腺病毒介导hRAMP1基因转染血管平滑肌细胞的增殖和凋亡

背景：外源性受体活性修饰蛋白1基因转染可能对血管平滑肌细胞增殖调控具有重要的作用。目的：探讨腺病毒载体介导人受体活性修饰蛋白1基因对体外培养的兔血管平滑肌细胞增殖和凋亡的影响。方法：分离培养获得兔主动脉血管平滑肌细胞,分别以携带人受体活性修饰蛋白1基因的腺病毒和空腺病毒转染后,分成人受体活性修饰蛋白1组、空病毒组和对照组。结果与结论：腺病毒转染细胞后随时间延长,报告基因绿色荧光蛋白表达逐渐增加,72 h表达最强,感染率达80%,96 h时仍有绿色荧光蛋白表达。人受体活性修饰蛋白1组的受体活性修饰蛋白1蛋白表达增加,血管平滑肌细胞凋亡率增加,细胞增殖明显抑制,与空病毒组和对照组比较差异有显著性意义(P < 0.05)。提示人受体活性修饰蛋白1基因感染血管平滑肌细胞后明显抑制血管平滑肌细胞增殖,促进细胞凋亡。

Effect of adenovirus mediated human RAMP1 gene on proliferation and apoptosis of vascular smooth muscle cells

BACKGROUND:Hyperproliferation of vascular smooth muscle cells (VSMCs) is one of the most important mechanisms of atherosclerosis and restenosis. Receptor activity modifying protein-1 (RAMP1) is receptor subunit of calcitonin gene related peptide (CGRP) and decides the activity of receptor. Exogenous RAMP1 could play an important role in the regulation of VSMCs proliferation.  OBJECTIVE:To investigate the effects of adenovirus mediated human RAMP1 (hRAMP1) gene on proliferation and apoptosis of VSMCs derived from rabbit thoracic aorta. METHODS:VSMCs, isolated from rabbit thoracic aorta, were divided into three groups: hRAMP1 group, empty-adenovirus vector group and control group according to whether transferring Ad-EGFP-hRAMP1 or Ad-EGFP, and each group included four subgroups of 24 hours, 48 hours, 72 hours and 96 hours. RAMP1 protein was detected by immunocytochemistry, and the proliferation of VSMCs was determined by cell counting and MTT assay, and the apoptosis of VSMCs was determined by flow cytometry and TUNEL assay.RESULTS AND CONCLUSION:VSMCs were successfully infected by Ad-EGFP-hRAMP1 or Ad-EGFP, and EGFP expression was increased, peaked at 72 hours and lasted for 96 hours. In the hRAMP1 group, the VSMCs showed hRAMP1 protein expression, and the activity of cell proliferation was significantly inhibited in the hRAMP1 group compared with empty adenovirus vector group and control group (P < 0.05). Also, the level of VSMCs apoptosis was significantly higher in the hRAMP1 group compared with other groups (P < 0.05). These results showed that the over-expression of hRAMP1 mediated by adenovirus vector plays an important role in inhibition of VSMCs proliferation and promotion of VSMCs apoptosis, which may be the therapeutic target of atherosclerosis and restenosis.