钙离子ATP酶2a基因修饰骨髓间充质干细胞移植改善慢性心力衰竭大鼠的心功能

背景：目前仍缺乏有效手段修复心力衰竭后受损心肌，逆转心肌病理性重塑。作为一种新的治疗策略，用正常肌细胞和治疗性基因干预受损心肌正逐渐显示出其在改善心功能方面的优势。目的：观察腺病毒转染不同代骨髓间充质干细胞的有效性和稳定性。并以携带肌浆网钙离子ATP酶（sarcoplasmic reticulumCa（2＋）ATPase，SERCA2a）基因的腺病毒转染骨髓间充质干细胞，治疗心力衰竭大鼠，比较SERCA2a基因治疗，骨髓间充质干细胞移植以及骨髓干细胞基础的SERCA2a基因治疗慢性心力衰竭大鼠的效果。设计：随机对照实验。单位：解放军总医院老年心血管病科和北京医科大学生物化学系。材料：选取购于北京医科大学动物实验中心的4周龄雄性sD大鼠作为骨髓供体。选取体质量200～250g雌性成年SD大鼠作为细胞移植和基因治疗受体。以雄性大鼠Y染色体sry基因鉴定供体移植细胞是否在雌性大鼠受体心肌内存活。实验所用Ad—SERCa2a，Ad—EGFP的构建由我科鲁小春博士完成；第3和8代骨髓间充质干细胞自行分离培养。方法：实验于2004—07／2005-12在北京医科大学生物化学系周春燕实验室完成。对30只雌性SD大鼠进行左冠状动脉结扎，制作急性心肌梗死后慢性心力衰竭大鼠模型。将造模成功的29只大鼠随机分为4组：基因治疗组7只，干细胞移植治疗组7只，基因修饰的干细胞移植组8只，腺病毒空载体对照组7只，分别予以单纯SERCA2a基因、MSC移植、SERCA2a基因修饰的骨髓间充质干细胞移植及腺病毒空载体干预。分离培养大鼠骨髓间充质干细胞，用携带SERCA2a及绿色荧光蛋白的腺病毒（Ad-SERCA2a—GFP）转染第3和8代骨髓间充质干细胞。主要观察指标：采用流式细胞仪检测不同代骨髓间充质干细胞的Ad-SERCA2a—GFP转染率。分别在治疗前及治疗后14，21d采用超声心动图

Enhancement of cardiac function of chronic heart failure rats by marrow stromal cell-based sarcoplasmic reticulum Ca^2＋ adenosine triphosphatase gene therapy

BACKGROUND： There are still few effective methods to repair injured myocardium after myocardial failure and pathologically rebuild reverral myocardium. As a new therapy, normal myocytes and therapeutic gene to interfere injured myocardium have advantageous effects in improving heart function.
OBJECTIVE： To observe the efficiency and stability of adenovirus-medicated gene transferred into different passages of bone marrow mesenchymal stem cell （MSC） and investigate the effect of MSC-based sarcoplasmic reticulum Ca^2＋ ATPase gene （SERCA2a） gene therapy for rats with chronic heart failure. To compare the effects of gene therapy, cell transplantation and MSC-based SERCA2a gene therapy for chronic heart failure. DESIGN： Randomized controlled study.
SETTING： Department of Senile Angiocardiopathy, General Hospital of Chinese PLA; Department of Biochemistry, Beijing Medical University.
MATERIALS： Male Sprague-Dawley （SD） rats with 4 weeks old, clean grade and weighing 45-50 g provided by the Animal Experimental Center, Peking Medical University were used as donators of bone marrow. Other female SD rats of 12 weeks old, clean grade and weighing 200-250 g were used as receptors of cell transplantation and gene therapy. Sry gene of Y chromosome in male rats was used to evaluate whether transplanted cells of donators lived in myocardium of receptor rats. Ad-SERCa2a and Ad-EGFP were constructed by Doctor Lu Xiao-chun; MSC in the 3^rd and 8^th generations was isolating cultured on its own.
METHODS： The experiment was carried out in the Zhou CY Laboratory （BSL-2）, Department of Biochemistry, Beijing Medical University from July 2004 to December 2005. Thirty female SD rats received ligation at the left coronary artery to make models with chronic cardiac failure following acute myocardial infarction. And then, 29 rats were randomly divided into four groups, including gene therapy group （n=7）, MSC group （n=7）, gene-modified MSC group （n=8） and control group （n=7）. Rats in the f