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目的:探讨E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)在高糖引起的人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVECs)损伤中的作用,为寻找糖尿病血管病变新的治疗靶点提供实验依据。方法:采用胶原酶消化法分离原代HUVECs,并用内皮细胞标志物CD31免疫荧光染色进行鉴定。分别用含有5.5 mmol/l葡萄糖(正常糖对照组)、5.5 mmol/l葡萄糖+27.5 mmol/l甘露醇(渗透压对照组)或33 mmol/l葡萄糖(高糖组)的培养液培养HUVECs 48 h。Western Blot检测剪切体caspase-3表达;Annexin V/PI双染后流式细胞术检测细胞凋亡。通过感染表达CREG基因的腺病毒获得CREG过表达的HUVECs,Western Blot及流式细胞术评价CREG过表达对HUVECs凋亡的影响。结果:高糖处理48 h后,HUVECs内剪切体caspase-3的蛋白表达增加,细胞凋亡率增加;过表达CREG后,高糖处理的HUVECs内剪切体Caspase-3表达和凋亡细胞比例均明显降低,但仍高于正常糖对照组。结论:CREG过表达可抑制高糖引起的HUVECs凋亡。
Objective: To investigate the role of E1A-stimulated genes repressor (CREG) in the injury of human umbilical vein endothelial cells (HUVECs) induced by high glucose, Therapeutic targets provide experimental evidence. Methods: Primary HUVECs were isolated by collagenase digestion and identified by endothelial cell marker CD31 immunofluorescence staining. HUVECs 48 were cultured in a medium containing 5.5 mmol / l glucose (normal sugar control), 5.5 mmol / l glucose + 27.5 mmol / l mannitol (osmotic control) or 33 mmol / l glucose h The expression of caspase-3 was detected by Western Blot and the apoptosis was detected by flow cytometry with Annexin V / PI double staining. HUVECs overexpressing CREG were infected with adenovirus expressing CREG gene. Western Blot and flow cytometry were used to evaluate the effect of overexpression of CREG on apoptosis of HUVECs. RESULTS: After 48 h of high glucose treatment, the protein expression of caspase-3 in HUVECs was increased and the apoptosis rate was increased. After over-expression of CREG, the expression of Caspase-3 in spliced HUVECs and the expression of apoptotic cells The proportion was significantly lower, but still higher than the normal sugar control group. Conclusion: Overexpression of CREG can inhibit the apoptosis of HUVECs induced by high glucose.