不同钙离子浓度可通过内质网应激影响人腹膜间皮细胞上皮间质转化  

Different calcium ion concentrations affect epithelial mesenchymal transformation of human peritoneal mesothelial cells via endoplasmic reticulum stress

作　　者：郭宝珠 程锦绣[1] 靳鑫 贺玉桃 孙羡敏 Guo Baozhu;Cheng Jinxiu;Jin Xin;He Yutao;Sun Xianmin(Department of Nephrology,the First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,Hebei,China)

机构地区：[1]河北北方学院附属第一医院肾内科,河北张家口075000

出　　处：《中华危重病急救医学》2024年第1期50-55,共6页Chinese Critical Care Medicine

基　　金：河北省医学科学研究计划项目(20231411);河北省张家口市科技计划项目(2221090D)。

摘　　要：目的探讨不同钙离子浓度通过内质网应激(ERS)对人腹膜间皮细胞(HPMC)上皮间质转化(EMT)的影响。方法体外培养HPMC细胞株HMrSV5并分组处理,对照组、高钙1组、高钙2组分别用含1.25、1.75、2.25 mmol/L钙离子浓度的培养基处理,溶剂对照组用含1.25 mmol/L生理钙离子浓度及0.1%二甲基亚砜(DMSO)的培养基处理,高钙+溶剂组用含2.25 mmol/L钙离子浓度及0.1%DMSO的培养基处理,高钙+4-苯基丁酸(4-PBA)组用含2.25 mmol/L钙离子浓度及1 mmol/L ERS抑制剂4-PBA的培养基处理。各组处理48 h后,光镜下观察细胞形态学变化;采用免疫荧光染色观察细胞中上皮细胞表型标志蛋白紧密连接蛋白-1(ZO-1)和间质细胞表型标志蛋白α-平滑肌肌动蛋白(α-SMA)的表达;采用荧光定量聚合酶链反应(PCR)检测细胞中EMT标志基因E-钙黏蛋白(E-cadherin)、ZO-1、α-SMA和波形蛋白(Vimentin)的表达;采用蛋白质免疫印迹试验(Western blotting)检测细胞中ERS标志蛋白磷酸化蛋白激酶R样内质网激酶(p-PERK)、磷酸化真核翻译起始因子2α(p-eIF2α)、转录激活因子4(ATF4)和C/EBP同源蛋白(CHOP)的表达。结果与对照组比较,高钙1组和高钙2组HMrSV5细胞变细长、呈纤维化样改变,ZO-1的荧光强度增加、α-SMA的荧光强度减弱,提示细胞从上皮细胞向间质细胞转化;细胞中E-cadherin、ZO-1的mRNA表达明显降低,α-SMA、Vimentin的mRNA表达及p-PERK、p-eIF2α、ATF4、CHOP的蛋白表达明显升高,且高钙2组上述标志基因或蛋白表达较高钙1组变化更明显〔E-cadherin mRNA(2^(-ΔΔCt)):0.53±0.05比0.75±0.09,ZO-1 mRNA(2^(-ΔΔCt)):0.42±0.06比0.69±0.06,α-SMA mRNA(2^(-ΔΔCt)):1.81±0.16比1.32±0.14,Vimentin mRNA(2^(-ΔΔCt)):2.05±0.22比1.48±0.16,p-PERK蛋白(p-PERK/β-actin):0.81±0.09比0.59±0.06,p-eIF2α蛋白(p-eIF2α/β-actin):0.87±0.10比0.50±0.06,ATF4蛋白(ATF4/β-actin):0.93±0.10比0.72±0.06,CHOP蛋白(CHOP/β-actin):0.79±0.09比0.46±0.04,均P<0.05〕。与溶�Objective To study the effects of different calcium ion concentrations on epithelial mesenchymal transformation(EMT)of human peritoneal mesothelial cell(HPMC)via endoplasmic reticulum stress(ERS).Methods HPMC cell line HMrSV5 was cultured in vitro and treated in groups.The cells in the control group,high calcium group 1,and high calcium group 2 were treated with medium containing calcium ion concentrations of 1.25,1.75,and 2.25 mmol/L,respectively.The solvent control group was treated with medium containing 1.25 mmol/L physiological calcium ion concentration and 0.1%dimethyl sulfoxide(DMSO),the high calcium+solvent group was treated with medium containing 2.25 mmol/L calcium ion concentration and 0.1%DMSO,the high calcium+4-phenylbutyric acid(4-PBA)group was treated with medium containing 2.25 mmol/L calcium ion concentration and 1 mmol/L ERS inhibitor 4-PBA,and each group was treated for 48 hours.Morphological changes of cells in each group were observed under light microscope.The expressions of epithelial cell phenotype marker zonula occluden-1(ZO-1)and mesenchymal cell phenotype markerα-smooth muscle actin(α-SMA)in the cells were observed by immunofluorescence staining.The expressions of EMT marker genes E-cadherin,ZO-1,α-SMA and Vimentin were detected by fluorescence quantitative polymerase chain reaction(PCR).The expressions of ERS marker proteins phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK),phosphorylated eukaryotic initiation factor 2α(p-eIF2α),transcription activating factor 4(ATF4)and C/EBP homologous protein(CHOP)were detected by Western blotting.Results Compared with the control group,the morphology of HMrSV5 cells became slender and fibrotic,the fluorescence intensity of ZO-1 increased,and the fluorescence intensity ofα-SMA decreased in high calcium 1 and high calcium 2 groups,indicating that the cells transformed from epithelial cells to mesenchyme cells.The mRNA expressions of E-cadherin and ZO-1 were significantly decreased,while the mRNA expressions ofα-SMA and

关 键 词：腹膜透析 腹膜纤维化 人腹膜间皮细胞 上皮间质转化 钙离子浓度 内质网应激 

分 类 号：R692.5[医药卫生—泌尿科学]