生胃酮对大鼠颈动脉损伤后新生内膜过度增殖的影响

1、生胃酮对大鼠颈动脉损伤后新生内膜过度增殖的影响         10-08-11 14:25:00     编辑：studa20             作者：宋明宝，于学军，崔馨，陈剑飞，卢来春，赵刚，于世勇，董红梅，黄岚【摘要】  目的：观察生胃酮对大鼠颈动脉损伤后新生内膜过度增殖的影响。方法：采用组织贴块法培养大鼠主动脉平滑肌细胞，Weste

2、rn blot检测生胃酮对大鼠主动脉平滑肌细胞缝隙连接蛋白43表达的影响。采用球囊损伤建立大鼠颈动脉损伤模型，对照组给予生理盐水2 mL、生胃酮组给予生胃酮3 mg/kg 腹腔注射，1次/d，HE染色观察新生内膜形成情况，免疫荧光染色检测新生内膜中缝隙连接蛋白43的表达，伊文思蓝DAPI双染色以评价新生内膜形成情况。结果：所培养的细胞抗SM actin免疫荧光染色阳性。50 mol/L生胃酮处理平滑肌细胞24 h后对缝隙连接蛋白43的表达无影响(0.85±0.06 vs 0.83±0.03，n=3，P>0.05)。球囊损伤大鼠颈动脉14 d后可见血管管腔狭窄、新生内膜

3、增生明显。免疫荧光染色显示形成的新生内膜中缝隙连接蛋白43表达丰富。经生胃酮干预后新生内膜增生明显减轻，新生内膜细胞计数显著低于对照组 (89±28.40 vs 236±15.04，n=5，P<0.01)。结论:缝隙连接在血管损伤后新生内膜形成过程中扮演重要角色，而缝隙连接阻断剂生胃酮能抑制新生内膜过度增生。 【关键词】  新生内膜;血管;缝隙连接;生胃酮;大鼠Abstract Objective: To observe the effect of carbenoxolone on neointimal hyperplasia after rat carot

4、id balloon injury. Methods: Rat aortic SMCs (RASMCs) were cultured by explanted rat aortic wall tissue and identified with cell immunofluorescence staining for SMactin. The expression of Cx43 in RASMCs which were treated with carbenoxolone at a concentration of 50 mol/L for 24 h was detected with we

5、stern blot. The model of vascular injury was established with rat carotid balloon injury. And animals were administrated with intraperitoneal injections of carbenoxolone 3 mg/(kg·d) in the carbenoxolone group or with saline (2 mL/d) in the control group for 2 weeks after carotid balloon injury.

6、 After 2 weeks, HE staining and DAPIEvens blue double staining were applied to evaluate the neointimal formation of targeted vessels. And cell immunofluorescence staining was used to detect protein Cx43 expression on targeted vessels. Results: RASCMCs were cultured successfully with positive immunof

7、luorescence staining for SM actin. In this experiment, we found that carbenoxolone couldnt induce the expression of Cx43 protein in RASMCs (0.85±0.06 vs 0.83±0.03, n=3, P>0.05). Two weeks after carotid balloon injury, carbenoxolone could significantly reduce the neointimal formation and

8、 the stenosis of blood vessel lumen. When nuclei number of neointima was used to evaluate the neointimal formation, result suggested that nuclei number of neointima in carbenoxolone group was significantly lower than that in the control group (89±28.40 vs 236±15.04， n=5， P<0.01). The ex

9、pression of Cx43 protein in neointima was abundant. Conclusions: Gap junctions play a key role in neointimal hyperplasia, and carbenoxolone, a special blocker of gap junction, can inhibit neointimal hyperplasia after rat carotid balloon injury.Key words Neointima; Vessel; Gap junction; Carbenoxolone

10、; Rat血管损伤后不良修复反应是血管损伤性疾病如儿童过敏性紫癜、动脉粥样硬化、血管成形术后再狭窄等的主要病理生理学基础，其中关键环节是血管平滑肌细胞由血管壁中层向内膜迁移并增殖，从而导致新生内膜形成并导致血管狭窄。抑制新生内膜过度增殖是血管损伤性疾病防治的重要策略。缝隙连接是相邻细胞间的膜通道结构，它由细胞膜上的连接子相互衔接而成。每个连接子包含六个相同哑铃形蛋白质亚单位缝隙连接蛋白(connexin, Cx)、连接子即Cx的六聚体所形成的跨膜蛋白通道1。该跨膜通道可允许小的信号分子和离子通过，在维持组织动态平衡、调节细胞生长、发育及分化中具有重要作用1。近年来研究发现，Cx在血管损伤性疾病的发生、发展过程中具有重要作用。本研究在大鼠颈动脉损伤模型的基础上探讨缝隙连接特异性阻断剂生胃酮对血管损伤后新生内膜过度增殖的影响。1 材料与方法1.1 材料肌动蛋白(SM actin)单克隆抗体购于美国Sigma公司;羊抗缝隙连接蛋白43多克隆抗体购于美国Santa Cruz公司; FITC标记的兔抗山羊IgG抗体购于北京中杉公司