Effects of hypoxia inducible factor 1alpha gene modified cardiac stem cell transplantation on cardiac function and survival rate of transplanted cells in rats with myocardial infarction

doi:10.3969/j.issn.2095-4344.2017.01.019

Abstract

Abstract:

BACKGROUND: The biggest obstacle after stem cell transplantation is the extremely low survival rate of transplanted cells in the severe environments such as ischemia and hypoxia. In order to solve this problem, gene modification is expected to fundamentally improve the survival, metabolism and proliferation/differentiation of stem cells, namely by anti-apoptotic gene transfection or transduction prior to transplantation to vary the gene structure.
OBJECTIVE: To observe the survival rate of transplanted cells and cardiac function in rats undergoing hypoxia inducible factor-1α (HIF-1α) gene modified cardiac stem cell transplantation into the edge area of myocardial infarction.
METHODS: Twenty-four Sprague-Dawley rats were used to make myocardial infarction models by ligation of coronary arteries and then randomized into HIF-1α-transfected cell transplantation group, cell transplantation group and model group (n=8 per group). Two weeks after modeling, model rats were given the injection of Ad-HIF1α-eGFP-transfected cardiac stem cells, Ad-eGFP-transfected cardiac stem cells or PBS, respectively.
RESULTS AND CONCLUSION: Cardiac stem cell transplantation improved the rat cardiac function, and HIF-1α transfected cardiac stem cell transplantation showed better effects on cardiac function improvement than simple cell transplantation. Additionally, the improvement in left ventricular ejection fraction was associated with the density of cardiac stem cells in the marginal zone. To conclude, HIF-1α transfected cardiac stem cell transplantation can effectively improve the cardiac function in rats with myocardial infarction.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Myocardial Infarction, Hypoxia-Inducible Factor 1, alpha Subunit, Tissue Engineering

CLC Number:

R394.2

Li Sha, Li Shu-ren, Hao Qing-qing. Effects of hypoxia inducible factor 1alpha gene modified cardiac stem cell transplantation on cardiac function and survival rate of transplanted cells in rats with myocardial infarction[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 103-109.

Figures/Tables 2

2.1 培养细胞的生长情况及形态原代培养时接种两三天后单细胞散在贴壁，组织块初期仍游离在培养液中，四五天后开始贴壁，随后成纤维样细胞和多角形不规则细胞爬出，七八天后圆形、体积小、折光性强的细胞开始爬出(图1A)。原代培养的细胞经消化、接种进行传代培养，细胞容易生长，生长速度增快。传代细胞24 h即开始贴壁，六七天细胞80%-90%贴壁融合。光镜下可观察到体积小、圆形及折光性强的细胞，细胞数量增多。各种悬浮及贴壁的杂细胞数量减少，细胞形态主要以成纤维细胞样和折光性强的圆形小细胞为主，后者在传代培养中保持未分化状态(图1B)。 2.2 心肌干细胞的鉴定结果免疫荧光染色结果显示，培养的细胞主要为c-kit阳性心肌干细胞(图2)。 2.3 心肌干细胞的纯化结果流式细胞仪识别并收集c-kit阳性心肌干细胞，培养的细胞中心肌干细胞纯度在11%左右。纯化后心肌干细胞继续贴壁生长，少量心肌干细胞呈规则长梭形，向心肌细胞方向分化(图3)。 2.4 细胞转染效果选用生长状态良好的第4代心肌干细胞，病毒转染24-48 h后，荧光显微镜下可见带有绿色荧光蛋白标记的心肌干细胞，且细胞排列整齐，形态均一呈长梭状，经最佳MOI值为300(图4)。 2.5 实验动物数量分析 24只SD大鼠接受造模手术，术后共有20只存活。共死亡4只，缺氧诱导因子1α转染心肌干细胞组1只结扎前降支过程中导致气胸死亡，心肌干细胞组与缺氧诱导因子1α转染心肌干细胞组各1只于结扎前降支过程中诱发室颤死亡，模型组1只于二次开胸细胞移植后72 h死亡。 2.6 心脏超声影像结果造模前各组大鼠心功能指标正常，造模后2周各组大鼠左心室射血分数、左心室缩短分数、左心室舒张期末内径、左心室收缩末内径无明显差异(P < 0.05)；移植后1和2周，与模型组相比，缺氧诱导因子1α转染心肌干细胞组和心肌干细胞组大鼠左心室射血分数及左心室缩短分数均显著升高；而缺氧诱导因子1α转染心肌干细胞组与心肌干细胞组相比大鼠左心室射血分数及左心室缩短分数并无明显差别；移植后3和4周，与模型组相比，缺氧诱导因子1α转染心肌干细胞组和心肌干细胞组大鼠左心室射血分数及左心室缩短分数均显著升高；缺氧诱导因子1α转染心肌干细胞组与心肌干细胞组相比大鼠左心室射血分数及左心室缩短分数有显著差异；移植后4周，缺氧诱导因子1α转染心肌干细胞组与心肌干细胞组相比左心室舒张期末内径及左心室收缩末内径均明显降低，其中以缺氧诱导因子1α转染心肌干细胞组变化更为显著(P < 0.05；表1，图5)。 2.7 移植后心肌干细胞存活率荧光显微镜下，心肌干细胞存活密度最高处，取200倍视野中心肌干细胞数为心肌干细胞存活密度。缺氧诱导因子1α转染心肌干细胞组和心肌干细胞组可见绿色荧光蛋白标记的移植心肌干细胞(图6)，而模型组中未见绿色荧光蛋白标记的细胞。经统计学分析发现，缺氧诱导因子1α转染心肌干细胞组[(12.391± 1.881)/200倍视野]心肌干细胞存活密度显著高于心肌干细胞组[(6.756±1.181)/200倍视野](P < 0.05)，模型组未见到绿色荧光。梗死后左室射血分数的改善与梗死边缘区心肌干细胞存活密度呈正相关(r=0.853，P < 0.05)，回归方程为Y=-1.639+0.853X，其中Y代表左心室射血分数值，X代表心肌梗死边缘区心肌干细胞存活密度。 2.8 心肌病理学变化移植后4周，Masson染色显示梗死边缘区心肌细胞排列紊乱，红色心肌纤维部分断裂，纤维组织增生呈蓝色，细胞间可见多种炎性细胞浸润，梗死区正常心肌细胞形态消失，代之以成纤维细胞及粗大的胶原纤维束，排列疏松，其中以模型组胶原纤维粗大，排列杂乱；心肌干细胞组比缺氧诱导因子1α转染心肌干细胞组心肌细胞排列紊乱，红色心肌纤维断裂显著，细胞排列疏松；缺氧诱导因子1α转染心肌干细胞组红色心肌细胞在胶原纤维中散在再生(图7)。"

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