人热敏瞬时受体通道1基因转染对猫角膜内皮细胞P27蛋白表达的影响

Influence of human thermal transient receptor potential 1 gene transfection in expression of P27 protein in corneal endothelial cells in cats

目的:探讨人热敏瞬时受体通道1(TRP1)基因转染对猫角膜内皮细胞P27蛋白表达的影响,探讨热敏TRP1基因对角膜内皮细胞增殖周期的作用,为该基因应用于人角膜内皮细胞提供理论依据。方法:将20只健康足月幼猫随机分为实验组和对照组,每组10只,麻醉后取角膜内皮细胞进行培养,培养后实验组细胞通过脂质体介导的方法转染人热敏TRP1基因,对照组采用5.0μL PBS代替表达载体加入培养基。RT-PCR观察人热敏TRP1基因mRNA表达水平,Western blotting观察猫角膜内皮细胞内P27蛋白的表达。继续培养96h观察目的基因转染对细胞分裂再生活性的影响。结果:细胞培养、加热后钙离子流入明显增多;人热敏TRP1基因转染后RT-PCR结果显示。实验组条带密度显著高于对照组(t=2.21,P=0.037);Western blotting结果显示,实验组P27蛋白表达显著高于对照组,差异有统计学意义(t=2.53,P=0.021);实验组和对照组有丝分裂指数、G1期细胞比例差异均有统计学意义(χ2=3.26,χ2=4.18,P<0.05)。结论:人热敏TRP1基因转染可以增加猫角膜内皮细胞P27蛋白表达,抑制角膜内皮细胞增殖。

Objective To explore the influence of human thermal transient receptor potential 1（TRP1） gene transfection in expression of P27 protein in corneal endothelial cells in cats and the effect of termal TRP1 gene in proliferation cycles of corneal endothelial cells and to provide theoretical foundation for the application of the gene in human corneal endothelial cells.Methods 20 healthy full-term kittens were randomly divided into experiment group and control group.The corneal endothelial cells were collected after anesthesia and cultured.The cells in experiment group were transfected with thermal TRP1 gene mediated by liposome.The cells in control group were treated with 5.0 μL PBS instead of the expression vector into medium.The mRNA expression of thermal TRP1 gene was detected by RT-PCR.The expression of P27 protein in cat corneal endothelial cells was observed by Western blotting.The influence of target gene transfection in the activities of cell division and regeneration 96 h after culture was observed.Results The inflow of calcium ions in the cultured cells was increased significantly after heating.The RT-PCR results of thermal TRP1 gene transfection showed that the strip density in experiment group was higher than that in control group（t= 2.21,P= 0.037）.The expression of P27 protein in experiment group was significantly higher than that in control group（t=2.53,P= 0.021）.The differences of mitotic index and the proportion of the cells at G1 phase between experiment group and control group were statistically significant（χ2=3.26,χ2=4.18,P〈0.05）.Conclusion The human thermal TRP1 gene transfection in cat corneal endothelial cells can increase the expression of P27 protein and inhibit the proliferation of corneal endothelial cells.