敲低GALNT2基因对高糖培养的人视网膜血管内皮细胞凋亡的抑制作用及其机制  

Inhibitory effect of GALNT2 gene knockdown on apoptosis of human retinal vascular endothelial cells in high glucose culture and its mechanism

作　　者：孙天洋 格日勒图 张玉凤 李春宇 金琳 包岭君 王佳乐 Sun Tianyang;Geriletu;Zhang Yufeng;Li Chunyu;Jin Lin;Bao Lingjun;Wang Jiale(Graduate School,Baotou Medical College,Inner Mongolia University of Science&Technology,Baotou 014060,China;Department of Ophthalmology,Inner Mongolia Autonomous Region People's Hospital,Hohhot 010017,China;Department of Ophthalmology,The Second Norman Bethune Hospital of Jilin University,Changchun 130012,China;Inner Mongolia Medical College of Inner Mongolia Medical University,Hohhot 010110,China)

机构地区：[1]内蒙古科技大学包头医学院研究生院,包头014060 [2]内蒙古自治区人民医院眼科,呼和浩特010017 [3]长春市吉林大学白求恩第二医院眼科,长春130012 [4]内蒙古医科大学内蒙古临床医学院,呼和浩特010110

出　　处：《中华实验眼科杂志》2023年第9期846-853,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：内蒙古自治区自然科学基金项目(2021LHMS08064)。

摘　　要：目的探讨敲低多肽N-乙酰半乳糖胺转移酶2(GALNT2)对高糖环境中人视网膜血管内皮细胞(HRCECs)增生及凋亡的影响及可能的作用机制。方法以GALNT2基因为模板构建小发夹RNA(shRNA)干扰慢病毒载体。将HRCECs分为空白对照组、模型组、NC-shGALNT2组和shGALNT2组,分别于含5.5 mmol/L葡萄糖、25 mmol/L葡萄糖、shGALNT2阴性对照病毒+25 mmol/L葡萄糖和shGALNT2敲低病毒+25 mmol/L葡萄糖培养基中培养24 h。采用实时荧光定量PCR法检测各组细胞中GALNT2 mRNA相对表达量;采用Western blot法检测各组细胞中GALNT2、表皮生长因子(EGF)、EGF受体(EGFR)、磷酸化EGFR(p-EGFR)蛋白相对表达量;采用细胞计数试剂盒8(CCK8)法检测各组细胞增生值;采用流式细胞术检测各组细胞凋亡率。结果模型组GALNT2 mRNA及蛋白相对表达量明显高于空白对照组,shGALNT2组GALNT2 mRNA及蛋白相对表达量明显低于空白对照组,差异均有统计学意义(均P<0.05)。模型组中细胞增生值较空白对照组明显降低,shGALNT2组中细胞增生值较模型组和NC-shGALNT2组明显升高,差异均有统计学意义(均P<0.05)。空白对照组、模型组、NC-shGALNT2组和shGALNT2组细胞凋亡率分别为(4.73±0.26)%、(8.66±0.25)%、(9.26±1.12)%和(5.47±0.18)%,总体比较差异有统计学意义(F=342.921,P<0.001),其中模型组细胞凋亡率明显高于空白对照组,shGALNT2组细胞凋亡率明显低于模型组和NC-shGALNT2组,差异均有统计学意义(均P<0.05)。模型组中EGFR蛋白相对表达量较空白对照组明显升高,p-EGFR蛋白相对表达量较空白对照组明显降低,差异均有统计学意义(均P<0.05)。shGALNT2组中p-EGFR蛋白相对表达量较模型组明显升高,差异均有统计学意义(均P<0.05)。结论敲低GALNT2可以提高高糖培养下HRCECs的增生能力,减少HRCECs凋亡,其可能与EGFR信号通路的激活有关。Objective To investigate the effect of polypeptide N-acetylgalactosaminaminyltransferase 2(GALNT2)on the proliferation and apoptosis of human retinal vascular endothelial cells(HRCECs)cultured in high glucose and its possible mechanism.Methods The small hairpin RNA(shRNA)targeting GALNT2 gene was constructed to interfere with the lentiviral vector and infect HRCECs.HRCECs were divided into blank control group,model group,NC-shGALNT2 group and shGALNT2 group,which were cultured in medium containing 5.5 mmol/L glucose,25 mmol/L glucose,shGALNT2 negative control virus 25 mmol/L glucose and shGALNT2 knockdown virus 25 mmol/L glucose for 24 hours,respectively.The relative expression of GALNT2 mRNA in the four groups was detected by real-time fluorescence quantitative PCR.The relative expression levels of GALNT2,epidermal growth factor(EGF),EGF receptor(EGFR)and phosphorylated EGFR(p-EGFR)were detected by Western blot.The proliferative values of HRCECs were detected by cell counting kit-8 method.The apoptosis rate of different groups was detected by flow cytometry.Results The relative expression levels of GALNT2 mRNA and protein were significantly higher in model group than in blank control group,and were significantly lower in shGALNT2 group than in blank control group(all at P<0.05).The cell proliferation value was significantly lower in model group than in blank control group,and was significantly higher in shGALNT2 than in model group and NC-shGALNT2 group(all at P<0.05).The apoptosis rates of blank control group,model group,NC-shGALNT2 group and shGALNT2 group were(4.73±0.26)%,(8.66±0.25)%,(9.26±1.12)%and(5.47±0.18)%,respectively,with a significant overall difference(F=342.921,P<0.001).The apoptosis rate was significantly higher in model group than in blank control group,and was significantly lower in shGALNT2 group than in model group and NC-shGALNT2 group(all at P<0.05).The relative expression level of EGFR protein was significantly higher and the relative expression level of p-EGFR protein was significantly

关 键 词：糖尿病视网膜病变 多肽N-乙酰半乳糖胺转移酶2 人视网膜血管内皮细胞 表皮生长因子受体 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]