永生化猪乳腺上皮细胞系的建立  

Establishment of Immortalized Porcine Mammary Epithelial Cell Line

作　　者：曹家铭 阚尧 李志远 钟友刚[2] 李秋艳[1] CAO Jia-Ming;KAN Yao;LI Zhi-Yuan;ZHONG You-Gang;LI Qiu-Yan(College of Biological Sciences,China Agricultural University,Beijing 100193,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学生物学院,北京100193 [2]中国农业大学动物医学院,北京100193

出　　处：《农业生物技术学报》2023年第12期2644-2653,共10页Journal of Agricultural Biotechnology

基　　金：国家现代农业产业技术体系(CARS-36)。

摘　　要：猪乳腺上皮细胞(porcine mammary epithelial cells,PMECs)在体外培养条件下依然具有高度分化的特性,可以维持其特有的形态特征及合成和分泌乳蛋白的功能。因此,建立可以维持猪乳腺上皮细胞特性的稳定的永生化细胞系对猪乳腺发育、分化及泌乳机制研究有着重要意义。本研究采用组织块培养法和酶消化法分离纯化妊娠末期母猪(Sus scrofa)乳腺上皮细胞,并对细胞培养方法进行优化。将人端粒酶逆转录酶基因(human telomerase reverse transcriptase,hTERT)电转染导入原代培养的猪乳腺上皮细胞,通过PCR检测获得永生化猪乳腺上皮细胞系hTERT-PMECs。对连续传代培养60代的hTERTPMECs进行细胞形态学观察,初步证明永生化后细胞类型没有发生改变;免疫荧光细胞染色结果表明,hTERT-PMECs可表达角蛋白;经催乳素、氢化可的松和胰岛素-转铁蛋白-硒(Insulin-Transferrin-Selenium,ITS)的诱导,反转录-PCR(reverse transcription-PCR,RT-PCR)鉴定结果显示,hTERT-PMECs仍具有分泌乳蛋白的功能。综上所述,本研究成功获得了永生化的猪乳腺上皮细胞系hTERT-PMECs,且永生化细胞维持了与原代细胞相同的特性。本研究为猪乳腺发育以及泌乳机制的研究提供了基础资料。Porcine mammary epithelial cells(PMECs)are highly differentiated in vitro and can maintain their unique morphological characteristics and function of producing and secreting milk proteins.Therefore,it is of great significance to establish a stable immortable PMECs line that can maintain the characteristics of PMECs for the study of the development,differentiation and lactation mechanism of porcine mammary gland.In this study,tissue block culture method and enzymatic digestion method were used to isolate and purify mammary epithelial cells from sows(Sus scrofa)at the end of pregnancy,and the cell culture method was optimized.Human telomerase reverse transcriptase(hTERT)gene was transfected into primary cultured pig mammary epithelial cells,by PCR detection,the immortalized hTERT-PMECs was obtained.The morphological observation of hTERT-PMECs cultured continuously for 60 generations showed that the type of hTERTPMECs cells did not change after immortalization.The results of immunofluorescence staining showed that hTERT-PMECs could express keratin.After the induction of prolactin,hydrocortisone and Insulin-Transferrin-Selenium(ITS),hTERT-PMECs was identified by reverse transcription-PCR(RT-PCR),the result showed that hTERT-PMECs still had the function of secreting lactoprotein.In summary,this study successfully obtained the immortalized porcine mammary epithelial cell line hTERT-PMECs,and the immortalized cells maintained the same characteristics as the primary cells.This study provides basic data for the study of mammary gland development and lactation.

关 键 词：永生化 人端粒酶逆转录酶基因(hTERT) 电转染 猪乳腺上皮细胞(PMECs) 

分 类 号：S828.99[农业科学—畜牧学]