间充质干细胞移植实现AT2R基因在体可调控表达在再狭窄形成的作用及机制

The role of regulatable expression of AT2R gene in vivo on restenosis after vascular injury by Dox-on MSCs transplantation

目的研究间充质干细胞(mesenchymal stem cells,MSCs)细胞移植实现AT2R基因在体可调控表达在大鼠颈动脉损伤后新生内膜形成中的作用及机制。方法采用常规分子生物学方法连续两个回合转染体外培养的MSCs,获得受到强力霉素(Dox)调控的低背景、高诱导表达AT2R基因的双重稳定MSCs系;建立大鼠颈动脉球囊损伤动物模型,将双重稳定MSCs系种植于动脉损伤局部,通过尾静脉注射强力霉素,分别于术后14天、28天行病理切片,采用免疫组化法及RT-PCR等技术检测AT2R基因在新生内膜中的表达及其对新生内膜形成(intima/media ratio,I/M)及纤维连接蛋白(FN)的影响。结果成功建立低背景、高诱导表达AT2R基因的双重稳定MSCs系。该MSCs系受到Dox给予/去除的紧密调控,诱导后48h就可使AT2R明显表达,在Dox干预72h后AT2R表达进一步增强;这种表达的可诱导性至少在8周内维持稳定。免疫组织化学及RT-PCR检测显示:球囊损伤大鼠血管后14天及28天,Dox组新生内膜中AT2R的表达显著高于对照组、MSC组、MSC转染组(mRNAF=335.746;蛋白F=51.277,P<0.01),而其内膜/中膜面积比(I/M)较其他各手术组显著降低(F=72.417,P<0.01)。同时Dox组新生内膜层及中膜层中FN阳性染色较对照组、MSC组和MSC转染组显著减弱。结论血管损伤局部导入双重稳定MSCs系后AT2R表达受到Dox的良好调控,可有效抑制球囊损伤后大鼠颈动脉新生内膜增生。同时Dox诱导AT2R表达后,新生内膜中FN表达减少,可能是MSCs细胞移植实现AT2R基因在体可调控表达有效抑制新生内膜增生的机制之一。

Objective To evaluate the role of regulatable expression of AT2R gene in vivo on restenosis after vascular injury by Dox-on mesenchymal stem cells（MSCs）transplantation.Methods Two successive rounds of transfection of cultured MSCs were performed by conventional molecular biological methods.MSCs with low background expression and high deoxycycline（Dox）induced expression of AT2R gene were deemed double-stable MSCs.Animal models for balloon-induced carotid injury were established in rats,Double-stable MSCs were transplanted into the site of carotid injury,and Dox was injected via vena caudalis into rats.Pathological study was carried out at 14d and 28d after cell transplant.AT2R gene expression in the neointima and its effect on intima/media ratio（I/M）and protein expression of Fibronectin（FN）were analyzed by immunohistochemistry and RT-PCR.Results MSCs with low background expression and high Dox induced expression of AT2R gene were established successfully.Significant AT2R expression was observed after 48 h of induction and further increased after 72h and could keep up over 8 weeks.Immunohistochemistry and RT-PCR indicated that AT2R expression in the neointima of Dox group was significantly upregulated than control group,MSCs group and MSCs transfection group（mRNA F=335.746 and protein F=51.277,P0.01）,but the intima/tunica media ratio significantly decreased in Dox group than other groups（F=72.417,P0.01）.and also was the protein expression of FN in Dox group（P0.05）.Conclusion AT2R expression at the site of carotid injury were well controled by DOX and effectively suppressed rat carotid neointimal hyperplasia following balloon-induced injury.The decreased expression of FN in neointima may be one of mechanisms of increasing AT2R expression in injured vessels prevents vascular restenosis.