乳小鼠心肌成纤维细胞和心肌细胞的分离培养及荧光鉴定

Isolation,primary culture and identification of cardiac fibroblasts and cardiac myocytes of neonatal mouse

目的探讨和建立适用于乳小鼠心肌成纤维细胞和心肌细胞体外分离、培养及鉴定的技术方法。方法乳小鼠心脏剪成组织块,用Ⅱ型胶原酶加胰蛋白酶联合消化法分离细胞,再通过差速贴壁分离法分别收集、培养乳小鼠心肌成纤维细胞和心肌细胞。光学显微镜下分别观察成纤维细胞和心肌细胞的基本形态特征的变化,并对2种细胞行苏木素-伊红(HE)染色,用第2代心肌成纤维细胞行波形蛋白免疫荧光鉴定,而原代心肌细胞行α-横纹肌肌动蛋白、心肌肌钙蛋白和心肌肌球蛋白重链免疫荧光鉴定。结果差速贴壁分离法心肌成纤维细胞原代培养90 min基本贴壁,贴壁较早,培养第3～5代细胞生长良好,72 h心肌成纤维细胞波形蛋白免疫荧光鉴定纯度高达97%;而心肌细胞贴壁较晚,24 h贴壁后部分心肌细胞出现自发性搏动现象,48 h后细胞逐渐展开,72 h后逐渐形成细胞簇并出现同步搏动,心肌肌球蛋白重链、心肌横纹肌肌动蛋白、心肌肌钙蛋白免疫荧光鉴定心肌细胞纯度分别为95%、93%、95%。结论差速贴壁分离法结合免疫荧光鉴定可分离乳小鼠心肌成纤维细胞和心肌细胞,此方法成熟、稳定、有效,为研究心脏疾病的基础与临床研究提供了理想的实验模型。

Objective To build the isolation,culture and identification techniques of cardiac fibroblasts and cardiac myocytes of neonate mouse. Methods The heart of neonate mouse was cut into pieces,then type Ⅱ collagenase plus trypsinase digestion method were used for isolating cell and then cardiac fibroblasts and cardiac myocytes were collected and cultured by different speed adherence.The basic shape change of cardiac fibroblasts and cardiac myocytes were observed under the opitical microscope and the two kinds cells were given hematoxylin-eosin staining.The second generation cardiac fibroblasts were assayed by vimentin immunofluorescence and cardiac myocytes were assayed by α-sarcomeric-actin,cardiac troponin and cardiac myoglobulin heavy chain immunofluorescence. Results Fibroblasts were adhered after 90 minutes of primary culture by the method of different speed adherence,and the third generation to the fifth generation cells growth well.The expression of vimentin of 97% fibroblasts were positive after 72 hours′ culture.But cardiac myocytes were adhered after 24 hours of culture and there was spontaneous beating phenomenon of part cardiac myocytes.The cells gradually spread after 48 hours.The cells clusters were formed and there was synchronous pulsation after 72 hours.The positive expression rate of cardiac myoglobulin heavy chain,α-sarcomeric-actin and cardiac troponin was 95%,93%,95%,respectively after 72 hours′ culture. Conclusion Different speed adherence method and immunoflurescence were effective and stable to isolate the cardiac fibroblasts and cardiac myocytes.This method provide an ideal experimental model for the basic and clinical research of heart disease.