Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells
Abstract Background Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-05-01
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| Series: | BMC Veterinary Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12917-022-03288-3 |
| _version_ | 1811341296900505600 |
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| author | Long Guo Zhihao Wang Jun Li Jianji Li Luying Cui Junsheng Dong Xia Meng Chen Qian Heng Wang |
| author_facet | Long Guo Zhihao Wang Jun Li Jianji Li Luying Cui Junsheng Dong Xia Meng Chen Qian Heng Wang |
| author_sort | Long Guo |
| collection | DOAJ |
| description | Abstract Background Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses. Results In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated. Conclusions In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening. |
| first_indexed | 2024-04-13T18:53:53Z |
| format | Article |
| id | doaj.art-adfbcc7f5f5248f484829e0e71890df2 |
| institution | Directory Open Access Journal |
| issn | 1746-6148 |
| language | English |
| last_indexed | 2024-04-13T18:53:53Z |
| publishDate | 2022-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Veterinary Research |
| spelling | doaj.art-adfbcc7f5f5248f484829e0e71890df22022-12-22T02:34:19ZengBMCBMC Veterinary Research1746-61482022-05-0118111010.1186/s12917-022-03288-3Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cellsLong Guo0Zhihao Wang1Jun Li2Jianji Li3Luying Cui4Junsheng Dong5Xia Meng6Chen Qian7Heng Wang8College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityCollege of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou UniversityAbstract Background Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses. Results In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated. Conclusions In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening.https://doi.org/10.1186/s12917-022-03288-3CanineCorneal epithelial cellSV40TImmortalizationStaphylococcus pseudintermediusInflammation |
| spellingShingle | Long Guo Zhihao Wang Jun Li Jianji Li Luying Cui Junsheng Dong Xia Meng Chen Qian Heng Wang Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells BMC Veterinary Research Canine Corneal epithelial cell SV40T Immortalization Staphylococcus pseudintermedius Inflammation |
| title | Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells |
| title_full | Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells |
| title_fullStr | Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells |
| title_full_unstemmed | Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells |
| title_short | Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells |
| title_sort | immortalization effect of sv40t lentiviral vectors on canine corneal epithelial cells |
| topic | Canine Corneal epithelial cell SV40T Immortalization Staphylococcus pseudintermedius Inflammation |
| url | https://doi.org/10.1186/s12917-022-03288-3 |
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