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

摘要/Abstract

摘要： 【目的】试验旨在建立永生化蒙古马胎儿成纤维细胞（equine fetal fibroblasts，EFFs）系，为马繁殖与发育生物学研究提供参考。【方法】采用1 mg/mL胶原酶Ⅳ消化分离原代EFFs，利用表达人端粒酶逆转录亚基（hTERT）和潮霉素B抗性的pLV-hTERT-IRES-hygro慢病毒感染EFFs，经潮霉素B筛选得到阳性细胞hTERT-EFFs，以第3代EFFs为对照，采用实时荧光定量PCR检测hTERT mRNA的表达水平，CCK-8法绘制生长曲线检测细胞增殖能力，流式细胞仪检测细胞凋亡情况，β-半乳糖苷酶染色检测细胞衰老情况。使用DNAMAN软件比对分析人、马、小鼠、牛、绵羊、猪多物种TERT核苷酸、蛋白和端粒酶RNA组分（TERC）序列相似性。【结果】原代EFFs在实验室培养体系下可传至17代，但第17代细胞发生了衰老。hTERT基因可成功转入EFFs，并至少稳定表达20代。然而hTERT-EFFs增殖能力显著低于EFFs （P<0.05），hTERT-EFFs凋亡早期细胞率显著高于EFFs （P<0.05）。细胞衰老检测结果表明，hTERT-EFFs β-半乳糖苷酶染色呈阳性。序列相似性比对结果显示，马TERT与hTERT mRNA保守性较高，相似性为73.2%；马TERT与hTERT蛋白保守性较其他物种高，相似性为76.3%，表明马TERT与hTERT在结构和功能上是高度相似；马TERC与人TERC相似性较高，在进化树上位于同一分支，但马TERC与TERT的结合及与端粒酶活性相关的关键核苷酸中存在突变。【结论】单独过表达hTERT可以延长EFFs体外传代次数不超过30代，但仍不足以使蒙古马EFFs细胞高质量永生化，这可能与hTERT与eTERC不相容有关。

关键词: 蒙古马; 马胎儿成纤维细胞(EFFs); 永生化; 人端粒酶逆转录亚基(hTERT); 细胞增殖

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

中图分类号:

S821

田书岳, 神英超, 王希生, 王敏, 伊敏娜, 才文道力玛, 陶力, 赵毕力格, 芒来, 格日乐其木格. 蒙古马胎儿成纤维细胞永生化细胞体系建立的研究[J]. 中国畜牧兽医, 2023, 50(1): 46-57.

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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