新生树鼩心肌细胞与心肌成纤维细胞的分离培养、纯化及鉴定

Isolation, primary culture and purification of neonatal tree shrew cardiac fibroblasts and cardiac myocytes

目的研究应用树鼩建立人类心血管病理、毒理细胞模型的纯度较高、活力较强的心肌细胞和心肌成纤维细胞的方法。方法使用差速贴壁1．5h纯化心肌细胞进行培养，并比较5-溴-2-脱氧尿苷、阿糖胞苷及TGF—β抑制剂的纯化效率以获取最适纯化方案，培养过程在37℃、5％CO2条件下进行。使用MTT法测定细胞活力以确定方法可行性。利用α-横纹肌肌钙蛋白（α-SA）及心肌肌钙蛋白I（TnI）对树鼩心肌细胞进行免疫组化及免疫荧光鉴定，利用波性蛋白（Vimentin）对树购心肌成纤维细胞进行免疫荧光染色以达到鉴定目的。结果原代培养新生树鼩心肌细胞生长良好。在倒置显微镜下观察，培养10d左右的心肌细胞可以基本完成局部融合并伴有细胞搏动。MTT法检测结果显示，心肌细胞活力可以在最少15d内保持良好水平。细胞鉴定结果显示，树鼩心肌细胞α—SA免疫组化呈阳性，TnI免疫荧光鉴定呈阳性，心肌成纤维细胞呈阴性，符合一般心肌细胞特征；心肌成纤维Vimentin免疫荧光结果呈阳性。结论分离纯化得到的树鼢心肌细胞和心肌成纤维细胞纯度较高、结构完整并具有相对较高的细胞活力，是一种较为理想的、可以应用于树鼩建立人类心血管病理、毒理细胞模型的心肌细胞和心肌成纤维细胞。

Objective To explore the effective culture conditions and methods of primaryneonatal tree shrew eardiomyocytes, to culture cardiac cells with high purity, and long survival time. Methods Culturing the cardiomyocytes after purified by the method of different speed adherence for 1.5 hour at 37℃ under 5% CO2 con- ditions, then comparing the purification efficiency of bromo-2-deoxy-uridine, cytarabine and TGF-β inhibitor to obtain optimum purification protocol. Cell viability was measured using the MTT assay to determine the feasibility of the method. Immunofluorescence used α-SA and TnI antibody in order to achieve the purpose of identification for cardiomyoeytes. Immunofluorescence used Vimentin antibody in order to achieve the purpose of identification for cardiac fibroblasts. Results Neonatal tree shrew cardiomyocytes in primary cultured grow well. When observed under inverted microscope, the cells which cultured for 10 days showd connection and began tobeat spontaneously. The MTT assay results showed that the detection of myocardial viability can maintain a good level in 15 d at least. Tree shrew Cardiomyocytes by immunofluorescence staining of α-SA and TnI monoclonal antibodies showed positive.Tree shrew Cardiac fibroblasts by immunofluoreseence staining of Vimentin monoclonal antibodies showed positive. Conclusion Cardiomyocyteswhich can survive for a long time with high purity can be obtained by this method, is a stable and reliable method of primary cultured tree shrews cardiomyocytes.