MSCSHH心肌移植对大鼠心肌梗死后血管新生的作用机制研究

目的 探讨Sonic hedgehog（SHH）基因转染骨髓间充质干细胞并移植大鼠心梗区后,血管新生的变化及作用机制.方法 以结扎法制作大鼠急性心肌梗死模型,并按随机数字表法分为5组（每组40只）.分别在梗死周边部位移植BMMSCSHH（转染组）、等量的BMMSC（细胞组）、BMMSC和pcDNA3.1-Shh DNA的混合物（混合组）、单纯pcDNA3.1-Shh DNA质粒（基因组）、等容积的低糖DMEM培养基（对照组）.移植后第1、2、4、8周每组分别取10只为标本,qPCR检测移植后各组间移植部位Ptc1、Gli-2、COUP-TFⅡ等SHH信号传导通路下游基因以及血管内皮生长因子（VEGF）、Ang-1促血管再生因子的表达差异.结果 移植后第7天,转染组移植部位SHH下游基因Ptc1、Gli-2、COUP-TFⅡ表达分别较对照组、细胞组、基因组、混合组上调（Ptc1：P＜0.01,P＜0.01,P＜0.05,P＜0.05; COUP-TF Ⅱ：P＜0.01,P＜0.01,P＜0.05,P＜0.05;Gli-2：P＜0.01,P＜0.01,P＜0.05,P＜0.05）;转染组VEGF、Ang-1等促血管再生因子分别较对照组、细胞组、基因组表达上调（VEGF：P＜0.01,P＜0.05,P＜0.05;Ang-1：P＜0.01,P＜0.05,P＜0.05）,而与混合组比较差异无统计学意义,但有表达上升的趋势（VEGF：P＝0.147,Ang-1：P＝0.15）.而在后续第2、4、8周检测上述因子表达差异无统计学意义（P＞0.05）.结论 转染SHH基因后的骨髓间充质干细胞移植大鼠心梗区后通过上调其下游基因表达促进血管新生,其作用随时间的推移逐渐减弱.

Study on angiogenesis mechanism-induced by BMMSCSHH transplantation after myocardial infarction in rats

Objective To investigate the changes in and mechanism of angiogenesis in myocardium induced by transplantation of the sonic hedgehog （Shh） gene in transfected bone marrow mesenchymal stem cells （BMMSC） after myocardial infarction. Methods A rat model of acute myocardial infarction was made by coronary artery ligation. The rats were randomly divided into five groups （ n = 40 rats per group）. These were further subdivided into groups of 10 rats. The peripheral regions of the infarcts were injected with ei- ther BMMSCsHH （ transfection group）, equivalent BMMSC （ cell only group）, BMMSC and pcDNA3. 1-Shh DNA mixture （ mixture group）, peDNA3.1-shh DNA alone （gene only group）, or equal volumes of low-sugar DMEM medium （control group）. A the 1st, 2nd, 4th, and 8th week after transplantation, specimens were harvested from the transplantation site to determine the expression of SHH signaling pathway downstream genes Ptc1, Gli-2, COUP-TF II, angiogenesis promoting factor vascular endothelial growth factor （ VEGF）, and Ang-1 using polymerase chain reaction（PCR）. Results Seven days after transplantation, the expressiona of SHH sig- naling pathway downstream genes （Ptc1, Gli-2, and COUP-TF II） were significantly more pronounced in the transfection group than in the control group, cell only group, gene only group, or mixture group （ Ptcl ： P 〈 0. 01, P 〈 0. 01, P 〈 0.05, and P 〈 0. 05, respec- tively;COUP-TF II： P 〈0. 01, P 〈0. 01, P 〈0.05, and P 〈0.05, respectively; Gli-2： P 〈0.01, P 〈0. 01, P 〈0. 05, and P 〈 0. 05, respectively）. The expressions of angiogenesis-promoting genes Vegf and Ang-1 were significantly more pronounced than in the control group, cell only group, or gene only group （ Vegf： P 〈 0. 01, P 〈 0.05, P 〈 0. 05 respectively; Ang-1 ： P 〈 0.01, P 〈 0. 05,and P 〈0. 05, respectively）. There was a slight but not significant increase in expression relative to themixture group （Vegf： P = 0. 147, Ang-1： P = 0. 15）. Subsequent tests at the 2nd, 4th, and 8th week after transplantation showed no significant differences in the levels of expressions of these genes （ P 〉 0. 05）. Conclusions Transplantation of the shh-gene-transfected bone marrow mesen- chymal stem cells to the peripheral regions of myocardial infarcts promoted angiogenesis by upregulating downstream gene expressions. This effect was gradually weakened over time.