蒙古马胎儿成纤维细胞永生化细胞体系建立的研究

doi:10.16431/j.cnki.1671-7236.2023.01.005

Abstract

Abstract: 【Objective】 The aim of this study was to establish immortalized equine fetal fibroblasts (EFFs) cell line in Mongolian horse, and lay a foundation for the study of horse reproduction and developmental biology.【Method】 Primary EFFs were isolated by digestion with 1 mg/mL collagenase Ⅳ.EFFs were infected with pLV-hTERT-IRES-hygro lentivirus expressing human telomerase reverse transcriptase (hTERT) and hygromycin B resistance.The third generation EFFs was used as the control, the positive cells of hTERT-EFFs were screened by hygromycin B, the expression of hTERT mRNA was detected by Real-time quantitative PCR.The growth curve was drawed to detect the cell proliferation by CCK-8 method.Cell apoptosis was detected by flow cytometry, the cell senescence was detected by β-galactosidase staining.DNAMAN software was used to compare and analyze the similarity of TERT nucleotide, protein and telomerase RNA component (TERC) sequences of Homo sapiens, Equus caballus, Mus musculus, Bos taurus, Ovis aries and Sus scrofa.【Result】 The primary EFFs could be passaged to 17 generations in the laboratory culture system, but the 17-generation cells experienced senescence.hTERT gene could be successfully transferred into EFFs and stably expressed for at least 20 generations.However, the proliferation ability of hTERT-EFFs was significantly lower than that of EFFs (P<0.05), and the apoptosis rate of hTERT-EFFs in early stage was significantly higher than that of EFFs (P<0.05).The detection of cell senescence proved the β-galactosidase positive staining was positive.Sequence alignment results showed that the mRNA conservation between horse TERT and hTERT was high, the similarity was 73.2%, and the protein conservation of horse TERT and hTERT was higher than that of other species, and the similarity was 76.3%, which indicated that horse TERT and hTERT were highly similar in structure and function.Horse TERC was highly similar to human TERC, and which were in the same branch on the homologous tree.However, there were mutations in the key nucleotides associated with TERT binding and telomerase activity in horse TERC.【Conclusion】 Overexpression of hTERT alone could prolong the number of in vitro passages of EFFs to less than 30 passages, but it was still not enough to immortalize EFFs cells of Mongolian horse with high quality, which might be related to the incompatibility between hTERT and eTERC.

Key words: Mongolian horse; equine fetal fibroblasts (EFFs); immortalization; human telomerase reverse transcriptase (hTERT); cell proliferation

CLC Number:

S821

TIAN Shuyue, SHEN Yingchao, WANG Xisheng, WANG Min, YI Minna, Tseweendolmaa, TAO Li, ZHAO Bilig, Manglai, Gerelchimeg. Study on Establishment of Immortalized Cell System of Equine Fetal Fibroblasts in Mongolian Horse[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 46-57.

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Study on Establishment of Immortalized Cell System of Equine Fetal Fibroblasts in Mongolian Horse

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