酶联合消化法和贴块法分离小鼠肺动脉平滑肌细胞及其生物学特性的研究

中国生物工程杂志

2013, Vol. 33

Issue (9): 10-16

研究报告

酶联合消化法和贴块法分离小鼠肺动脉平滑肌细胞及其生物学特性的研究

辛毅, 龚达, 习昕, 石洪涛, 刘飒, 许秀芳, 李娜, 黄益民

首都医科大学附属北京安贞医院北京市心肺血管疾病研究所北京 100029

Combined Enzymatic Digestion Method and Explants Culture Method Used on Primary Culture and Biological Characteristic Identification of Pulmonary Artery Smooth Muscle Cells of mice

XIN Yi, GONG Da, XI Xin, SHI Hong-tao, LIU Sa, XU Xiu-fang, LI Na, HUANG Yi-min

Capital Medical University Affiliated Beijing Anzhen Hospital, Beijing Institute of Heart, Lung ＆ Blood Vessel Diseases, Beijing 100029, China

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摘要： 目的:探讨适用于小鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)分离培养及鉴定的技术方法。方法:采用Ⅰ型胶原酶、木瓜蛋白酶联合消化法及组织贴块2种方法对PASMCs进行体外培养。在倒置显微镜下观察PASMCs的生长特点;台盼蓝法测定细胞传代成活率;分别对PASMCs行生长曲线、MTT法、DNA周期检测比较增殖情况;免疫荧光染色法鉴定细胞。结果:酶消化法分离培养的细胞1 d后,在倒置相差显微镜下可见细胞贴壁呈椭圆形生长,4d后生长迅速呈典型的谷-峰状生长,7d后达80%融合即可传代;贴块法培养的细胞3d后,可见细胞从组织块边缘长出,7d后细胞数量逐渐增多,10d后细胞生长迅速呈单层致密排列,细胞传代成活率均为96%。2种方法获得的第2代PASMCs经平滑肌ɑ-肌动蛋白(ɑ-SM actin)免疫荧光染色鉴定,其阳性率均为90%以上;生长曲线、MTT法显示细胞增殖及细胞DNA周期无明显差异。结论:2种方法均可高效获得PASMCs在体外稳定培养,为研究肺血管疾病提供较为理想的种子细胞。

关键词： 肺动脉平滑肌细胞; 原代培养; 生物学特性

Abstract: Objective:To investigate a technical method to isolate, cultivate and identify the mice pulmonary arterial smooth muscle cells(PASMCs). Methods:Isolation of pulmonary arterial smooth muscle cells (PASMCs) has been performed using Trypsin-typeⅠ collagen and papain digestion method and tissue explants culture method. The cell growth of PASMCs was observed under invert microscope. Cell viability rate of the different cells passage was evaluated by trypan blue staining. The PASMCs proliferation profile was analyzed by curve of growth, MTT assay and DNA cell cycle analysis respectively,and were identified by immunofluorescence as well. Result:After one day culture in the enzyme digested method, the adherent cells were observed to grow quickly with a typical "peak-valley" after 4 days under invert microscope. The cells can be passaged 7-8d after the rapid growth. But in the explants culture method, the cells started to amplify to grow gradually from the center to the periphery. Cells began to grow rapidly after 7d culture and single compact arrangement then fused 80% after 10 day. The cell viabilities from both isolation methods were more than 96% tested by trypan blue staining. There was over 90% positive expression in intracellular a-smooth muscle actin (a-SMA) in immunofluorescence staining.The shape of the second passage cell growth curves were similar in both methods ; There were also no significant differences in cell cycle and MTT assay in both methods. In cell cycle over 85% of cells were in G0/G1 phase. Conclusion:The PASMCs can be effectively isolated both by enzyme digestion method and explant methods which would provide us ideal seeding cells for pulmonary vascular diseases.

Key words: Pulmonary arterial smooth muscle cells Primary culture Biological characteristics

收稿日期: 2013-03-20 出版日期: 2013-09-25

ZTFLH:

R332

基金资助: 国家自然科学基金资助项目(81270217)

通讯作者: 许秀芳drxuxiufang@aliyun.com E-mail: drxuxiufang@aliyun.com

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引用本文:

辛毅, 龚达, 习昕, 石洪涛, 刘飒, 许秀芳, 李娜, 黄益民. 酶联合消化法和贴块法分离小鼠肺动脉平滑肌细胞及其生物学特性的研究[J]. 中国生物工程杂志, 2013, 33(9): 10-16.

XIN Yi, GONG Da, XI Xin, SHI Hong-tao, LIU Sa, XU Xiu-fang, LI Na, HUANG Yi-min. Combined Enzymatic Digestion Method and Explants Culture Method Used on Primary Culture and Biological Characteristic Identification of Pulmonary Artery Smooth Muscle Cells of mice. China Biotechnology, 2013, 33(9): 10-16.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I9/10

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