集含NAP蛋白的上清液,将其加入兔视网膜神经上皮细胞的培养基中,观察细胞的生长状况,同时以未加上清液的兔视网膜神经上皮细胞为对照。结果经斑点杂交证实rAAV-GFP滴度为2.25×1011cfu/mL,rAAV-NAP滴度为2.25×1010cfu/mL。感染rAAV-GFP的兔IPECs表达出明显的绿色荧光。在加入含有NAP蛋白上清液的培养基中,兔视网膜神经上皮细胞生长良好,存活细胞数目多,细胞突起粗而长,培养14d时其突起长度〔(14.6±1.1)μm〕与对照组〔(3.1±0.6)μm〕相比差异有统计学意义(P<0.05)。结论神经短肽NAP对体外培养的兔视网膜神经上皮细胞有促进生长的作用。

Objective With the rabbit iris pigment epithelial cells （IPECs）, which were containing the NT4-NAP fusion gene, taken as the substituting secreting cells producing the neuropeptide NAP, the effect of neuropeptide NAP on the growth status of retinal neuroepithelial cells of rabbit was examined and explored. Methods The iris pigment epithelial cells and retinal neuroepithelial cells of rabbit were cultured; rAAV-GFP and rAAV-NAP （containing NT4-NAP fusion gene） were eonstrueted; the rabbit IPECs were infected with rAAV-GFP and rAAV-NAP; the infections of viruses were detected by GFP fluorescence expression; the supernatant of culture from rabbit IPECs with NAP was collected and added into the culture medium of rabbit retinal neuroepithelial cells, and the growth state of retinal neuroepithelial cell was observed. Results Compared to control cells, the rabbit IPECs could express the GFP fluorescence. The rabbit retinal neuroepithelial cells with NAP supernatant could survive more, and the survival cells showed longer and stronger axons. On 14^th day of cell culture, the mean axon length of NAP group was （14.6±1.1） μm, while that of control group was only （3.1± 0. 6 ） μm. Obviously, a significant difference existed between two groups （P〈0. 05）. Conclusion The rabbit IPECs with NT4-NAP fusion gene can secrete NAP, and the NAP can promise the growth of rabbit retinal neuroepithelial cell.