Comparison of different isolation and culture methods and identification of rhesus monkey lung fibroblasts
10.19405/j.cnki.issn1000-1492.2022.07.006
- Author:
Zhen Xu
1
;
Haifeng Jiang
1
;
Lei Zhang
1
;
Xiaoyi Liu
1
;
Tingyu Dong
1
;
Xuewen Tan
1
;
Shangxue Yan
1
;
Yan Chang
1
;
Wei Wei
1
Author Information
1. Institute of Clinical Pharmacology，Anhui Medical University，Key Lab of Anti-inflammatory and Immune Medicine，Ministry of Education，Anhui Collaborative Innovation Center of Anhui-inflammatory and Immune Medicine，Hefei 230032
- Publication Type:Journal Article
- Keywords:
rhesus monkey;
lung;
primary culture;
lung fibroblast
- From:
Acta Universitatis Medicinalis Anhui
2022;57(7):1041-1047
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To compare the efficiency of different methods for extracting rhesus monkey lung fibroblasts and their effects on functions, so as to provide a method for obtaining primary lung fibroblasts that are closer to human fibroblasts.
Methods:Two extraction methods for rhesus monkey lung fibroblasts were used, direct tissue block adhesion method and collagenase combined digestion with tissue block adhesion method. The cell morphology was observed with the inverted microscope, the purity of isolated rhesus monkey lung fibroblasts was identified by immunofluorescence, cell viability was detected by CCK-8, the expression of α-SMA was detected by flow cytometry and the effect of long-term in vitro culture on cell apoptosis was detected by apoptosis kit. Western blot was used to detect the expression of α-SMA protein.
Results:The combined digestion with collagenase and tissue block adhesion method could see small and bright cells crawling out in 24 hours, and cells could be seen crawling out in a large area after 48 hours. The cells were in a long spindle shape, after 4 days to 5 days, a single layer of cells could be formed. Identified by immunofluorescence, all cells expressed α-SMA. Tissue adhesion method showed small and bright cells crawling out after 72 hours. After 4 days to 5 days, the cells crawled out in a small area and showed a long spindle shape. After a week, the cells crawled out in a large area and formed a single layer of cells and the cells are all expressed α-SMA by immunofluorescence. The experimental results showed that the cell viability of the cells crawled out by the collagenase digestion method was significantly higher than that of the tissue adhesion method. After TGF-β1 stimulates the cells, the cells extracted by collagenase digestion method proliferated faster and expressed α-SMA more obviously.
Conclusion:Both methods can isolate rhesus monkey lung fibroblasts in vitro, but the collagenase digestion method extracts cells in a shorter time and in better condition. The expression of related proteins is more stable after stimulation by stimulants, which is an effective method to obtain rhesus monkey lung fibroblasts, and it is also an effective method to obtain primary lung fibroblasts that are closer to human.
- Full text:2025022616540285186猕猴肺成纤维细胞的不同分离培养方法及鉴定比较_许振.pdf
