裸鼹鼠乳鼠心脏成纤维细胞的分离、培养及鉴定

Isolation,Culture and Identification of Cardiac Fibroblasts from Suckling Rat of Naked Mole Rats

目的建立裸鼹鼠乳鼠心脏成纤维细胞的体外分离、培养及鉴定方法。方法选取出生1~3 d的裸鼹鼠乳鼠,在冰上分离出乳鼠心脏,用眼科剪将心脏剪成约1 mm×1 mm×1 mm小块,采用胰蛋白酶和II型胶原酶先后消化法分离心脏成纤维细胞。在光学显微镜下观察心脏成纤维细胞形态特征变化,CCK-8法检测细胞增殖活性,蛋白质印迹和免疫荧光法检测细胞波形蛋白表达情况。结果酶消化法分离的心脏成纤维细胞原代培养4 h已经完全贴壁,36 h即可铺满整个35 mm培养皿。传代培养2~3 d时心脏成纤维细胞增殖活力最强,蛋白质印迹和免疫荧光均证实细胞表达波形蛋白。结论胰蛋白酶和II型胶原酶先后消化法分离裸鼹鼠乳鼠心脏成纤维细胞,可获得活力好且纯度高的心脏成纤维细胞。

Objective To establish the method for the isolation,culture and identification of cardiac fibroblasts from naked mole rat suckling mice in vitro.Method Neonatal rat hearts were isolated on ice from naked mole rats born 1 to 3 days after birth.The hearts were cut into 1 mm×1 mm×1 mm pieces with ophthalmic scissors.Cardiac fibroblasts were isolated by digestion with trypsin and type II collagenase successively.Morphological characteristics of cardiac fibroblasts were observed under light microscope,proliferation activity was detected by CCK-8 assay,vimentin expression was detected by Western blot and immunofluorescence assay.Result Cardiac fibroblasts isolated by enzyme digestion were completely adherent in primary culture for 4 h,and the whole 35 mm dish could be covered in 36 h.The proliferation activity of cardiac fibroblasts was the highest after 2 to 3 days of subculture,and the expression of vimentin was confirmed by Western blot and immunofluorescence.Conclusion Cardiac fibroblasts isolated by trypsin and type II collagenase digestion can obtain cardiac fibroblasts with good viability and high purity.