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。
ObjectiveTo 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.
MethodsThe 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.
ResultsThe 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 lower in model group than in blank control group (all at P<0.05). The relative expression of p-EGFR protein was significantly higher in shGALNT2 group than in model group (all at P<0.05).
ConclusionsKnocking down GALNT2 can improve the proliferative ability of HRCECs under high glucose culture and reduce apoptosis, which may be related to the activation of EGFR signaling pathway.
孙天洋,格日勒图,张玉凤,等. 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 [J]. 中华实验眼科杂志,2023,41(09)：846-853.
DOI:10.3760/cma.j.cn115989-20221207-00576版权归中华医学会所有。
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孙天洋：实验操作、论文撰写；张玉凤、李春宇、金琳、包岭君、王佳乐：数据整理、统计分析；格日勒图：实验操作、论文撰写、论文智力性内容审阅及修改
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