摘要
目的:探讨水通道蛋白1(AQP1)对人角膜内皮细胞(HCE)增殖、迁移及凋亡影响的机制。方法:以质粒互补脱氧核糖核酸3.1(pcDNA3.1)为载体，将AQP1过表达质粒(pcDNA3.1－AQP1)、空载对照质粒(pcDNA3.1－NC)、AQP1小干扰核糖核酸序列(siRNA－AQP1)及对照序列(siRNA－NC)分别转染于HCE细胞内。按其转染类别分为非转染对照组(Control组)、pcDNA3.1－AQP1组、pcDNA3.1－NC组、siRNA－AQP1组及siRNA－NC组。采用实时荧光定量聚合酶链反应法、5－溴－2－脱氧尿嘧啶实验、Transwell小室法及流式细胞术分别检测细胞的AQP1信使核糖核酸表达量、增殖能力、迁移能力及凋亡情况。为明确p38丝裂原活化蛋白激酶(p38 MAPK)在其机制中的作用，在pcDNA3.1－AQP1组添加p38 MAPK激活剂10 μmol/L茴香霉素，并将其命名为pcDNA3.1－AQP1+茴香霉素组，并采用蛋白印迹法检测紧密连接蛋白1(TJP1)、钠钾三磷酸腺苷酶、p38 MAPK及磷酸化p38 MAPK蛋白的表达含量。各组细胞AQP1信使核糖核酸相对表达量、细胞增殖活性、迁移率、凋亡率、TJP1、钠钾三磷酸腺苷酶蛋白表达量及磷酸化p38 MAPK/p38 MAPK比值均满足方差齐性和正态分布，以±s表示，组间比较采用独立样本t检验。结果:Control组、pcDNA3.1－NC组、siRNA－NC组、pcDNA3.1－AQP1组及siRNA－AQP1组HCE细胞AQP1信使核糖核酸相对表达量分别为(1.00±0.08)、(1.05±0.07)、(0.97±0.07)、(2.14±0.12)及(0.33±0.02)。与Control组比较，pcDNA3.1－NC组和siRNA－NC组比较的差异均无统计学意义(t＝0.653，0.684；P>0.05)，说明pcDNA3.1－NC组和siRNA－NC组未影响细胞，故后续指标检测不再检测该两组。pcDNA3.1－AQP1组HCE细胞增殖活性、细胞迁移率、细胞凋亡率、TJP1、钠钾三磷酸腺苷酶蛋白表达量及磷酸化p38 MAPK/p38 MAPK比值分别为(63.31±7.35)%、(74.28±7.04)%、(3.64±1.48)%、(1.73±0.13)、(2.04±0.15)及(0.18±0.02)；siRNA－AQP1组分别为(22.15±3.26)%、(35.73±3.86)%、(20.35±2.83)%、(0.23±0.02)、(0.21±0.02)及(0.75±0.09)；pcDNA3.1－AQP1+茴香霉素组分别为(52.35±4.38)%、(58.74±5.42)、(6.73±0.53)%、(1.38±0.21)、(1.18±0.19)及(0.33±0.03)。pcDNA3.1－AQP1组与Control组各指标比较的差异均有统计学意义(t＝3.844，3.534，3.253，4.253，4.753，4.321；P<0.05)；siRNA－AQP1组与Control组各指标比较的差异均有统计学意义(t＝4.136，3.741，3.621，3.426，4.122，4.894，3.795；P<0.05)；pcDNA3.1－AQP1+茴香霉素组与pcDNA3.1－AQP1组各指标比较的差异均有统计学意义(t＝3.251，3.363，3.053，3.242，3.674，4.264；P<0.05)。结论:AQP1过表达能够促进HCE细胞增殖和迁移，抑制细胞凋亡，并参与维持HCE细胞功能，其作用机制可能与抑制p38 MAPK信号通路激活有关。
Abstract
Objective:The aim of this study is to investigate the effects of aquaporin 1 (AQP1) on proliferation, migration and function maintenance of human corneal endothelial (HCE) cells and its related mechanisms.Methods:pcDNA3.1 as a carrier, AQP1 overexpression plasmid (pcDNA3.1-AQP1), empty control plasmid (pcDNA3.1-NC), AQP1 small interfering ribonucleic acid sequence (siRNA-AQP1), and control sequence (siRNA-NC) were transfected into HCE cells, respectively. According to transfection types, cells were divided into non transfection control group (Control group), pcDNA3.1-AQP1 group, pcDNA3.1-NC group, siRNA-AQP1 group, and siRNA-NC group. The effect of AQP1 on the proliferation, migration, and apoptosis of HCE cells were evaluated.The expression of AQP1 mRNA in cells was detected by qRT-PCR. The ability of cell proliferation was detected by EdU assay. The cell migration ability was detected by Transwell chamber method. The apoptosis was detected by flow cytometry.To clarify the role of p38 mitogen activated protein kinase (p38 MAPK) signaling pathways in its mechanisms, p38 MAPK activator 10 μmol/L anisomycin was added to the pcDNA3.1-AQP1 group and called the pcDNA3.1-AQP1+ anisomycin group. The protein expression levels of human tight junction protein 1 (TJP1), sodium potassium triphosphate adenylase (Na+ /K+ -ATPase), p38 MAPK, and phosphorylated p38 MAPK in HCE cells were detected by Western blot.The relative expression level of AQP1 mRNA, cell proliferation activity, migration rate, apoptosis rate, the expression level of TJP1 and sodium potassium triphosphatase protein, and phosphorylation p38 MAPK/p38 MAPK ratio in each group of cells conformed to the homogeneity of variance and normal distribution, represented by ±s, and independent sample t-test was used for intergroup comparison.Results:The relative expression of AQP1 mRNA of Control group, pcDNA3.1-NC group, siRNA-NC group, pcDNA3.1-AQP1 group and siRNA-AQP1 group were (1.00±0.08), (1.05±0.07), (0.97±0.07), (2.14±0.12) and (0.33±0.02). Compared with the Control group, there were no both signifcant differences between pcDNA3.1-NC group and siRNA-NC group (t=0.653, 0.684; P>0.05), indicating that cells with pcDNA3.1-NC and siRNA-NC were not affected, thus other indices of these cells were not detected again. The cell proliferation activity, the cell migration ratio, the apoptosis rate, the expression TJP1, Na+ /K+ -ATPase protein, and the ratio of phosphorylated p38 MAPK/p38 MAPK of pcDNA3.1-AQP1 group were (63.31±7.35)%, (74.28±7.04)%, (3.64±1.48)%, (1.73±0.13), (2.04±0.15) and (0.18±0.02), respectively; those of siRNA-AQP1 group were(22.15±3.26)%, (35.73±3.86)%, (20.35±2.83)%, (0.23±0.02), (0.21±0.02) and (0.75±0.09), respectively; those of pcDNA3.1-AQP1+ anisomycin group were (52.35±4.38)%, (58.74±5.42), (6.73±0.53)%, (1.38±0.21), (1.18±0.19) and (0.33±0.03), respectively. There were signifcant differences in all indices between pcDNA3.1-AQP1 group and Control group (t=3.844, 3.534, 3.253, 4.253, 4.753, 4.321; P<0.05); between siRNA-AQP1 group and Control group (t=4.136, 3.741, 3.621, 3.426, 4.122, 4.894, 3.795; P<0.05); between pcDNA3.1-AQP1 group and pcDNA3.1-AQP1+ anisomycin group(t=3.251, 3.363, 3.053, 3.242, 3.674, 4.264; P<0.05).Conclusions:AQP1 overexpression can promote HCE cell proliferation and migration, inhibit apoptosis, and participate in the maintenance of HCE cell function, which may be relevant with the inhibition of p38 MAPK signaling pathway activation.
基金项目
新疆维吾尔自治区自然科学基金(2022D01C217)
万方数据