钙化性心脏瓣膜间质细胞培养方法的应用与改进

Application and improvement of calcific heart valve interstitial cell culture method

目的比较胶原酶消化两段处理法与传统酶消化法培养获得的细胞质量。方法Ⅱ型胶原酶两段处理法为实验组（2g／L），传统酶消化法为对照组（2g/L）。观察两组细胞贴壁时间和24h贴壁细胞数量的差异；免疫细胞化学染色和流式细胞术分析钙化性瓣膜间质细胞免疫表型。结果胶原酶消化两段处理法培养24h后贴壁细胞数量为传统酶消化法获得细胞数量的4倍，两组细胞获得量差异有统计学意义（P〈0．05），通过流式细胞术证实实验组具有活性的原代瓣膜间质细胞量为94．39％，细胞免疫表型检测提示99．2％呈波形蛋白（vimentin）阳性，53．3％呈α-平滑肌肌动蛋白（α-SMA）阳性。结论应用改进后的培养技术获得的细胞数量、细胞活性及细胞表型均优于传统酶消化法且可以应用于体外模型的建立等实验。

Objective To compare the quality of cells obtained by two different methods： the modified two-stage collagenase digestion method versus the traditional enzyme digestion method. Methods Cells obtained by the modified two-stage eollagenase digestion method were studied as the experiment group, and those obtained by the traditional enzyme digestion method served as the control group. The time when the cells began to adhere and the quantity of adherent cells after 24 h were observed. Immunocytochemical staining and flow cytometry were performed to analyze the phenotype of calcific valvular interstitial cells. Results The quantity of adherent cells after 24 h in the experiment group was four times of that in the control group. The difference was statistically significant （ P 〈 0. 05 ）. Flow cytometry confirmed alive primary valve interstitial cells accounted for 94. 39% in the experimental group. Cell phenotype detection revealed 99.2% were positive for vimentin, and 53.3% were positive for α-smooth muscle actin （α-SMA）. Conclusion In terms of the quantity of cells, cell viability and cell phenotype, the cells obtained by the modified two-stage eollagenase digestion method are superior to those obtained by traditional enzyme digestion and can be used in vitro experiments.