西南医科大学学报2026，Vol.49Issue(4)：448-454,7.DOI:10.3969/j.issn.2096-3351.2026.04.006
一种稳定高效的成年小鼠心脏成纤维细胞原代培养方法建立及功能验证
Establishment and Functional Validation of a Stable and Efficient Primary Culture Method for Adult Mouse Cardiac Fibroblasts
摘要
Abstract
Objective Establish an efficient and stable in vitro isolation and culture system for primary cardiac fibroblasts from adult mice.Methods Heart tissues from adult C57BL/6J mice were minced into approximately 1 mm3 fragments and digested with a 2 mg/mL type Ⅱ collagenase solution.Tissue fragments were subjected to two rounds of water bath oscillation combined with mechanical trituration using a pipette.Primary cardiac fibroblasts were isolated and purified via differential adhesion.Cell morphology and growth were observed under an inverted microscope.Cell purity and activation status following transforming growth factor-β1(TGF-β1)induction were assessed using immunofluorescence staining.The effects of TGF-β1 stimulation on the expression of fibrosis-related proteins[such as α-smooth muscle actin(α-SMA),periostin,collagen Ⅰ(Col-1),collagen Ⅲ(Col-3)]and fibrosis-associated sig-naling molecules(including p-Smad3,p-p38,p-ERK1/2,p-Akt,p-Stat3)were analyzed by Western blot.Results After differen-tial adhesion,a large number of bright,round adherent cells were observed microscopically.After 2 days in culture,most cells had transitioned from a round to a long spindle shape and exhibited rapid proliferation.By day 4,cell numbers had increased significantly,forming confluent monolayers without gaps.Immunofluorescence staining for Vimentin/α-SMA/CD31 indicated that the purity of pas-sage 1(P1)cardiac fibroblasts exceeded 95％.α-SMA immunofluorescence staining suggested that TGF-β1 stimulation for 24 hours significantly induced fibroblast activation.Western blot results demonstrated that prolonged TGF-β1 stimulation(24～48 hours)sig-nificantly promoted the expression of α-SMA,periostin,Col-1,and Col-3.In contrast,short-term stimulation(15 minutes～3 hours)induced phosphorylation of Smad3,p38,ERK1/2,Akt,and Stat3 to varying degrees.Conclusion This study successfully established a simple,efficient,and reproducible system for the isolation and culture of cardiac fibroblasts from adult mice,thereby pro-viding a reliable cellular experimental model for research related to cardiovascular diseases.关键词
成年小鼠/原代心脏成纤维细胞/细胞培养与鉴定/转化生长因子-β1/纤维化相关蛋白/纤维化信号分子Key words
Adult mice/Primary cardiac fibroblasts/Cell culture and identification/Transforming growth factor-β1/Fibrosis-related proteins/Fibrotic signaling molecules分类
医药卫生引用本文复制引用
王智,杨宇,秦何霖,谷家琴,张艺瀚,范忠才,杜延飞..一种稳定高效的成年小鼠心脏成纤维细胞原代培养方法建立及功能验证[J].西南医科大学学报,2026,49(4):448-454,7.基金项目
国家自然科学基金(82200287) （82200287）
核医学与分子影像四川省重点实验室开放基金(HYX22001) （HYX22001）
西南医科大学临床医学专项(2024LCYXZX46) （2024LCYXZX46）
泸州市人民政府-西南医科大学科技战略合作项目(2025LZXNYDJC09) （2025LZXNYDJC09）
