供心过表达微小RNA-499减轻心脏缺血再灌注损伤的研究

Over-expression of microRNA-499 within donor heart reduces cardiac ischemia-reperfusion injury

目的 观察供心过表达微小RNA-499 （miR-499）对心脏缺血再灌注损伤的保护作用.方法 采用聚合酶链反应（PCR）行miR-499转基因小鼠鉴定.分别获取野生型C57BL/6小鼠（WT）和同系（C57BL/6背景）miR-499转基因小鼠（Tg）来源的供心,均置于4℃生理盐水中保存16h,检测保存液中心肌酶谱：肌酸激酶（CK）和乳酸脱氢酶（LDH）的释放量.实验分为WT供心组和Tg供心组,建立同系小鼠腹部异位心脏移植模型,通过视诊和触诊观察不同小鼠供心经过冷保存,移植给相同受体后心功能的恢复,并于移植后24h获取供心,行苏木素-伊红（HE）染色,观察移植心脏组织病理学改变.结果 PCR结果显示miR-499转基因小鼠能检测到miR-499基因及相关载体片段,在图中呈现阳性条带;WT小鼠来源心脏的CK和LDH释放值分别为（5 043.0±523.8） U/L和（1 068.0±150.0）U/L,Tg小鼠来源心脏的CK和LDH释放值分别为（3 667.0±194.7） U/L和（684.0±19.1） U/L,Tg小鼠心脏在冷缺血过程中,两种心肌酶谱的释放值均明显低于对照组,差异有统计学意义（P＜0.05）.移植后相同时间点观测结果显示,WT小鼠来源心脏的临床功能评分明显低于Tg小鼠,15 min时的平均评分分别为0.9分和2.3分,24 h的平均评分分别为2.1分和3.4分（P＜0.05）.移植后24 h,WT小鼠来源供心有大量炎性细胞浸润并伴有心肌细胞坏死,而Tg小鼠供心的炎性细胞浸润和心肌细胞坏死较少（P＜0.05）.结论 供心过表达miR-499能减轻心脏在冷缺血和热灌注过程中造成的炎性损伤和心肌细胞凋亡,有利于再灌注后心功能的恢复,在心脏移植过程中具有保护作用.

Objective To explore the role of over-expression of microRNA-499 （miR-499） within donor heart against ischemia-reperfusion injury.Methods Polymerase chain reaction （PCR） was used to identify the miR-499 transgenic mice （Tg）.Donor hearts were obtained from wild-type C57BL/6 （WT） and Tg mice and stored in 4 ℃ saline for 16 h.The release of creatine kinase （CK） and lactate dehydrase （LDH） were detected.Both stored donor hearts were transplanted heterotopieally into syngeneie WT recipient mice.The recovery of graft function indicated by its contraction or beating,was assessed by direct palpation and inspection.The transplanted hearts were harvested at 24 h,then histopathology was observed by haematoxylin-eosin staining.Results Tg mice showed positive stripe,which highly expressed miR-499 gene.Donor hearts from Tg mice significantly reduced the release of CK and LDH as compared to WT donors （3 667.0±194.7） vs.（5 043.0±523.8） U/L; （684.2 ±19.1） vs.（1 068.0±150.0） U/L,respectively） and exhibited lower degree of perivascular inflammation,leukocyte infiltration and myocyte necrosis 24 h after transplantation.The scores of graft function for WT heart grafts were significantly lower than those for Tg heart grafts at the same time-points （0.9 vs.2.3 at 15 min and 2.1 vs.3.4 at 24 h） （P 〈 0.05）.Conclusion Over-expression of miR-499 within donor heart can attenuate inflammatory injury and myocardial cell apoptosis caused by cold ischemia and hot reperfusion,benefit for heart function recovery and have a protective role in heart transplantation.