基质金属蛋白酶组织抑制因子3基因转染血管平滑肌细胞移植对急性心肌梗死后早期心肌重塑的影响

目的研究基质金属蛋白酶组织抑制因子3（tissue inhibitor-3of matrix metalloproteinases,TIMP-3）基因转染血管平滑肌细胞（vascular smooth muscle cells,VSMCs）移植,对急性心肌梗死（acute myocardialinfarction,AMI）后早期心脏结构变化的影响,并探讨其可能的机制。方法取Wistar大鼠胸主动脉采用组织块贴壁法培养VSMCs。另取61只Wistar雌性大鼠建立左冠状动脉远端结扎的AMI动物模型,将存活的54只大鼠模型随机分为三组,每组18只。于冠状动脉结扎后立即向缺血部心室壁内注射含有1×10^6个TIMP-3基因转染VSMCs（A组）、1×10^6个VSMCs（B组）或不含细胞的PBS液（C组）各0.5ml。术后1周,进行心脏形态学检测观察大鼠心脏结构变化,免疫组织化学染色验证TIMP-3基因转染VSMCs在缺血心肌中的存活情况,Western blot法测定大鼠缺血心肌TIMP-3蛋白和基质金属蛋白酶9（matrix metalloproteinase9,MMP-9）蛋白的含量。结果成功分离、培养VSMCs,纯度达98%,TIMP-3基因成功转入VSMCs中。术后1周,A组大鼠梗死心肌面积占左室游离壁面积的百分比为28.73%±1.56%,小于B组39.63%±1.84%和C组46.32%±2.16%（P〈0.01）;B组小于C组（P〈0.01）。A组大鼠左室容积指数为5.27±0.21mm3/g,小于B组6.69±0.34mm3/g和C组9.67±0.88mm3/g（P〈0.01）;B组小于C组（P〈0.01）。免疫组织化学染色见TIMP-3基因转染VSMCs被成功植入缺血心肌并在其中存活。Western blot法示A组大鼠缺血心肌TIMP-3蛋白含量为300704.8±3692.8,高于B、C组的195548.8±3014.2及177991.1±2502.1（P〈0.01）;B组高于C组（P〈0.01）。A组MMP-9蛋白含量为594827.4±5708.5,低于B、C组的921461.4±8887.4及1044445.0±8788.6（P〈0.01）;B组低于C组（P〈0.01）。结论将TIMP-3基因转染的VSMCs移植入心肌缺血区可明显抑制AMI后早期心肌重塑。     

利用人骨髓基质干细胞构建组织工程心脏瓣膜的体外实验

BACKGROUND:Nowadays, mechanical or biological valve recipients used in the clinic are still at the risk of infection, hemorrhage, thrombosis and reoperation owing to valve stenosis. Tissue-engineered heart valve with biological activity can overcome the disadvantages above. While, the optimal choice of scaffolds and seeding cells remains disputable. OBJECTIVE:To explore the feasibility to construct tissue-engineered heart valve with acellularized porcine aortic valve scaffold and human bone marrow stromal stem cells in vitro. METHODS:The porcine aortic valves were decellularized with the detergent and enzymatic extraction process to remove the cellular components. Human bone marrow stromal stem cells were aspirated from sternum of the patients with simple congenital heart malformation, and then the cells were seeded on the acellularized porcine aortic valve scaffold and cultured for 5 days. RESULTS AND CONCLUSION:Flow cytometry identified that the characteristics of surface antigen of the inoculated seed cells were in line with those of human bone marrow stromal stem cells. Light microscopy and electron microscopy confirmed that the cellular components in the porcine aortic valves could be removed to obtain the complete acellular fiber mesh stent. There was no significant difference in biomechanical property between before and after acellularization. The human bone marrow stromal stem cells implanted on the acellularized porcine aortic valve scaffold could form a continuous cell layer on the surfaces of the scaffold. The inoculated bone marrow stromal stem cells could be differentiated into fibroblasts. The implantation of human bone marrow stromal stem cells on the acellularized porcine aortic valve scaffold can construct the tissue-engineered heart valve.     

高龄和青年大鼠骨髓间质细胞移植对缺血心脏心功能的影响

目的　探讨高龄和青年大鼠自体骨髓间质细胞移植是否能改善心功能。方法　从高龄和青年大鼠中获取骨髓间质细胞 ,部分细胞培养后克隆计数 ,其余培养时加以诱导。冠状动脉结扎后制作同源大鼠心肌梗死模型 ,3周后分为高龄细胞移植组、青年细胞移植组和对照组。 5周后再次行心脏超声检查 ,以及检查细胞存活和疤痕区血管增生情况。结果　与青年组相比 ,高龄组骨髓间质干细胞数目以及体外诱导后结蛋白表达率明显下降。移植的高龄细胞可以在疤痕区存活 ,诱导血管新生 ,但未发现心功能改善。结论　高龄大鼠骨髓间质细胞移植未能发现可以改善梗死心肌的心功能 ,可能由于高龄骨髓间质细胞缺乏足够的干细胞数目和较低的诱导分化率。