不同体外培养条件对缺氧诱导原代心肌成纤维细胞活化的影响  

Effect of different in vitro culture conditions on hypoxia-induced activation of primary cardiac fibroblasts

作　　者：付可威 曹梦菲 罗洁 袁伟[1] Fu Kewei;Cao Mengfei;Luo Jie;Yuan Wei(Department of Cardiovascular Medicine,Af filiated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu Province,China)

机构地区：[1]江苏大学附属医院心血管内科,镇江212001

出　　处：《中华老年心脑血管病杂志》2025年第4期497-503,共7页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：江苏省重点研发计划项目(BE2021694)。

摘　　要：目的探讨不同浓度葡萄糖、血清体外培养对缺氧诱导的原代心肌成纤维细胞活化及Yes相关蛋白(Yes-associated protein,YAP)表达的影响。方法采用酶消及差速贴壁法分离并纯化原代乳小鼠细胞,随机分为6组:低糖高血清组(1.0 g/L葡萄糖+10%血清),高糖高血清组(4.5 g/L葡萄糖+10%血清),低糖中血清组(1.0 g/L葡萄糖+5%血清),高糖中血清组(4.5 g/L葡萄糖+5%血清),低糖无血清组(1.0 g/L葡萄糖+0%血清),高糖无血清组(4.5 g/L葡萄糖+0%血清)。体外培养24 h观察细胞形态,Western blot检测α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、转化生长因子(transforming growth factor beta,TGF-β)、Ⅰ型胶原及YAP表达,免疫荧光法观察α-SMA、YAP表达及定位,5-乙炔基-2′脱氧尿嘧啶核苷染色以及划痕实验分别评估细胞增殖、迁移能力。结果经1%O_(2)培养24 h后,随着培养液血清水平下降及葡萄糖浓度升高,细胞形态逐渐向肌成纤维细胞变化,以高糖无血清组细胞形态变化最为显著。与高糖高血清组比较,高糖无血清组Ⅰ型胶原、TGF-β、α-SMA及YAP表达明显升高,差异有统计学意义(P<0.05);与低糖高血清组比较,低糖无血清组α-SMA荧光强度明显升高[(9.23±2.45)%vs(2.40±2.04)%,P<0.05];与低糖无血清组比较,高糖无血清组Ⅰ型胶原、TGF-β、α-SMA及YAP表达明显升高,差异有统计学意义(P<0.05);与高糖高血清组和低糖无血清组比较,高糖无血清组α-SMA荧光强度、YAP入核、细胞迁移明显升高,差异有统计学意义(P<0.05)。结论在1%O_(2)缺氧条件下,选用高糖无血清培养对于构建缺氧诱导活化的体外细胞模型最为合适,并且YAP激活程度最为显著。Objective To investigate the effects of in vitro culture with different concentrations of glucose and serum on hypoxia-induced activation and Yes-associated protein(YAP)expression in primary cardiac fibroblasts(CF).Methods Primary CF were isolated from ICR suckling mice with enzyme digestion and then purified using differential adhesion technique.The cells were randomly assigned into 6 groups:low glucose high serum group(1.0 g/L glucose+10%serum),high glucose high serum group(4.5 g/L glucose+10%serum),low glucose medium serum group(1.0 g/L glucose+5%serum),high glucose medium serum group(4.5 g/L glucose+5%serum),low glucose serum-free group(1.0 g/L glucose+0%serum)and high glucose serum-free group(4.5 g/L glucose+0%serum).In 24 h after above culture,the cell morphology was observed.Western blotting was used to detect the expression ofα-smooth muscle actin(α-SMA),transforming growth factor beta(TGF-β),type I collagen(Col I).and YAP.Immunofluorescence assay was employed to observe the expression and localization ofα-SMA and YAP.EDU staining and cell scratch assay were applied to measure cell proliferation and migration ability,respectively.ResultsAfter culture in 1%O_(2) for 24 h,the cell morphology was gradually altered toward myofibroblasts as the concentration of glucose rose and the serum level fell in the culture medium,especially in the group of high glucose and serum-free.The expression levels of Col I,TGF-β,α-SMA and YAP were significantly higher in the high glucose no serum group than in the high glucose high serum group(P<0.05).The fluorescence intensity ofα-SMA was obviously increased in the low glucose no serum group than the low glucose high serum group[(9.23±2.45)%vs(2.40±2.04)%,P<0.05].Compared to the low-sugar serum-free group,the high glucose serum-free group showed a significant increase in the levels of Col I,TGF-β,α-SMA and YAP(P<0.05).Notably higherα-SMA fluorescence intensity,more YAP nuclear translocation,and enhanced cell migration were observed in the high glucose serum-free gro

关 键 词：缺氧 成纤维细胞 体外培养技术 Yes相关蛋白 

分 类 号：R73[医药卫生—肿瘤]