Identification and functional study of immortalized mouse thymic epithelial cells
Autor: | Jian-Li Gao, Kai He, Jia-Man Shen, Hong-Bin Zheng, Wen-Qin Guo, Robert D. Hoffman, Bing-Qian He, Li Ma, Chuan Ding |
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Rok vydání: | 2020 |
Předmět: |
0301 basic medicine
SV40 large T antigen T-Lymphocytes education Biophysics Thymus Gland Cell morphology Biochemistry Flow cytometry Cell Line 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine Annexin medicine Animals Humans Propidium iodide Molecular Biology Cells Cultured Cell Proliferation Mice Inbred BALB C medicine.diagnostic_test Chemistry Cell Cycle hemic and immune systems Cell Differentiation Epithelial Cells Cell Biology Cell cycle Coculture Techniques Cell biology 030104 developmental biology Apoptosis 030220 oncology & carcinogenesis tissues Immortalised cell line |
Zdroj: | Biochemical and biophysical research communications. 525(2) |
ISSN: | 1090-2104 |
Popis: | As the key cells in a three-dimensional scaffold within the thymus, Thymic epithelial cells (TECs) play critical roles in the homing, migration and differentiation of T cell precursors through adhesive interactions and the release of various cytokines. In this study, primary cultures of mouse TECs were isolated and identified with TEC-specific antibodies CK5 and CK8. These TECs were immortalized by retroviral transduction of simian virus (SV) 40 large T antigen. We then compared the functions of TECs and immortalized TECs (iTECs). Cell morphology and the proliferative capacity of TECs and iTECs were observed by inverted microscope photography and crystal violet assay after passage. A soft agar assay was then performed to observe their clone formation ability. The expression levels of epithelial cell related factors, such as IL-7, Lptin, Pax-9, Sema3A and et al., were detected by IF and qPCR. TECs were co-cultured with human acute monocytic leukemia cells (THP-1), and the effect of TECs on promoting THP-1 proliferation was observed with flow cytometry and CFSE labeling. Senescence-associated β-galactosidase assay was measured to detect the anti-aging capabilities of the cells. Cell cycle distribution was analyzed by propidium iodide (PI) staining, and paclitaxel (PTX)-induced apoptosis was detected by Annexin V-PI staining to evaluate the anti-apoptotic ability of the cells. Throughout, we found that the immortalized TECs still retain the characteristics of primary TECs, such as the morphology, function and epithelial characteristics; however, iTECs have stronger capabilities in proliferation and anti-aging. Our research suggests that the iTECs were successfully immortalized by SV40 large T antigen, and that the biological characteristics and functions of iTECs were similar to the original TECs. This immortalized cell can be used as an efficient cell model in functional research of the thymus substituting primary TECs with iTECs. |
Databáze: | OpenAIRE |
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