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目的建立适用于胚胎大鼠心脏成纤维细胞体外分离、培养及鉴定的技术方法。方法从SD孕19 d大鼠子宫中分离出胚胎鼠,再从胎鼠中分离出心脏,将心脏剪成肉糜状,用胰蛋白酶加上Ⅱ型、Ⅳ胶原酶联合消化法分离心脏成纤维细胞,再通过差速贴壁分离法分别收集、培养胚胎大鼠心脏成纤维细胞。在光学显微镜下观察心脏成纤维细胞形态特征的变化,用第2代心脏成纤维细胞用波形蛋白(vimentin)、结蛋白(desmin)、血管性血友病因子(vWF)、平滑肌肌动蛋白(α-SMA)免疫荧光鉴定。结果差速贴壁法分离的心脏成纤维细胞原代培养120 min已经完全贴壁,培养第3~5代细胞生长良好,24 h心脏成纤维细胞波形蛋白免疫荧光鉴定纯度高达97%,培养2~3 d时心脏成纤维细胞增殖活力最强。结论差速贴壁分离法结合免疫荧光鉴定可分离胚胎大鼠心脏成纤维细胞,此种方法可得到产量大,纯度高,活力好的心脏成纤维细胞,适用于细胞水平上的心血管疾病发病机制的研究。
Objective To establish a method for the isolation, culture and identification of embryonic rat cardiac fibroblasts in vitro. Methods The embryos were isolated from the uterus of SD pregnant rats at 19 d. The hearts were isolated from fetal rats and the heart was cut into the shape of minced meat. The cardiac fibroblasts were separated by trypsin combined with collagenase Ⅱ and Ⅳ The fibroblasts of embryonic rat hearts were collected and cultured respectively by differential adherent separation method. The morphological changes of cardiac fibroblasts were observed under a light microscope. The second generation of cardiac fibroblasts were stained with vimentin, desmin, vWF and smooth muscle actin α-SMA) immunofluorescence identification. Results The primary culture of cardiac fibroblasts isolated by differential adherence method had been completely adhered for 120 min. The cells grew well on the 3rd to 5th passages. The purity of vimentin immunofluorescence was 97% ~ 3 d, the strongest cardiac fibroblast proliferation activity. Conclusion The method of differential adherent separation combined with immunofluorescence identification can isolate embryonic rat cardiac fibroblasts. This method can produce cardiac fibroblasts with high yield, high purity and high activity, and is suitable for the pathogenesis of cardiovascular diseases at the cellular level Mechanism of research.