生胃酮對(duì)大鼠頸動(dòng)脈損傷后新生內(nèi)膜過度增殖的影響

1、生胃酮對(duì)大鼠頸動(dòng)脈損傷后新生內(nèi)膜過度增殖的影響         10-08-11 14:25:00     編輯：studa20             作者：宋明寶，于學(xué)軍，崔馨，陳劍飛，盧來春，趙剛，于世勇，董紅梅，黃嵐【摘要】  目的：觀察生胃酮對(duì)大鼠頸動(dòng)脈損傷后新生內(nèi)膜過度增殖的影響。方法：采用組織貼塊法培養(yǎng)大鼠主動(dòng)脈平滑肌細(xì)胞，Weste

2、rn blot檢測(cè)生胃酮對(duì)大鼠主動(dòng)脈平滑肌細(xì)胞縫隙連接蛋白43表達(dá)的影響。采用球囊損傷建立大鼠頸動(dòng)脈損傷模型，對(duì)照組給予生理鹽水2 mL、生胃酮組給予生胃酮3 mg/kg 腹腔注射，1次/d，HE染色觀察新生內(nèi)膜形成情況，免疫熒光染色檢測(cè)新生內(nèi)膜中縫隙連接蛋白43的表達(dá)，伊文思藍(lán)DAPI雙染色以評(píng)價(jià)新生內(nèi)膜形成情況。結(jié)果：所培養(yǎng)的細(xì)胞抗SM actin免疫熒光染色陽性。50 mol/L生胃酮處理平滑肌細(xì)胞24 h后對(duì)縫隙連接蛋白43的表達(dá)無影響(0.85±0.06 vs 0.83±0.03，n=3，P>0.05)。球囊損傷大鼠頸動(dòng)脈14 d后可見血管管腔狹窄、新生內(nèi)膜

3、增生明顯。免疫熒光染色顯示形成的新生內(nèi)膜中縫隙連接蛋白43表達(dá)豐富。經(jīng)生胃酮干預(yù)后新生內(nèi)膜增生明顯減輕，新生內(nèi)膜細(xì)胞計(jì)數(shù)顯著低于對(duì)照組 (89±28.40 vs 236±15.04，n=5，P<0.01)。結(jié)論:縫隙連接在血管損傷后新生內(nèi)膜形成過程中扮演重要角色，而縫隙連接阻斷劑生胃酮能抑制新生內(nèi)膜過度增生。 【關(guān)鍵詞】  新生內(nèi)膜;血管;縫隙連接;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血管損傷后不良修復(fù)反應(yīng)是血管損傷性疾病如兒童過敏性紫癜、動(dòng)脈粥樣硬化、血管成形術(shù)后再狹窄等的主要病理生理學(xué)基礎(chǔ)，其中關(guān)鍵環(huán)節(jié)是血管平滑肌細(xì)胞由血管壁中層向內(nèi)膜遷移并增殖，從而導(dǎo)致新生內(nèi)膜形成并導(dǎo)致血管狹窄。抑制新生內(nèi)膜過度增殖是血管損傷性疾病防治的重要策略。縫隙連接是相鄰細(xì)胞間的膜通道結(jié)構(gòu)，它由細(xì)胞膜上的連接子相互銜接而成。每個(gè)連接子包含六個(gè)相同啞鈴形蛋白質(zhì)亞單位縫隙連接蛋白(connexin, Cx)、連接子即Cx的六聚體所形成的跨膜蛋白通道1。該跨膜通道可允許小的信號(hào)分子和離子通過，在維持組織動(dòng)態(tài)平衡、調(diào)節(jié)細(xì)胞生長(zhǎng)、發(fā)育及分化中具有重要作用1。近年來研究發(fā)現(xiàn)，Cx在血管損傷性疾病的發(fā)生、發(fā)展過程中具有重要作用。本研究在大鼠頸動(dòng)脈損傷模型的基礎(chǔ)上探討縫隙連接特異性阻斷劑生胃酮對(duì)血管損傷后新生內(nèi)膜過度增殖的影響。1 材料與方法1.1 材料肌動(dòng)蛋白(SM actin)單克隆抗體購(gòu)于美國(guó)Sigma公司;羊抗縫隙連接蛋白43多克隆抗體購(gòu)于美國(guó)Santa Cruz公司; FITC標(biāo)記的兔抗山羊IgG抗體購(gòu)于北京中杉公司