简易灌注装置同时提取成年小鼠心肌细胞和心脏成纤维细胞  

Simplified perfusion device for simultaneous isolation of adult mouse cardiomyocytes and cardiac fibroblasts

作　　者：师雷 金振晓 谭延振 张冰 崔君 冯乐乐 宋宇杰 刘英英[2] 张苗 易蔚 Shi Lei;Jin Zhenxiao;Tan Yanzhen;Zhang Bing;Cui Jun;Feng Lele;Song Yujie;Liu Yingying;Zhang Miao;Yi Wei(Department of Cardiovascular Surgery of Xijing Hospital,Air Force Military Medical University,Shaanxi Xi’an 710032,China)

机构地区：[1]空军军医大学第一附属医院心血管外科,西安710032 [2]西北大学,西安710032 [3]陕西中医药大学,咸阳712046

出　　处：《中国体外循环杂志》2023年第3期172-178,共7页Chinese Journal of Extracorporeal Circulation

基　　金：国家自然科学基金面上项目(82170336)。

摘　　要：目的简化Langendorff灌注装置,并且同时提取成年小鼠心肌细胞(AMCM)和心脏成纤维细胞(AMCF)用于实验。方法采用简易灌注装置进行成年小鼠主动脉逆行灌注,同时提取AMCM和AMCF。对其进行形态学观察,并分别通过L-苯肾上腺素(PE)和转化生长因子(TGF)-β1刺激诱导AMCM肥厚和AMCF向肌成纤维细胞分化,以评估其生物学功能。结果复钙后耐钙杆状AMCM占比(62.65±3.12)%,并且可以在体外培养超过1周,与对照组相比,PE刺激后AMCMβ-肌球蛋白重链、心钠肽蛋白表达水平显著升高(P<0.05),杆状AMCM长宽比显著缩小(P<0.05)。AMCF纯度较高,在体外可以传代3~4次,与对照组相比,TGF-β1刺激后AMCF Collagen I、Collagen III、α-平滑肌肌动蛋白(α-SMA)蛋白表达水平显著升高(P<0.05)和荧光强度显著增强(P<0.05)。结论简易灌注装置可以简单、稳定、高效地同时提取满足实验需求、具备生物学功能的AMCM和AMCF。Objective To simplify the Langendorff perfusion device and isolate adult mouse cardiomyocytes(AMCM)and cardiac fibroblasts(AMCF)simultaneously for experiment.Methods AMCM and AMCF were isolated at the same time by retrograde perfusion through the aorta of adult mouse with simplified perfusion device.The extracted cells were cultured and observed in vitro.Their biological functions were evaluated by L-phenylephrine(PE)induced cardiomyocyte hypertrophy and transforming growth factor-β1(TGF-β1)induced fibroblast differentiation into myofibroblast.Results Calcium-tolerant rodshaped cardiomyocytes accounted for(62.65±3.12)%after calcium reintroduction,and these cells could be cultured in vitro for at least one week.Compared with the control group,AMCM after PE stimulation were characterized by a significant increase inβ-myosin heavy chain(β-MHC)and atrial natriuretic peptide(ANP)protein expression levels(P<0.05),and a significant decrease in Length-Width ratio(P<0.05).AMCF had high purity and could be passaged 3 to 4 times in vitro.Compared with the control group,a significant increase in Collagen I、Collagen III、α-Smooth muscle actin(α-SMA)protein expression levels(P<0.05)andα-SMA fluorescence intensity(P<0.05)were observed in AMCF after TGF-β1 stimulation.Conclusion The simplified perfusion device is able to simultaneously isolate biologically functional AMCM and AMCF easily,stably,and efficiently.

关 键 词：Langendorff灌注装置 心脏灌注 成年小鼠心肌细胞 成年小鼠心脏成纤维细胞 细胞提取 细胞培养 

分 类 号：R-33[医药卫生]