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

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