血管生成素1基因修饰骨髓间质干细胞移植后心肌梗死大鼠的心功能变化

Changes in heart function in myocardial infarction rats following angiopoietin 1 gene modified bone marrow mesenchymal stem cell transplantation

背景:基因修饰的骨髓间质干细胞在表达自身特性的同时,可促进骨髓间质干细胞的分化并提高其存活率。以慢病毒为载体的血管生成素1(angiopoietin1,Ang1)基因转导的骨髓间充质干细胞移植对大鼠心肌梗死心功能的影响目前尚无报道。目的:探讨Ang1基因修饰的大鼠骨髓间质干细胞(rat mesenchymal stem cells,rMSCs)移植对急性心肌梗死后血管再生和心功能的影响。设计、时间及地点:随机对照动物实验,于2007-08/2009-08在福建医科大学附属第一医院中心实验室完成。材料:清洁级雄性近交系F344大鼠48只,由中科院上海实验动物中心提供。慢病毒载体质粒pNL-IRES2-EGFP、包装质粒pHELPER、包膜质粒pVSVG和293T细胞由美国杜兰大学陈一平教授惠赠;Ang1基因修饰质粒pNL-Ang1-IRES2-EGFP、rMSCs由福建省神经病学研究所张志坚教授惠赠。方法:包装、浓缩慢病毒,将携带Ang1基因慢病毒转导的rMSCs命名为Ang1-rMSCs,将未携带目的基因(空载体)慢病毒转导的rMSCs命名为Mock-rMSCs。48只大鼠结扎冠状动脉左前降支制作心肌梗死模型,2周后随机分为3组,分别于心肌梗死区注入5×1010L-1的Ang1-rMSCs,Mock-rMSCs,rMSCs。主要观察指标:RT-PCR检测Ang1 mRNA的表达,取心脏组织切片观察移植细胞的存活情况,通过超声心动图检测心功能,免疫荧光检测新生血管密度。结果:成功构建Ang1基因修饰的rMSCs,Ang1 mRNA在移植后28d仍稳定表达。移植后28d荧光显微镜下Ang1-rMSCs组和Mock-rMSCs组心脏组织冰冻切片均可见大量EGFP阳性细胞,而rMSCs组未见EGFP阳性细胞,即移植的Ang1-r MSCs存活率明显增高。与Mock-rMSCs、rMSCs组比较,移植后7,28dAng1-rMSCs组左室射血分数、短轴缩短速率均显著增加(P<0.05),心功能明显改善;心肌梗死区毛细血管数明显增多(P<0.05)。结论:Ang1基因修饰的rMSCs移植治疗大鼠心肌梗死后可促进血管再生,改善心功能。

BACKGROUND： Gene modified bone marrow mesenchymal stem cells （BMSCs） not only can express the gene characteristic, but also can promote the differentiate and survival rate of BMSCs. BMSCs modified angiopoietin-1 gene by the vehicle of lentiviral vector transplantation in the effect of rat heart function after myocardial infarction has not been reported. OBJECTIVE： To investigate the influence of grafted angiopoietin 1 （Ang1） gene modified rat BMSCs on angiogenesis and heart function following acute myocardial infarction. DESIGN, TIME AND SETTING： The randomized controlled animal study was performed at the Central Laboratory, First Affiliated Hospital, Fujian Medical University from August 2007 to August 2009. MATERIALS： A total of 48 clean male inbred line F344 rats were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. Lentivirus vector plasmid pNL-IRES2-EGFP, packaging plasmid pHELPER, and envelop plasmid pVSVG and 293T cells were gifted by Professor Chen Yi-ping from Tulane University, USA. Ang1 gene modified plasmids pNL-Ang1-IRES2-EGFP and rMSCs were presented by Professor Zhang 7hi-jian from Fujian Institute of Neurology. METHODS： Three plasmids pNL-Ang1-IRES2-EGFP （pNL-IRES2-EGFP）, pHELPER and pVSVG were cotransfected into 293T to package lentiviral vector particles. The supernatant was collected after transfection and was concentrated by centrifugation. Then BMSCs were transduced with Angl gene modified lentiviral vector （Angl-rMSCs） or lentiviral empty vector （Mock-rMSCs）. Myocardial infarction model was established by ligating the left anterior descending coronary artery of the 48 F344 rats. Two weeks after surgery, 3 groups were set up, and rats in each group were respectively injected with Ang1-rMSCs, Mock-rMSCs and rMSCs into the border zone surrounding the infarct （5×10^10/L）. MAIN OUTCOME MEASURES： RT-PCR was used to determine Angl mRNA expression. Survival rate of BMSCs in the heart section was observed. Echocardiogram analysis was utilized to detect rat heart function. Immunofluorescence was employed to measure angiogenesis density. RESULTS： Angl gene modified rMSCs were successfully obtained, and Angl mRNA was stably expressed at 28 days following transplantation. Under the fluorescence microscope, heart tissue frozen section was positive for EGFP in the Ang1-rMSCs and Mock-rMSCs groups, but negative in the rMSCs group at 28 days following transplantation. That is, the survival rate of Ang1-rMSCs significantly increased. Compared with the Mock-rMSCs and rMSCs groups, left ventricular ejection fraction and shortening fraction significantly increased in the Ang1-rMSCs group at 7 and 28 days following transplantation （P 〈 0.05）, and heart function was significantly improved. Capillary density around the infarct area significantly increased （P 〈 0.05）. CONCLUSION： Angl gene modified rMSCs may effectively promote angiogenesis and improve heart function in myocardial infarction rats.