风湿性心脏病患者心房成纤维细胞的原代培养

The primary culture of atrial fibroblasts in rheumatic heart disease patients

目的建立一种人心房成纤维细胞原代培养简单有效的方法。方法新鲜右心耳组织取自心外科风湿性心脏病手术患者,采用0.25%胰蛋白酶和0.2%Ⅱ型胶原酶消化法分离细胞,用含2O%胎牛血清的DMEM培养液进行培养并传代,显微镜下观察心房成纤维细胞生长状况。波形蛋白(Vimentin)、α平滑肌肌动蛋白(α-SMA)、Ⅷ因子(VWF)抗体对细胞进行免疫荧光鉴定。结果获取大量细胞,形态典型;Vimentin、α-SMA表达阳性,VWF阴性,鉴定为心房肌成纤维细胞。结论胰蛋白酶和Ⅱ型胶原酶消化法是一种简单有效的人心房成纤维细胞原代培养方法。

Objective To develope a simple and effective method of culturing primary human atrial fibroblasts.Methods Aseptic right atrial appendage tissue specimen were obtained from adult patients who underwent surgery for rheumatic heart disease.Fresh atrial appendage tissue specimen were cut into small pieces and digested with 0.25% trypsin and 0.2% collagen II.Cell suspension was cultured in DMEM containing 20% fetal bovide serum.Cultured human atrial fibroblasts were characterized by microscope.Cells were analyzed by immunofluorescence technique with the following antibodies:vimentin,α-smooth muscle alpha-actin and VWF.Results The appearance of cell was typical and a great number of cells were collected.The cultured human atrial fibroblasts were positive both for vimentin antigens and for α-SMA,negative for VWF.Conclusions Trypsin and collagen II digestion is simple and effective for isolation of human atrial fibroblasts.