人生长激素基因修饰成肌细胞移植对心肌梗死细胞凋亡及心功能的影响(英文)

Effects of human growth hormone gene-modified myoblast transplantation on myocardial cellular apoptosis and cardiac function in rats with myocardial infarction

背景:骨骼肌细胞移植于损伤心肌可以改善心脏功能,但大部分细胞在移植早期因损伤心肌的缺血及炎性氧化应激环境而发生死亡。研究证实通过细胞预处理或联合基因治疗可以改善移植细胞的生存。目的:探讨人生长激素基因修饰的成肌细胞移植对心肌梗死大鼠心肌细胞凋亡和心功能的影响。设计、时间及地点:细胞学体内实验,于2007-05/2008-12在山西省长治医学院和华中科技大学同济医学院附属协和医院完成。材料:SD大鼠30只,随机分为3组:人生长激素组、绿色荧光蛋白组、模型对照组,10只/组。另取1～3d龄SD乳鼠用于骨骼肌成肌细胞培养。pLghGHSN质粒、pLgGFPSN质粒由本实验室提供。方法:分别取携带人生长激素基因的pLghGHSN质粒和携带绿色荧光蛋白基因的对照质粒pLgGFPSN,用二步转染法转染E86和PT67包装细胞,G418筛选并扩增阳性克隆。各组大鼠均结扎左冠状动脉前降支制作心肌梗死模型,2周后人生长激素组将转染pLghGHSN的成肌细胞悬液150μL(含6×106个成肌细胞)分3点注射于心肌梗死区边缘,绿色荧光蛋白组同法注射等量转染pLgGFPSN的成肌细胞悬液,模型对照组注射等体积无血清培养基。主要观察指标:细胞移植4周后,超声心动图和血流动力学检查各组心功能变化;苏木精-伊红染色检测心肌梗死面积,天狼星红染色检测胶原纤维容积分数;Westernblot法检测心肌组织Bax,Bcl-2,人生长激素和caspase3蛋白的表达。结果:与模型对照组比较,人生长激素组左室腔变小,左室游离壁厚度增加,左室舒张末期内径和左室收缩末期内径均显著减小(P<0.05),短轴缩短率显著增加(P<0.05)。与绿色荧光蛋白组、模型对照组比较,人生长激素组大鼠左室压力最大上升速率、左室压力最大下降速率绝对值、左室收缩末压均显著升高(P<0.05),左室舒张末压显著降低(P<0.05);血清中类胰岛素样生长因子1质量浓度显著升高(P<0.05);心肌梗死面积明显减小(P<0.05);bax,caspase3蛋白表达水平降低,Bcl-2蛋白表达水平增加,人生长激素蛋白呈阳性表达。结论:人生长激素基因修饰的成肌细胞移植可以抑制心肌细胞凋亡,与单独成肌细胞移植相比能够更有效地缩小心肌梗死面积,更好地改善心肌梗死大鼠的心功能。

BACKGROUND： Skeletal myoblast transplantation can improve cardiac function, but most of cells exhibit apoptosis in the early stage of transplantation due to myocardial ischemia and inflammatory oxidative stress environment. Cell pretreatment or combined gene therapy has been shown to improve the survival of transplanted cells. OBJECTIVE： To investigate the effects of human growth hormone （hGH）-modified myoblast transplantation on myocardial cellular apoptosis and cardiac function in rats with myocardial infarction. DESIGN, TIME AND SETTING： A cytological experiment in vivo was performed in the Heping Hospital Affiliated to Changzhi Medical College ＆ Union Hospital Affiliated to Tongji Medical Collage, Huazhong University of Science and Technology between May 2007 and December 2008. MATERIALS： Thirty Sprague-Dawley （SD） rats were randomly divided into 3 groups （n = 10）： hGH, green fluorescent protein （GFP）, and control. An additional number of SD neonate rats aged 1-3 days were included for culture of myoblasts. Plasmids pLghGHSN and pLgGFPSN were provided by our laboratories. METHODS： Plasmid pLghGHSN carrying hGH gene and plasmid pLgGFPSN carrying GFP were used to respectively transfect E86 and PT67 packaging cells by a two-step transfection protocol, followed by G418 screening and positive clone amplification. Rat models of myocardial infarction were established in each group by ligating the left anterior descending branch coronary artery. Two weeks after myocardial infarction model induction, 150 μL pLghGHSN-transfected myoblast suspension （6× 10^6 myoblasts）, pLgGFPSN-transfected myoblast suspension, or serum-free culture medium was injected into the peripheral infarcted regions through 3 points in the hGH, GFP, and control groups, respectively. MAIN OUTCOME MEASURES： At 4 weeks after cell transplantation, cardiac function was examined by echocardiography and hemodynamics, myocardial infarct area was determined by hematoxylin-eosin staining, collagen volume fraction was detected by Sirius red staining, and expression levels of bax, bcl-2, hGH and caspase-3 protein in the myocardial tissue were detected by Western blot method. RESULTS： Compared with control group, left ventricular cavity became small, left ventricular free wall was thickened, left ventricular end diastolic diameter and left ventricular end systotic diameter were significantly reduced, and shortening fraction was significantly increased in the hGH group. The maximal change rate of left ventricular pressure rise or decline （±dp/dt max） and left ventricular end-systolic pressure were significantly increased （P 〈 0.05）, left ventricular end-diastolic pressure was significantly decreased （P 〈 0.05）, serum level of insulin-like growth factor 1 was significantly increased （P 〈 0.05）, myocardial infarct area was significantly reduced （P 〈 0.05）, protein levels of bax and caspase-3 were significantly decreased, but protein level of bcl-2 was significantly increased in the hGH group than in the GFP and control groups. In addition, hGH protein-positive expression was found in the hGH group. CONCLUSION： hGH gene-modified myoblast transplantation can inhibit myocardial cell apoptosis and it can more effectively reduce infarct area and better improve the cardiac function of rats with myocardial infarction than simple myoblast transplantation.