沉默GFAP基因调控高糖诱导的视网膜微血管内皮细胞增殖与凋亡

Silencing GFAP gene regulates the proliferation and apoptosis of high glucose-induced retinal microvascular endothelial cells

目的:研究沉默胶质纤维酸性蛋白(GFAP)基因对高糖诱导的人视网膜微血管内皮细胞(hRMECs)增殖和凋亡的影响及其机制。方法:采用qRT-PCR法检测高糖(30mmol/mL)和低糖(5mmol/mL)处理的hRMECs中GFAP的表达。通过慢病毒介导法建立GFAP基因稳定沉默的高糖诱导的hRMECs细胞模型,用SRI-011381(TGF-β信号通路特异性激活剂)、二甲基亚砜(DMSO)处理该细胞模型,采用Western blot法检测细胞中GFAP、转化生长因子-β1(TGF-β1)、转录激活因子2(Smad2)、Smad3蛋白的表达,并分别采用CCK-8法和流式细胞术检测细胞增殖和凋亡。结果:高糖处理的hRMECs中GFAP表达水平显著升高。通过慢病毒介导法可成功构建GFAP基因沉默的高糖诱导的hRMECs细胞模型,且沉默GFAP后,细胞的增殖能力明显提高,凋亡率明显抑制,TGF-β信号通路关键基因TGF-β1、Smad2、Smad3的蛋白表达均明显抑制,而激活TGF-β信号通路可逆转沉默GFAP对高糖诱导的hRMECs细胞的增殖促进和凋亡抑制作用。结论:沉默GFAP基因可促进高糖诱导的人视网膜微血管内皮细胞增殖,抑制细胞凋亡,其机制可能与TGF-β信号通路失活相关。

AIM:To investigate the effect of silencing glial fibrillary acidic protein gene(GFAP)on proliferation and apoptosis of high glucose-induced human retinal microvascular endothelial cells(hRMECs)and its mechanism.METHODS:Expression of GFAP in hRMECs treated with high sugar(30mmol/mL)and low sugar(5mmol/mL)was detected by qRT-PCR.The high glucose-induced hRMECs cells of silencing GFAP gene was established by lentiviral-mediated method.High glucose-induced hRMECs cells were treated with SRI-011381(TGF-βsignaling pathway activator)and dimethyl sulfoxide(DMSO);Expression of GFAP,transforming growth factor-1(TGF-β1),activating transcription factor2(Smad2),Smad3 proteins were measured by Western blot,and cell proliferation and apoptosis were detected by CCK-8 and flow cytometry,respectively.RESULTS:Expression of GFAP was significantly increased in high glucose treated hRMECs.The high glucose induced hRMECs cell model of GFAP gene silencing was successfully constructed by lentivirus mediation,and the cell proliferation ability was significantly improved,the apoptosis rate is significantly inhibited,and expression of TGF-1,Smad2 and Smad3 proteins in the TGF-βsignaling pathway was significantly inhibited after silencing GFAP,while activation of TGF-βsignaling pathway could reverse the inhibitory effect of silencing GFAP on the proliferation and apoptosis in high glucose hRMECs.CONCLUSION:Silencing GFAP gene can promote the proliferation of high glucoseinducedhuman retinal microvascular endothelial cells and inhibit cell apoptosis,the mechanism may be related to the inactivation of TGF-βsignaling pathway.