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OA

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.