论文部分内容阅读
目的晚期糖基化终产物AGEs联合高糖(AGE-BSA+高糖)刺激人阴茎海绵体血管内皮细胞(HCECs)诱导糖尿病环境,观察其对内皮功能相[关的miR-1 55、miR-15a和miR-181c及其靶基因的表达变化影响。方法利用人阴茎海绵体分离鉴定的HCECs,随机分为两组:正常+BSA组(NC组)、AGE-BSA+高糖(DM组)。采用蛋白印迹实验检测RAGE蛋白表达确定诱导体外糖尿病环境;采用实时定量PCR来检测miR-155、miR-15a和miR-181c及对应靶基因eNOS、TXNIP、VEGFA、IRS1和KLF6的mRNA表达变化:采用蛋白印迹实验验证靶基因的蛋白表达变化。结果与NC组相比,DM组在AGE-BSA+高糖刺激下,RAGE基因的mRNA水平和蛋白水平均明显升高;同时观察到miR-155表达明显升高而miR-15a和miR-181c的表达明显降低。miR-155对应的靶基因eNOS的mRNA水平和蛋白水平明显降低;miR-15a对应的靶基因TXNIP的mRNA水平和蛋白水平明显升高,靶基因IRSI的mRNA水平明显升高但蛋白水平变化不明显,靶基因VEGFA的mRNA水平没有显著变化;miR-181c对应的靶基因KLF6的mRNA水平和蛋白表达水平明显升高,而靶基因IRS1的mRNA水平明显升高但蛋白水平变化不明显。结论 HCECs在AGE-BSA+高糖刺激的糖尿病环境下,miR-155、miR-1 5a和miR-181c均有显著的表达差异,并负调控它们对应的靶基因eNOS、TXNIP及KLF6发生不同程度的表达变化。结果提示糖尿病环境下的HCECs中多种miRNAs可能参与了糖尿病内皮功能障碍的发生发展过程,其具体的机制有待进一步深入研究。
Objective To investigate the effect of AGEs combined with high glucose (AGE-BSA + high glucose) on human corpus cavernosal endothelial cells (HCECs) induced diabetic environment and its effect on endothelial function [miR-1 55, miR-15a And miR-181c and its target gene expression changes. Methods HCECs isolated from human corpus cavernosum were randomly divided into two groups: normal + BSA group (NC group) and AGE-BSA + high glucose group (DM group). Western blotting was used to detect the expression of RAGE protein to induce the in vitro diabetic environment. Real-time quantitative PCR was used to detect the mRNA expression of miR-155, miR-15a and miR-181c and corresponding target genes eNOS, TXNIP, VEGFA, IRS1 and KLF6: Western blotting experiments to verify the target gene protein expression changes. Results Compared with NC group, the mRNA and protein levels of RAGE in DM group were significantly increased under the stimulation of AGE-BSA + high glucose, and the expression of miR-155 and miR-181c were significantly increased The expression was significantly reduced. The mRNA and protein levels of eNOS, a target gene of miR-155, were significantly decreased. The mRNA and protein levels of TXNIP, a target gene of miR-15a, were significantly increased. The mRNA level of target gene IRSI was significantly increased but the level of protein was not significantly changed , While the mRNA level of target gene VEGFA did not change significantly. The mRNA and protein expression of KLF6, a target gene of miR-181c, was significantly increased, while the mRNA level of target gene IRS1 was significantly increased but the protein level did not change significantly. Conclusions HCECs showed significant difference in the expression of miR-155, miR-1a and miR-181c in AGE-BSA + high glucose-stimulated diabetes mellitus, and negatively regulated their corresponding target genes eNOS, TXNIP and KLF6 to some extent Change of expression The results suggest that a variety of miRNAs in HCECs under diabetic conditions may be involved in the development and progression of diabetic endothelial dysfunction, and its specific mechanism needs further study.