Construction of tissue engineering adult cardiac myocytes

doi:10.3969/j.issn.2095-4344.2014.20.003

Abstract

Abstract:

BACKGROUND: Construction of tissue engineering adult cardiac myocytes has been a new research hotspot in cardiovascular fields.
OBJECTIVE: To explore a simple, fast method for the separation of adult rat cardiomyocytes, and preliminarily explore the construction methods of tissue engineering adult cardiac myocytes.
METHODS: Segmented enzymatic digestion method was used to isolate cardiac myocytes from adult rats. Subsequently, cardiac myocytes were transfected with adenovirus and liposome-mediated red fluorescent protein gene. Construction efficiency of tissue engineering cells was qualified using inverted fluorescence microscopy and flow cytometry. Finally, cardiac myocytes were transfected with adenovirus-mediated hypoxia-inducible factor-1a, and the expression of hypoxia-inducible factor-1a was examined by western blot analysis.
RESULTS AND CONCLUSION: A lot of cardiac myocytes were collected using the segmented enzyme digestion method. Flow cytometric analysis showed that the survival of adult cardiomyocytes was (87.03±0.70)%. Compared with liposomal transfection (transfection efficiency was 0), adenovirus infection efficiency was (70.31± 1.39)%, and the cells expressed red fluorescence under fluorescence microscope. After 4 days of adenovirus infection, transfected cells expressed hypoxia-inducible factor-1a protein. These results showed that segmented enzyme digestion is a fast way to isolate adult cardiac myocytes, and recombinant adenovirus vector is a good vector to transfect cardiac myocytes.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: myocytes, cardiac, adenoviruses, human, hypoxia-inducible factor 1, alpha subunit, anoxia, tissue engineering

CLC Number:

R318

Bai Chen-guang, Liu Xiao-hong. Construction of tissue engineering adult cardiac myocytes[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(20): 3127-3132.

Figures/Tables 2

2.1 成年大鼠心肌细胞的形态倒置显微镜下，新分离的成年大鼠心室肌细胞呈杆状或矩形，横纹清晰，菱角分明，长宽比为4-5∶1。当细胞贴壁后，苏木精-伊红染色可见明显的细胞横纹(图1)。 2.2 细胞存活效率由于活性细胞的细胞膜完整，所以PI不能进入细胞内而拒染。死亡或损伤严重的心肌细胞则由于细胞膜缺乏完整性，为PI所着色。根据流式细胞检测结果，实验观察到应用此分离方法可获得(87.03±0.70)%的活性成年大鼠成年心肌细胞(图2)。 2.3 组织工程化成年心肌细胞的构建效率实验中制备的重组腺病毒以MOI值为50感染大鼠心肌细胞后，细胞未出现肿胀、漂浮、死亡等病态反应；在荧光倒置显微镜下，携带RFP的心肌细胞发出红色的荧光(图3A)；流式细胞仪检测结果显示，当MOI值为50时，红色荧光细胞的百分比为(70.31±1.39)%(图3C)。说明重组腺病毒体外转染效率高，目的基因能得以有效地表达。按照Lipofectamine 2000脂质体转染说明书转染pcDNA3.0质粒，在荧光倒置显微镜下观察细胞转染效率，一直到第5天，均未见表达红色荧光的细胞(图3B)；流式细胞仪检测结果亦显示红色荧光细胞比例为0(图3D)，与Ad-RFP转染组相比，差异有显著性意义(P < 0.05)。 2.4 组织工程化成熟心肌细胞构建为观察腺病毒转染的基因在成熟心肌细胞中是否能够有效表达，采用Western blot检测缺氧诱导因子1a蛋白的表达。如图4所示，当心肌细胞感染Ad-HIF-1a后，可大量表达Mr 25 000的缺氧诱导因子1a，而当心肌细胞感染Ad-GFP后，未有目标蛋白表达，说明重组腺病毒体外转染心肌细胞后，可有效表达目标蛋白，提示携带缺氧诱导因子1a基因的组织工程细胞构建成功。"

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