转染基质细胞衍生因子-1α基因预防显微血管吻合术后血栓形成

Local transfer of stromal cell-derived factor-1α gene inhibits microsurgical anastomotic thrombosis

目的 探讨局部转染基质细胞衍生因子-1α（SDF-1α）基因预防显微血管吻合术后血栓形成的效果. 方法 2014年6月至2015年4月,利用基因工程技术构建SDF-1α基因重组质粒.使用体质量300～350 g的SD大鼠60只,其中15只大鼠左下肢肌肉内分别注射重组SDF-1α质粒、空载质粒及生理盐水,注射前及注射后第3、7、14、28天,利用ELISA试剂盒检测血浆SDF-1α水平;另外45只大鼠随机分为3组,分别注射重组质粒、空载质粒及生理盐水.注射后第7天,将所有大鼠左侧股动脉挤压离断后行端端血管吻合.术后15 min、30 min及2h,利用通畅实验检测吻合口是否堵塞;术后2h取吻合口股动脉标本采用HE染色、扫描电镜检测血栓形成情况,RT-PCR检测相关基因转录水平. 结果 大鼠下肢局部注射重组SDF-1α质粒后外周血SDF-1α水平短暂升高,第7天达到高峰,第28天基本恢复正常水平.术后2h,重组质粒组通畅率（60％）高于空载质粒组及生理盐水组（均为20％）,差异具有统计学意义（P＜0.05）.苏木素-伊红（HE）染色显示重组质粒组血栓堵塞面积百分比为（52.00±36.27）％,小于[生理盐水组（84.30±22.59）％和质粒对照组[（85.40±18.93％）],差异有统计学意义（P＜0.05）.RT-PCR结果发现术后2h,重组质粒组内皮型一氧化氮合酶基因转录水平升高,差异有统计学意义（P＜0.05）.扫描电镜提示重组质粒组血栓明显少于对照组.结论 局部转染SDF-1α基因能够抑制显微血管吻合术后吻合口血栓形成,提高吻合口通畅率,可能与局部内皮型一氧化氮合酶转录水平增高有关.上述结果为显微外科血管吻合术后血栓的预防提供新的方法.

Objective To evaluate the effect of local transfer of stromal cell-derived factor-1α （SDF-1α） gene on inhibiting microsurgical anastomotic thrombosis in a crushing model of rat femoral artery.Methods From June,2014 to April, 2015, sixty Sprague-Dawley rats were used in this study.Plasmid DNA encoding SDF-1α gene, empty plasmid, or saline was injected into the left femoral muscles in 15 rats.Plasma level of SDF-1α was tested by ELISA at 3, 7, 14 and 28 days after injection.The other 45 rats were divided randomly into 3 groups.Plasmid DNA encoding SDF-1α, empty plasmid or saline was injected in each group respectively.Seven days after injection, the left femoral artery was crushed, transected and repaired by end-to-end microsurgical anastomosis in each rat.Vascular patency was assessed by milk test at 15, 30 and 120 minutes after reperfusion.Thrombosis formation was assessed using H＆amp;E staining and scanning electron microscopy.RT-PCR was used to detect the expression of endothelial nitric oxide synthase, thrombomodulin, yon Willebrand factor and tissue type plas minogen activator.Results The plasma level of SDF-1α increased transiently, and peaked at 7 days after injection （P 〈 0.05）.The patency rates in SDF-1α group were significantly higher than that in control groups at 120 minutes （20%, 20% and 60% in three groups respectively） （P 〈 0.05）.Treatment of SDF-1α significantly reduced the area of thrombotic occlusion compared with controls （84.30 ± 22.59%, 85.40 ± 18.93%, 52.00 ± 36.27% in saline, empty plasmid and SDF-1α groups, respectively） （P 〈 0.05）.RT-PCR results revealed that endothelial nitric oxide synthase was overexpressed in SDF-1α group than control groups （P 〈 0.05）.Conclusion The current study showed that local transfer of SDF-1α gene increases arterial patency and inhibits microsurgical anastomotic thrombosis in a crush model of rat femoral artery.This beneficial effect might be mediated through up-regulation of endothelial nitric oxide synthase.These findings provide a novel approach for inhibition of microsurgical anastomotic thrombosis.