局部miR-126基因治疗促进小鼠缺血后肢血管新生的观察

The observation of Local miR-126 gene therapy to promote neovascularization in mouse ischemic hindlimb

目的:通过体外构建重组腺病毒mi R-126,应用于小鼠缺血后肢腓肠肌局部治疗,观察mi R-126对缺血局部具有促血管新生作用。方法:重组腺病毒mi R-126包装、纯化、滴度的鉴定;C57小鼠随机分为A组(C57左侧缺血后肢手术组)、B组(空病毒C57左侧缺血后肢手术组)、C组(重组腺病毒mi R-126 C57左侧缺血后肢手术组)三组,制作小鼠缺血后肢模型,术后即刻将重组腺病毒mi R-126和腺病毒各50μl局部注射于小鼠缺血左后肢腓肠肌。分别于3、7、14天各组(3只)取左后肢腓肠肌做HE染色、CD31免疫组化染色、west ern bl ot检测Akt、ERK1/2、pAkt、pERK1/2蛋白水平以及实时定量PCR检测等。结果:各种检测结果显示C组较A、B两组血管内皮细胞增生明显,新生血管数目计数明显增多,mi R-126表达水平明显增高,尤其在第7天升高最为明显,以及VEGF、bFGF等介导的I P3和MAPK信号通路中ERK1、pERK1、AKT和pAKT蛋白水平表达明显增高。结论:mi R-126局部应用于缺血后肢,通过激活Akt、ERK1/2的相关通路,促进血管新生,利于缺血后肢功能恢复。

Objective To investigate local Ad-miR-126 gene in vitro constructiong therapy enhance angiogenesis in mouse ischemic hindlimb. Methods Recombination Ad-miR-126 gene was constructed,packaged,purificated, and identificated of titer in vitro.Mice models of left ischemic hindlimb were established by ligation of left Femoral artery.Mice animals were randomly devided into A group （the left ischemic hindlimb operation group） ,B group （the adenovirus left ischemic hindlimb oeration group ）,C group （the recombination Ad-miR-126 left ischemic hindlimb oeration group）.The ischemic gastrocnemius muscles were injected 50 I^l/each mice with recombination Ad-miR-126 and adenovirus immediately after operation. HE dyeing,histological study,western blot and Real time quantitative PCR detection were performed at 3,7,14 days. Results Capillary density of the ischemic gastrocnemius muscles was significantly improved in the C group compared to the B groupand the A group in HE dyeing and CD31 histological study. A striking increase with the level of miR-126 in the C group was observed particularly at 7 clays when compared with the A group and the B group in Real time quantitative PCR detction. Western blot showed that ERK1, pERK1, AKT and pAKT gene which are important members of the IP3 and MAPK signal lines mediated by VEGF and bFGF ,were dramatically enhanced in the C group compared to the B group and the A group. Conclusion Local miR-126 gene therapy can improve ischemia-induced neovascularization,and be benificiate to the recover of ischemic hindlimb＇s function , which may be due to stimulation of the AKT/ERK-related pathway.