人心脏干细胞提取技术的研究

A new technology of extraction of human cardiac stem cells in vitro

目的研究体外分离、培养人左心耳c-kit+(CD117)心脏干细胞(c-kit+CSCs)的技术方法,为进一步研究心脏干细胞生物学特性、药物筛选和以后的临床应用提供技术支持。方法心脏手术中切取部分左心耳组织,经Ⅱ型胶原酶消化,接种培养、传代后进行流式细胞表面标志鉴定和无菌分选。结果通过单纯的酶消化方法可以成功地从人左心耳组织中分离出单个干细胞,流式细胞仪鉴定其表型为经典的心脏干细胞表型——c-kit+,多次传代后c-kit+持续高表达,无菌分选后可获得高纯度的c-kit+心脏干细胞。结论本研究技术可从人左心耳组织中分离培养出阳性率较高的c-kit+心脏干细胞,无菌分选后可获得高纯度的c-kit+心脏干细胞,进一步培养短期内可获得大量的用于实验和临床的c-kit+心脏干细胞。

Objective To isolate and culture human c-kit ~ cardiac stem ceils （c-kit ＋-CSCs） from the left atrial appendage in vitro for providing technical assistance for further research of biological characteristics, drug screening and clinical application of cardiac stem ceils. Methods Myocardial tissues obtained from the left atrial appendage of patients during surgery were digested with collagenase II. The isolated cells were inoculated in culture flask and passaged when they covered at the bottom of culture flask. Then, the cells were harvested for identification of surface marker and sterile sorting with flow cytometry. Results We can successfully isolate single stem cells from the atrial appendage of patients through the simple enzyme digestion, and demonstrate that the phenotype of these cells were classic heart stem cell marker--c-kit ＋ by flow cytometry. The percentages of c-kit ＋ cells continuously keep high expression during many times culture. We can obtain a large number of high purity of c-kit＋ cardiac stem cells through sterile sorting with flow cytometry. Conclusions We can isolate and culture high positive rate of c-kit＋ cardiac stem ceils from the atrial appendage of patients. A lot of c-kit ＋ cardiac stem cells with high purity were gained after sterile sorting, which can provide abundant c-kit＋ CSC in a short time for scientific research and clinical practice.