Egr-1特异脱氧核酶调控大鼠血管平滑肌细胞Egr-1及PCNA的表达

The specific DNA enzyme targeting Egr-1 regulated the expression of Egr-1 and PCNA in rats' vascular smooth muscle cells

目的采用早期生长反应因子-1(Egr-1)特异脱氧核酶(ED5)转染大鼠主动脉平滑肌细胞,观察其对Egr-1及PCNA表达的影响,从而探讨ED5抑制血管平滑肌细胞增殖的作用机制。方法组织块贴壁法进行血管平滑肌细胞(VSMCs)原代培养,采用形态学观察和α-平滑肌肌动蛋白(α-SM-actin)免疫细胞化学染色进行细胞鉴定,然后对VSMCs转染ED5及ED5乱序杂码寡核苷酸(ED5SCR)。实验分三组:对照组、ED5组、ED5SCR组。观察转染后Egr-1及PCNA蛋白表达情况,并分析计算各组积分光密度值大小。结果成功完成VSMCs原代培养并转染ED5及ED5SCR。经10%胎牛血清刺激后,三组内Egr-1蛋白1h表达最强,随着时间的延长呈下降趋势;PCNA蛋白4h开始表达,24h到最高峰,之后略呈下降趋势。与对照组及ED5SCR组相比,ED5均在一定程度上抑制了Egr-1及PCNA的表达(P<0.05)。结论 ED5可以在一定程度上抑制VSMCs中Egr-1及PCNA的蛋白表达,进而抑制体外培养的VSMCs增殖,这种新的基因治疗方法将为动脉粥样硬化、再狭窄等血管增殖性疾病的治疗提供一种新的途径。

Objective The specific DNA enzyme(ED5) targeting the early growth response factor-1(Egr-1) was transfected into the vascular smooth muscle cells (VSMCs) of rats,to observe the expression changes of Egr-1 and proliferating cell nuclear antigen(PCNA)and explore the mechanism of ED5 inhibits VSMCs' proliferation.Methods Primary cultured VSMCs,which were identified by morphological observation and α-SM-actin immunocytochemical stain,were transfected with ED5 and ED5SCR.There were three groups:the Control group,ED5 group and ED5SCR group.We detected the protein levels of Egr-1 and PCNA,analyzed and calculated the integral light density values in each group.Results The primary VSMCs were cultured and then transfected with ED5 or ED5SCR.With the treatment of 10% fetal bovine serum,the expression of Egr-1 and PCNA were increased most one hour after transfection,and then decreased.They expressed from the fourth hour and reached the top at the twenty-fourth hour and then went down in PCNA group.The expression of Egr-1 and PCNA were inhibited to certain extent in compared with other groups in ED5 group(P<0.05).Conclusions The expression of Egr-1 and PCNA protein are inhibited in VSMCs after the transfection of ED5.Therefore it could inhibit VSMCs' proliferation in vitro culture.This new gene therapy approach may provide a new way for treatment of atherosclerosis and restenosis.