Neuronatin对人视网膜色素上皮细胞增殖迁移的影响

目的:研究胚胎时期表达部位广泛、丰度高,而成年后分化表达的印记基因Neuronatin(Nnat)的两种剪接形式Nnatα和Nnatβ对人视网膜色素上皮细胞(RPE)增殖、迁移的影响。方法:构建Nnatα、β两种剪接形式的表达质粒,转染RPE获得表达该基因的稳定表达细胞株;CCK-8实验检测稳定表达细胞株的增殖能力,流式细胞仪分析细胞周期,细胞划痕实验检测其迁移能力。结果:成功构建了Nnatα和Nnatβ表达质粒,并获得了Nnatα和Nnatβ基因稳定表达PRE细胞株。CCK-8实验结果显示cNNATα组与对照组相比较,增值率为23.33%(P0.05),cNNATβ组相较于对照组无显著性差异,细胞周期分析cNNATα组和cNNATβ组细胞在G2-S期的百分率分别为18.60%、11.11%,对照组细胞的为9.94%;相较于对照组,cNNATα组的细胞迁移能力显著增强,cNNATβ组的细胞迁移能力微弱增强。结论:Nnatα对RPE有一定的增殖作用,其影响主要在S期;同时,Nnatα显著促进RPE细胞的迁移能力。

The Effect of Neuronatin on Proliferation and Migration in Human Retinal Pigment Epithelial Cells

Objective:To investigate the effect of two splicing forms of Neuronatin (Nnat-alpha and Nnat-beta), which are expressed widely and abundantly in the fetal period and differentially expressed mainly in the pituitary gland in the adulthood on the proliferation and migration in human retinal pigment epithelium (RPE) cellline.Methods:To construct two splicing forms (Nnat alpha and Nnat-beta)expressing plasmids and obtain stable RPE celllines after transfecting RPE cellline with the constructs containing the targeting gene; proliferation, cell cycle analysis and its ability to migrate were evaluated by CCK-8 assay, flow cytometry and cell wound scratch assay respectively.Results:Nnat-alpha and Nnat-beta expressing plasmids were successfully constructed, Nnat alpha and Nnat-beta gene stable expressing RPEcelllines were obtained. CCK-8 test results showed that pNNAT-alpha increased cell proliferation by 23.33% (P<0.05) compared with the control pSNAV transfected cellline. Further analysis through flow cytometry revealed that the percentage of G2S-phase cells in the control pSNAV transfected cellline was 9.94 %, and in pNNAT-alpha and pNNAT beta stable celllines were 18.60% and 11.11% respectively. Compared with the control cellline, the migration of pNNAT alpha cellline was enhanced significantly, and the ability of migration of pNNAT beta was enhanced weakly.Conclusion:NNAT-alpha could enhance the proliferation of RPE cellline significantly and the influence mainly happened on S period and NNAT-a''phacould enhance the migration of RPE celllines significantly.