Isolation, Identification and Subculture of Pulmonary Fibroblasts in Tree Shrew

doi:10.3969/j.issn.1674-5817.2019.02.007

Abstract

Abstract: Objective To establish a reliable method for isolation, identification and subculture of pulmonary fibroblasts in tree shrew. Methods Pulmonary fibroblasts of neonatal tree shrew were isolated and cultured by tissue adherence culture and trypsin digestion respectively. The fibroblasts was observed by inverted microscope and identified by immunofluorescence with Vimentin. Then the pulmonary fibroblasts was subcultured, and cryopreservation and recovery experiments were carried out. The cell growth curve was drawn by MTS method, and human embryonic lung diploid fibroblasts (KMB-17) were used as control. Results Tree shrew pulmonary fibroblasts could be obtained by both tissue adherence culture and trypsin digestion. The cells were mainly spindle-shaped, and vimentin immunofluorescence assay were generally positive, which were consistent with KMB-17. Tree shrew pulmonary fibroblasts can be subcultured 4-5 times continuously. After cryopreservation and recovery, the cells can still remain viable and enter the logarithmic growth phase about 3 days later with a cell density of 5×10⁴cells/mL. Conclusion The pulmonary fibroblasts in tree shrew can be effectively obtained by tissue adherence and trypsin digestion, which will be very helpful for the study of lung-related diseases in vitro.

Key words: Standardization and Application Research in Tree Shrew, Kunming 6500118, China)

CLC Number:

Q95-33

WANG Wen-guang,KUANG De-xuan,LU Cai-xia,et al. Isolation, Identification and Subculture of Pulmonary Fibroblasts in Tree Shrew[J]. Laboratory Animal and Comparative Medicine, 2019, 39(2): 105-110. DOI: 10.3969/j.issn.1674-5817.2019.02.007.

References

[1] Yao YG.Creating animal models, why not use the Chinese tree shrew (Tupaia belangeri chinensis)[J]. Zool Res, 2017, 38(3):118-126.
[2] Xiao J, Liu R, Chen CS.Tree shrew (Tupaia belangeri) as a novel laboratory disease animal model[J]. Zool Res, 2017, 38(3):127-137.
[3] 张顶, 高丽, 张远旭, 等. 树鼩肝细胞体外分离培养体系的复建及主要影响因素分析[J]. 动物学研究, 2009, 30(1):24-30.
[4] 阮萍, 杨春, 欧超, 等. 树鼩肝枯否细胞的分离及鉴定[J]. 临床与实验病理学杂志, 2014, 30(2):159-163.
[5] 吴元桢, 欧阳轶强, 黄玲, 等. 新生树鼩海马神经干细胞的体外培养与鉴定[J]. 广西医学, 2012, 24(3):257-260.
[6] 龚敏, 李树清, 李凡. 树鼩脑星形胶质细胞的体外培养及纯化[J]. 生理学报, 2011, 63(1):89-92.
[7] 张理超, 张捷, 付必莽, 等. 树鼩淋巴细胞的快速分离和分析方法的初步建立[J].云南大学学报: 自然科学版, 2013,35(S2):359- 363+367.
[8] 苗雨润, 宋庆凯, 匡德宣, 等. 树鼩角膜原代上皮细胞的分离培养、纯化与鉴定[J]. 实验动物与比较医学, 2017, 37(2):130-135.
[9] 陆彩霞, 李晓飞, 王文广, 等. 树鼩骨髓间充质干细胞体外分离培养及成脂成骨诱导[J]. 中国比较医学杂志, 2014,24(3):10-13+84 -85.
[10] 陈小波, 贺猛, 李光剑, 等. 宣威烟煤粉尘诱发树鼩支气管黏膜上皮改变的研究[J]. 中国肺癌杂志, 2015, 18(8):469-474.
[11] Yang Z F, Zhao J, Zhu Y T, et al.The tree shrew provides a useful alternative model for the study of influenza H1N1 virus[J]. Virol J, 2013, 10(1):111.
[12] 高建, 刘干, 李俊. 肺成纤维细胞在肺纤维化进程中的作用[J]. 中国药理学通报, 2010, 26(9):1125-1129.
[13] 邓佳慧, 肖军花, 王嘉陵. 成年小鼠肺成纤维细胞分离纯化及原代培养[J]. 基础医学与临床, 2012, 32(6):713-714.
[14] 连敏, 叶燕青, 汪甜, 等. 新生大鼠肺成纤维细胞的原代培养改良法及细胞鉴定[J]. 氨基酸和生物资源, 2014, 36(1):27-31.

[1]	ZHANG Jiaoling, LIN Aping, XU Shiying, PAN Dequan, CAI Yijun. Optimization and Application of Mouse Embryo Transfer Technology [J]. Laboratory Animal and Comparative Medicine, 2026, (): 1-9.
[2]	. [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 611-613.
[3]	LI Chunyang, LIU Xingchen, LI Yuzhuo, LU Yong, WANG Yan, GAO Changshan, NIE Yanhong, BIAN Xinyan, HU Chao, ZHU Jiaqiao, SUN Qiang. Generation of Cynomolgus and Rhesus Monkey Models Carrying the BMPR1B^FecB Variant [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 467-475.
[4]	LEI Jianjun, SUO Chenhao, ZHANG He. Resveratrol Alleviates Blood‑Brain Barrier Damage in Cerebral Ischemia‑Reperfusion Rat Models through Modulation of the CCL3/CCR5 Signaling Pathway [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 487-497.
[5]	WANG Lu, WANG Qingjie, YANG Chunhong, ZHANG Yijian. Application of New Approach Methodologies in Biomedical Field and Their Implementation of "3R" Principle [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 573-582.
[6]	GONG Zhiqun, RAO Xiwu, ZHANG Tianyi, WU Rongchen, YE Mingnan, JIANG Chunxi, WANG Sumei, ZHU Yanjuan, CHAI Xiaoshu. Construction and Evaluation of Mouse Models Simulating Immune Checkpoint Inhibitor-Associated Pneumonitis [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 498-506.
[7]	LI Hongman, CHEN Yutong, SHI Yingpei, WANG Yijing, PAN Yan, XU Tong, ZHOU Yi, DENG Qiyue, LIU Xue. A New Perspective on Mental Health Assessment in Laboratory Animals: Stress Response Monitoring Based on Hair Characteristics [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 553-563.
[8]	MA Wenjing, LIU Yanping, LI Yifan, GUO Huan, XIE Lihui, DU Longji, MA Jiali, WANG Hongping, LI Jiamin. Practice in Validity Period Management of Disinfectants, Feed, and Bedding in Laboratory Animal Facilities under Good Laboratory Practice Management System [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 593-603.
[9]	LI Zifa, LIU Shan, SUN Wei, ZHAO Feng, WEI Sheng, LI Qin, XU Kaiyong. Reflections on Building a Science-Education Integration System for Laboratory Animal Science: A Case Study of Shandong University of Traditional Chinese Medicine [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 604-610.
[10]	QIANG Sujing, LI Dongting, XU Jinhua, CAI Li, WEI Siyu, ZOU Jialei, DING Yuqiang. Identification and Analysis of the Klebsiella pneumoniae Species Complex in Laboratory Rats and Mice [J]. Laboratory Animal and Comparative Medicine, 2026, 46(4): 564-572.
[11]	LIU Liang, CHEN Bin, MEN Li, ZHANG Huan, LIU Jinzhou, LIU Keliang, LI Yangyou, DUAN Jiahui, YAN Jintai, YANG Guolin, JIN Bo. Serving National Strategies and the Chengdu-Chongqing Biomedical Industry: Innovative Layout and Practical Pathways of Undergraduate Education in Laboratory Animal Science [J]. Laboratory Animal and Comparative Medicine, 2026, (): 1-12.
[12]	YANG Ran, FENG Jiwei, MA Fei, ZHANG Yu, LIU Zhiwei, JIIN Lilun. Fraxetin Inhibits Ferroptosis and Alleviates Cartilage Damage in Osteoarthritis Rat Through Nrf2/HO-1 Signaling Pathway [J]. Laboratory Animal and Comparative Medicine, 2026, (): 1-12.
[13]	AI Xiufeng, ZHANG Lizong, FANG Mingsun, LÜ Dongying, CHEN Chu, CAI Zhaowei, WANG Dejun. Analysis of Differences in the Intestinal Flora of Rats and Mice after Drinking Chlorinated Water Based on 16S rRNA Sequencing [J]. Laboratory Animal and Comparative Medicine, 2026, 46(3): 437-445.
[14]	ZHOU Yimin, ZHANG Xinyu, YANG Jianuo, LIU Mengjia, SI Cancan, YE Hailü, SUN Wenchao, LAN Tian. Development of a Triplex TaqMan Quantitative PCR Method for Simultaneous Detection of MHV, MPV, and Reo-3 [J]. Laboratory Animal and Comparative Medicine, 2026, 46(3): 408-415.
[15]	. [J]. Laboratory Animal and Comparative Medicine, 2026, 46(3): 464-466.