永生化绵羊瘤胃成纤维细胞系的建立

doi:10.12160/j.issn.1672-5190.2021.03.001

畜牧与饲料科学 ›› 2021, Vol. 42 ›› Issue (3): 1-6.doi: 10.12160/j.issn.1672-5190.2021.03.001

• 基础研究 • 下一篇

永生化绵羊瘤胃成纤维细胞系的建立

赵霏霏^1,2, 杨丹^1,2, 辛雅明^1,2, 杨银凤^1,2

1.内蒙古农业大学兽医学院,内蒙古呼和浩特 010018;
2.内蒙古自治区兽医学重点实验室,内蒙古呼和浩特 010018

收稿日期:2021-03-08 出版日期:2021-05-30 发布日期:2021-06-21
通讯作者: 杨银凤（1963—）,女,教授,博士,博士生导师,主要研究方向为动物解剖及黏膜免疫学。
作者简介:赵霏霏（1991—）,男,硕士研究生,主要研究方向为动物解剖及黏膜免疫学。
基金资助:
国家自然科学基金项目（32060780）

Establishment of Immortalized Ovine Ruminal Fibroblast Cell Line

ZHAO Fei-fei^1,2, YANG Dan^1,2, XIN Ya-ming^1,2, YANG Yin-feng^1,2

1. College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;
2. Inner Mongolia Key Laboratory of Basic Veterinary Medicine,Hohhot 010018,China

Received:2021-03-08 Online:2021-05-30 Published:2021-06-21

摘要/Abstract

摘要： [目的]建立永生化绵羊瘤胃成纤维细胞系,为基础研究和动物病毒的研究提供稳定的体外细胞模型。[方法]采用组织块贴壁法培养绵羊瘤胃成纤维细胞（ovine ruminal fibroblasts cells,ORFCs）,利用PEI试剂将带有hTERT基因的pCI-neo-hTERT质粒转染ORFCs,经G418筛选得到阳性克隆细胞。采用RT-PCR、Western Blot检测F₁₅和F₃₅代阳性克隆细胞的hTERT基因的转录和表达情况;利用血球计数法绘制原代ORFCs和F₃₅代阳性克隆细胞的生长曲线比较其增殖能力。[结果]将hTERT基因成功转入ORFCs并稳定表达;阳性克隆细胞的增殖能力显著高于原代ORFCs。[结论]该试验成功建立了永生化绵羊瘤胃成纤维细胞系。

关键词: 绵羊瘤胃成纤维细胞系, 永生化, 人源端粒酶逆转录酶

Abstract: [Objective] To establish immortalized ovine ruminal fibroblast cell line and to provide stable in vitro cell model for basic research and animal virus research. [Method] Ovine ruminal fibroblast cells（ORFCs） were cultured by using tissue fragment clinging method. pCI-neo-hTERT plasmid with hTERT gene was transferred into ORFCs using PEI reagents. The positive clone cells were screened by G418. The transcription and expression level of hTERT gene in 15^th and 35^th generation positive clone cells were detected by RT-PCR and Western Blot. The growth curves of primary cells and 35^th generation positive clone cells were drawn with blood cell count method to assess the proliferation ability. [Result] The hTERT gene was successfully transferred into ORFCs and stably expressed. The proliferation ability of positive clone cells was significantly higher than that of primary ORFCs. [Conclusion] Immortalized ovine ruminal fibroblast cell line is successfully established.

Key words: ovine ruminal fibroblast cell line, immortalization, human telomerase reverse transcriptase

中图分类号:

Q813

赵霏霏, 杨丹, 辛雅明, 杨银凤. 永生化绵羊瘤胃成纤维细胞系的建立[J]. 畜牧与饲料科学, 2021, 42(3): 1-6.

ZHAO Fei-fei, YANG Dan, XIN Ya-ming, YANG Yin-feng. Establishment of Immortalized Ovine Ruminal Fibroblast Cell Line[J]. Animal Husbandry and Feed Science, 2021, 42(3): 1-6.

参考文献

[1] 韦云芳,万九生,李静,等.昆明犬胎儿成纤维细胞系的建立及生物学特性分析[J].中国畜牧兽医,2014, 41(12):161-166.
[2] SIKORA E, BIELAK-ZMIJEWSKA A, MOSIENIAK G.What is and what is not cell senescence[J].Postepy Biochemii,2018,64(2): 110-118.
[3] KIM C W, GO R E, LEE G A, et al.Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization[J].Journal of Pharmacological and Toxicological Methods, 2016, 78:52-57.
[4] BAI H, SAKURAI T, BAI R, et al.Establishment and characterization of immortalized bovine endometrial epithelial cells[J].Animal Science Journal,2014,85(8):799-804.
[5] DE LANGE T.Shelterin: the protein complex that shapes and safeguards human telomeres[J].Genes and Development,2005,19(18):2100-2110.
[6] LI S.Cell-cycle-dependent telomere elongation by telomerase in budding yeast[J].Bioscience Reports, 2011,31(3):169-177.
[7] 吴庆侠,董海龙,刘忠艳,等.永生化山羊子宫内膜上皮细胞系的建立及其生物学特性[J].中国农业大学学报,2017,22(2):73-79.
[8] WANG J, HU G, GAO W, et al.Immortalized porcine intestinal epithelial cell cultures susceptible to porcine rotavirus infection[J].Journal of Virological Methods,2014,202:87-94.
[9] YANG J, TANG Y, CHEN W, et al.Establishment and characterization of an immortalized human chondrocyte cell line[J].Biotechnology Letters,2020,42:707-716.
[10] 李吉霞. 牛乳腺上皮细胞永生化及其多能性的研究[D].杨凌:西北农林科技大学,2010.
[11] 宋慧子,栾兆进,王兆琛,等.永生化绵羊附睾上皮细胞系的建立及其生物学特性分析[J].畜牧兽医学报,2019,50(9):1822-1831.
[12] 刘腾,董丹丹,张莉,等.永生化绵羊肺成纤维细胞系的构建及鉴定[J].中国动物传染病学报,2018,26(2):54-58.
[13] 马时荣,严蕊琳,李玉林,等.兔骨折创伤修复时成纤维细胞的增生与凋亡[J].吉林大学学报(医学版),2005(1):61-63.
[14] 陈小云,张敏,王磊,等.端粒酶在细胞永生化中的应用[J].中国兽药杂志,2013,47(4):53-57.

永生化绵羊瘤胃成纤维细胞系的建立

Establishment of Immortalized Ovine Ruminal Fibroblast Cell Line

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

本文评价

[1]	程明,刘开东,张宝旬,孙友德,李培培,杨培培,王建华,刘宗正. 崂山奶山羊骨髓间充质干细胞永生化细胞系的建立[J]. 畜牧与饲料科学, 2019, 40(1): 11-14.
[2]	辛守帅,张宝旬,刘开东,李培培,杨培培,王建华,刘宗正. 崂山奶山羊永生化骨髓间充质干细胞的诱导分化研究[J]. 畜牧与饲料科学, 2019, 40(1): 1-5.