摘要
背景:采用成年心肌细胞构建组织工程细胞已成为心脏疾病领域新的科研热点。目的:探讨简单、快捷的成年大鼠心肌细胞分离方法,并初步探索组织工程化成年心肌细胞的构建方式。方法:采用分段式酶消化方法消化成年大鼠心室肌心肌细胞。分别转染腺病毒和脂质体介导的红色荧光蛋白基因,倒置荧光显微镜、流式细胞仪检测组织工程细胞的构建效率。转染腺病毒介导的缺氧诱导因子1a,采用Western blot检测目标蛋白的表达。结果与结论:采用分段式酶消化方法可获得大量心肌细胞。流式细胞分析表明所获得的成年心肌细胞的存活率为(87.03±0.70)%。与脂质体转染相比(转染效率为0),腺病毒感染效率高,为(70.31±1.39)%,荧光显微镜下细胞发出红色荧光。在腺病毒转染第4天后,可有效表达目标蛋白缺氧诱导因子1a。以上结果表明分段式酶消化法是成熟心肌细胞的快速分离方式,并明确重组腺病毒载体是转染心肌细胞的最佳基因载体。
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. Final y, 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 col ected 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.
出处
《中国组织工程研究》
CAS
CSCD
2014年第20期3127-3132,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金项目(30800229)~~
