Abstract: Objective　To compare the differences between tissue explant adherence method and enzymatic digestion method for isolating and culturing rat pulmonary artery fibroblasts (PAFs), and to identify the optimal method for obtaining primary PAFs with favorable growth status and stable performance.Methods　Rat pulmonary arteries were processed using tissue explant adherence method and enzymatic digestion method, respectively. Cellular morphology and passage growth status were observed under microscopy. Immunofluorescence staining was employed to detect the expression levels of platelet-endothelial cell adhesion molecule (CD31), α-smooth muscle actin (α-SMA), and the specific marker Vimentin. The optimal isolation and culture method was comprehensively evaluated based on these observations. Results　Both method successfully obtained PAFs, but the enzymatic digestion method demonstrated significant superiority over tissue explant adherence method. PAFs prepared by enzymatic digestion exhibited rapid growth (50%-60% adherence rate by Day 7 and full confluence by Day 10) with typical spindle-shaped morphology, maintaining excellent proliferative capacity through passages 3-5. In contrast, PAFs from tissue explant adherence method showed slow growth (only 60% 70% confluence by Day 14) and apparent senescence with limited passage by passage 5. Immunofluorescence result revealed that PAFs from both method were negative expression for CD31, showed low α-SMA expression, and positive Vimentin expression, consistent with fibroblast characteristics. Notably, enzymatic digestion-derived PAFs displayed morphology and marker expression patterns more aligned with ideal fibroblast phenotype.Conclusion　Enzymatic digestion method efficiently produces PAFs with stable morphology, high proliferative activity, and superior passaging performance.

Key words: pulmonary artery fibroblasts, tissue explant adherence, enzymatic digestion, immunofluorescence

摘要： 目的　比较组织块贴壁法与酶消化法制备大鼠肺动脉成纤维细胞(PAFs)的差异,筛选最佳培养方法,以获 取生长状态良好、性能稳定的原代PAFs。方法　采用两种方法分别处理剥离的大鼠肺动脉,通过显微镜观察细胞 形态、传代生长状态,并通过免疫荧光技术检测血小板内皮细胞黏附分子(CD31)、α-平滑肌肌动蛋白(α-SMA)和 特异性标志物波形蛋白(Vimentin)表达水平,综合筛选最佳培养方法。结果　两种方法均可获得PAFs,但酶消化 法显著优于组织块贴壁法。酶消化法制备的PAFs生长迅速(第7天贴壁率达50%~60%,第10天铺满),形态呈 典型梭形,传代至第3~5代仍保持良好增殖能力;而组织块贴壁法制备的PAFs生长缓慢(第14天覆盖率仅60%~ 70%),传至第5代即出现明显老化,传代受限。免疫荧光结果显示,两种方法制备的PAFs CD31呈阴性表达,α SMA低表达,Vimentin呈阳性表达,均符合成纤维细胞特征,其中酶消化法的细胞形态与标志物表达更符合理想成 纤维细胞表型。结论　酶消化法可高效制备形态稳定、增殖活性高且传代性能优良的PAFs。

关键词: 肺动脉成纤维细胞, 组织块贴壁法, 酶消化法, 免疫荧光