摘要
目的 通过改良后的酶消化法进行小鼠主动脉血管平滑肌细胞(vascular smooth muscle cells,VSMCs)原代培养,提高VSMCs原代培养效率及质量,进而建立更快速有效的体外钙化模型。方法 剥离小鼠主动脉中膜并将其剪碎,分别采用改良组织贴块法及改良酶消化法进行小鼠VSMCs原代培养,免疫荧光染色鉴定细胞纯度后,分组(经典钙化组、改良钙化组)进行体外钙化诱导,采用vonKossa染色法评估各组钙化差异。结果 与改良组织贴块法相比,改良后的酶消化法培养原代VSMCs可在短时间内获得大量细胞,鉴定VSMCs纯度>98%,且细胞不易老化,传代存活率高。与传统体外钙化方法比,改良组的钙化更容易且稳定。结论 本研究通过改良的酶消化法提高了原代平滑肌细胞的存活率及培养效率,同时通过改良体外钙化诱导液配方,建立了良好的体外钙化模型。
AIM To improve the survival rate and culture efficiency of primary culture of mouse aortic vascular smooth muscle cells(VSMCs) through modified enzyme digestion method,and thus to establish a simple and efficient method of calcification model in vitro.METHODS The mice aortic medium membrane was peeled off and sheared,and the primary culture of mouse VSMCs was carried out by modified tissue-piece inoculation and modified enzyme digestion method.The purity of cells was determined by immunofluorescence staining and the VSMCs were divided into two groups(classical calcification group and modified calcification group) for in vitro calcification induction.Finally,vonKossa staining was adopted to evaluate the calcification differences between the groups.RESULTS Compared with the modified tissue-piece inoculation,the modified enzyme-combined digestion could obtain a large number of primary VSMCs in a shorter time and the purity of the identified VSMCs was more than 98%.Compared with traditional calcification methods,the modified method made the calcification model easier and more stable.CONCLUSION In this study,the survival rate and culture efficiency of primary smooth muscle cells are successfully improved by the modified enzyme digestion method and the preparation of the calcification model in vitro is optimized by improving the formula of the calcification induction solution.
作者
李晓阳
邱梦倩
李标
李渊
孙康云
马雪兴
韩震
LI Xiao-yang;QIU Meng-qian;LI Biao;LI Yuan;SUN Kang-yun;MA Xue-xing;HAN Zhen(Department of Cardiology,Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou 215000,Jiangsu,China)
出处
《心脏杂志》
CAS
2023年第6期643-647,共5页
Chinese Heart Journal
基金
镇江市创新能力建设计划临床医学研究中心项目(SS2018008-KF06)。
关键词
小鼠
血管平滑肌细胞
改良
酶消化法
原代培养
钙化
mouse
vascular smooth muscle cell
modifiy
enzyme digestion
primary culture
calcification
