永生化绵羊附睾上皮细胞系的建立及其生物学特性分析

doi:10.11843/j.issn.0366-6964.2019.09.009

Abstract

Abstract: This study aimed to establish sheep immortalized epididymal cell line and provide a basis for further studying the regulation mechanism of epididymis function in sheep. The primary sheep epididymal epithelial cells were isolated and cultured by enzymatic digestion. The pCI-neo-hTERT plasmid was transferred into the sheep epididymal epithelial cells(SEECs) by using liposome and the hTERT-SEECs was screened by G418. The cell expression of keratin 18 (CK18) and human telomerase reverse transcriptase(hTERT) were identified by immunofluorescence. The expression of hTERT mRNA was detected by RT-PCR.The cell growth curve was detected by CCK-8. The growth cycle and apoptosis status were detected by flow cytometry. The ploidy was detected by karyotype analysis. The mRNA and protein expression of glutathione peroxidase 5 (GPX5) and androgen receptor (AR) were detected. The results showed that the primary sheep epididymal epithelial cell exhibited native cobblestone morphology. hTERT was transferred successfully into the SEECs. After 45 passage, the hTERT-SEECs still showed cobblestone morphology, the CK18 and hTERT could be steadily expressed in hTERT-SEECs,and the cells were in normal diploid, the proliferation ability of hTERT-SEECs was higher than that of primary SEECs, the percentage of hTERT-SEECs in G1 phase were significantly less than that of SEECs (P<0.05), while the percentage of hTERT-SEECs in S phase was significantly higher than that of SEECs (P<0.05), the percentage of living hTERT-SEECs was significantly higher than that of SEECs (P<0.05), the apoptosis rate of hTERT-SEECs was significantly less than that of SEECs (P<0.05). There were no significant difference in GPX5 and AR protein concentrations between hTERT-SEECs and SEECs (P>0.05). After long term culture, the hTERT-SEECs established in this study can maintain normal epididymal epithelial cell morphology, have strong proliferative capacity and retain the biological characteristics of epididymal epithelial cells.

CLC Number:

S826.3

SONG Huizi, LUAN Zhaojin, WANG Zhaochen, DU Wei, ZHAO Yongchao, ZHANG Jiaxin. Establishment of Sheep Immortalized Epididymal Epithelial Cell Line and Analysis of Its Biological Characteristics[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1822-1831.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2019.09.009

https://www.xmsyxb.com/EN/Y2019/V50/I9/1822

References

[1] GERVASI M G,VISCONTI P E.Molecular changes and signaling events occurring in spermatozoa during epididymal maturation[J]. Andrology,2017,5(2):204-218.
[2] 肖娟,王讯,罗毅,等.附睾小体功能蛋白及sRNA研究进展[J].遗传,2018,40(3):197-206.
XIAO J,WANG X,LUO Y,et al.Research progress in sRNAs and functional proteins in epididymosomes[J]. Hereditas, 2018, 40(3):197-206.(in Chinese)
[3] SUlLIVAN R,MIEUSSET R.The human epididymis:its function in sperm maturation[J].Hum Reprod Update,2016, 22(5):574-587.
[4] ADAM C,CYR D G.Role of specificity protein-1 and activating protein-2 transcription factors in the regulation of the gap junction protein beta-2 gene in the epididymis of the rat[J].Biol Reprod,2016,94(6):120.
[5] 范晓梅,张通,粟瑞兰,等.绒山羊附睾上皮细胞培养方法的建立及其生长特性[J].畜牧兽医学报,2017,48(7):1212-1220.
FAN X M,ZHANG T,LI R L,et al.Establishment of culture method and growth characteristics for epididymal epithelial cells from cashmere goats[J].Acta Veterinaria et Zootechnica Sinica,2017,48(7):1212-1220.(in Chinese)
[6] 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].J Pharmacol Toxicol Methods,2016,78:52-57.
[7] DE LANGE T.Shelterin:the protein complex that shapes and safeguards human telomeres[J].Genes Dev, 2005, 19(18):2100-2110.
[8] CHILTON W,O'BRIEN B,CHARCHAR F.Telomeres,aging and exercise:guilty by association?[J].Int J Mol Sci,2017, 18(12):2573.
[9] WU X Q,HUANG C,HE X,et al.Feedback regulation of telomerase reverse transcriptase:new insight into the evolving field of telomerase in cancer[J].Cell Signal,2013,25(12):2462-2468.
[10] QIN S Q,KUSUMA G D,Al-SOWAYAN B,et al.Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta[J].Placenta,2016,39:134-146.
[11] 盛宏霞,吴庆侠,靳亚平,等.山羊子宫内膜基质细胞永生化研究[J].畜牧兽医学报,2009,40(6):818-823.
SHENG H X,WU Q X,JIN Y P,et al.Immortalization of goat endometrial stromal cells by telomerase reverse transcriptase transfection[J]. Acta Veterinaria et Zootechnica Sinica,2009,40(6):818-823.(in Chinese)
[12] WANG J,HU G D,GAO W J,et al.Immortalized porcine intestinal epithelial cell cultures susceptible to porcine rotavirus infection[J]. J Virol Methods,2014,202:87-94.
[13] 高雯,臧荣余,王雁,等.人类输卵管上皮永生化细胞系的建立及鉴定[J].中国癌症杂志,2013,23(4):241-247.
GAO W,ZANG R Y,WANG Y,et al.Immortalization of human fallopian tube epithelial cells[J].China Oncology,2013,23(4):241-247.(in Chinese)
[14] HONG H X,ZHANG Y M,XU H,et al.Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase[J].Mol Cells,2007,24(3):358-363.
[15] 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.
LI J X.Immortalization of bovine mammary epithelial cell with multipotency[D].Yangling:Northwest A&F University,2010.(in Chinese)
[16] KWAK S,JUNG J E,JIN X,et al.Establishment of immortal swine kidney epithelial cells[J].Anim Biotechnol,2006,17(1):51-58.
[17] FURIHATA T,ITO R,KAMⅡCHI A,et al.Establishment and characterization of a new conditionally immortalized human astrocyte cell line[J].J Neurochem,2016,136(1):92-105.
[18] HE Y L,WU Y H,HE X N,et al.An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer[J].Theriogenology,2009,71(9):1417-1424.
[19] LI Z H,KREINER M,EDRADA-EBEL R A,et al.Perfusion culture enhanced human endometrial stromal cell growth in alginate-multivalent integrin α5β1 ligand scaffolds[J].J Biomed Mater Res A,2011,99(2):211-220.
[20] BASSOLS J,KÁDÁR E,BRIZ M D,et al.In vitro culture of epithelial cells from the caput,corpus,and cauda epididymis of Sus domesticus[J].Theriogenology,2004,62(5):929-942.
[21] ARAKI Y,SUZUKI K,MATUSIK R J,et al.Immortalized epididymal cell lines from transgenic mice overexpressing temperature-sensitive simian virus 40 large T-antigen gene[J].J Androl,2002,23(6):854-869.
[22] HU H,ZHENG N,GAO H N,et al.Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro[J].Cell Biol Int,2016,40(8):861-872.
[23] PARK H J,LEE W Y,JEONG H Y,et al.Regeneration of bovine mammary gland in immunodeficient mice by transplantation of bovine mammary epithelial cells mixed with matrigel[J].Int J Stem Cells,2016,9(2):186-191.
[24] 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.
WU Q X,DONG H L,LIU Z Y,et al.Establishment of goat immortalised endometrium epithelial cell line and study on its biological characteristics[J].Journal of China Agricultural University,2017,22(2):73-79.(in Chinese)
[25] LI J X,SAID A,GE X G,et al.Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions[J].Cytotechnology,2017,70(1):67-82.
[26] GUCALP A,TRAINA T A.The androgen receptor:is it a promising target?[J].Ann Surg Oncol,2017,24(10):2876-2880.
[27] LEIR S H,BROWNE J A,EGGENER S E,et al.Characterization of primary cultures of adult human epididymis epithelial cells[J]. Fertil Steril,2015,103(3):647-654.e1.
[28] O'HARA L,WELSH M,SAUNDERS P T K,et al.Androgen receptor expression in the caput epididymal epithelium is essential for development of the initial segment and epididymal spermatozoa transit[J].Endocrinology,2011,152(2):718-729.
[29] LI R L,FAN X M,ZHANG T,et al.Expression of selenium-independent glutathione peroxidase 5(GPx5) in the epididymis of Small Tail Han sheep[J].Asian-Australas J Anim Sci,2018,31(10):1591-1597.
[30] TAYLOR A,ROBSON A,HOUGHTON B C,et al.Epididymal specific,selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation[J].Hum Reprod,2013,28(9):2332-2342.
[31] BARRANCO I,TVARIJONAVICIUTE A,PEREZ-PATIÑO C,et al.Glutathione peroxidase 5 is expressed by the entire pig male genital tract and once in the seminal plasma contributes to sperm survival and in vivo fertility[J].PLoS One,2016, 11(9):e0162958.

Establishment of Sheep Immortalized Epididymal Epithelial Cell Line and Analysis of Its Biological Characteristics

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[2]	SONG Pengyan, WANG Siwei, YUE Qiaoxian, ZHANG Yinliang, CHEN Xiaoyong, ZHOU Rongyan. Identification of oar-miR-200b Promoter and Effects on Mitochondrial Function in Follicular Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5066-5076.
[3]	HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.
[4]	SONG Meijun, HAO Kexing, HAI Siyu, CHEN Yan, WANG Jing, HU Guangdong. Effects of SRIF-14 on Proliferation and Apoptosis of Endometrial Epithelial Cells in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3325-3334.
[5]	LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.
[6]	HAN Xiuyuan, ZHAO Liang, WANG Chuang, QI Meiyu, YAO Yuchang. Nicotinic Acid Enhances Low Temperature Preservation of Sheep Sperm by Reducing Oxidative Stress Levels [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1979-1989.
[7]	YANG Chulei, ZHANG Yiyuan, TANG Hong, GUO Yanhua, REN Xiumeiao, WANG Limin, ZHOU Ping. Research Progress of FecB Gene on Reproductive Performance of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1370-1380.
[8]	LI Yu, DUAN Chunhui, SONG Zhipan, YUE Sicong, WANG Yuan, ZHANG Yingjie, LIU Yueqin. Effects of PGF2α on Luteal Tissue Morphology, Expression of PGF2α Receptors and Necroptosis-associated Genes During Luteal Phase in Hu Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1500-1510.
[9]	WANG Yahan, ZHENG Yujing, ZHONG Pei, LI Xiaodan, WANG Feng. Expression Patterns and Functional Analysis of YAP1 in Endometrium of Hu Sheep with Different Fertility during Estrus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 189-200.
[10]	WANG Mengyao, ZHAI Zhenhan, ZHAO Lu, WANG Saiqiao, WANG Yuqin. Identification and Functional Analysis of lncRNA in Ovaries of Sheep at Different Stages of Estrus Cycle [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4221-4231.
[11]	LIU Xinjie, WU Xiaoxue, LIU Suping, YUAN Liming, CHEN Ning, SAIWU Jiafu. Preparation of Polyclonal Antibody against PLC-γ1 and Preliminary Study on Its Effect on Early Embryo Development in Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 3842-3855.
[12]	LIU Xinjie,WU Xiaoxue,LIU Suping,YUAN Liming,CHEN Ning,SAIWU Jiafu. Effects of Sheep Phospholipase C-γ1 on in vitro Maturation of Sheep Oocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3459-3469.
[13]	WU Xiaoxue, YUAN Liming, LIU Xinjie, LIU Suping, CHEN Ning, SAIWU Jiafu. Effect of PLCγ1 Gene on the Development of Sheep Early Embryo in vitro [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2578-2585.
[14]	YAN Shuo, ZHAO Shanshan, ZHU Zhendong, PAN Qingjie, DONG Huansheng. Study on Nuclear-plasma Transporter KPNA4 of Sheep Sperm Cell [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2194-2201.
[15]	LI Yu, DUAN Chunhui, SONG Zhipan, YUE Sicong, WANG Yuan, ZHANG Yingjie, LIU Yueqin. Effects of PGF2α on Reproductive Hormones and Related Cytokines during Luteal Phase in Ewes [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1807-1818.