哈萨克羊胎盘原代滋养层细胞的分离、纯化与基本特征鉴定  

Isolation and purification primary trophoblast cells from Kazakh sheep plancnta and identification of their basic characteristics

作　　者：陈慧慧 郝科兴 龙德智 宋美君 海思妤 王静[1] 胡广东 CHEN Huihui;HAO Kexing;LONG Dezhi;SONG Meijun;HAI Siyu;WANG Jing;HU Guangdong(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000

出　　处：《畜牧与兽医》2022年第11期5-11,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(32060751);省部共建绵羊遗传改良与健康养殖国家重点实验室开放课题(MYSKLKF201905)。

摘　　要：为了获得纯度较高的哈萨克羊胎盘原代滋养层细胞,采用组织块培养法、胰酶消化法和胰酶+组织块法分离原代滋养层细胞,通过胰酶差时消化法对细胞进行纯化,显微镜下观察滋养层细胞的形态学特点和生长特性,利用免疫细胞化学染色法对滋养层细胞的特异性表达角蛋白7(cytokeratin 7,CK7)进行鉴定,通过RT-PCR对滋养层细胞标志基因干扰素-τ(IFN-τ)进行检测,同时使用孕酮(P_(4))、雌二醇(E_(2))或P_(4)、E_(2)和IFN-τ处理滋养层细胞,实时荧光定量PCR(qRT-PCR)检测胚胎附植相关基因的转录水平。结果显示,与其他方法相比,利用胰酶+组织块法细胞生长周期较短,第2天有细胞从组织块中迁出,5~6 d细胞呈单层铺开,细胞均呈上皮样生长。免疫细胞化学染色显示,细胞CK7染色阳性结果大于95%。RT-PCR检测到分离的滋养层细胞可正常表达IFN-τ。此外,通过P_(4)、E_(2)和IFN-τ处理后,胚胎附植相关基因ISG15、CXCL10、RSAD2、CTSL、SPP1和MUC1在滋养层细胞中的表达水平显著上调。本研究通过胰酶+组织块培养法结合胰酶差时消化法成功分离和纯化哈萨克羊胎盘原代滋养层细胞,为哈萨克羊胚胎附植相关调控机制的研究奠定了基础。In order to obtain primary trophoblast cells with higher purity from Kazakh sheep,the methods of tissue block culture,trypsin digestion and trypsin and tissue block were used to isolate and culture trophoblast cells which were then purified by trypsinization.Observation was performed of the morphological characteristics and growth characteristics of the trophoblast cells under microscope.The specific expression of cytokeratin 7(CK7)in the trophoblast cells was identified by immunocytochemical staining,and the marker gene interferon-τ(IFN-τ)of the cells was detected by RT-PCR.The trophoblasts were treated with progesterone(P_(4)),estradiol(E_(2))or P_(4),E_(2) and IFN-τsimultaneously,and the transcription levels of the embryonic implantation-related genes were detected by real-time quantitative PCR(qRT-PCR).The results showed that,among the three cell culture methods,the combined use of the trypsin and tissue block methods had a shorter cell growth cycle,with the cells migrating from the tissue block on the 2^(nd) day and spreading out in a single layer on the 5^(th) to 6^(th) days.The morphology of the cells was observed under the microscope and the cells showed epithelioid growth with different cell shapes.The cells were identified by immunocytochemical staining,and the results showed that the cells were positive for CK7,with a positive rate of more than 95%.RT-PCR revealed that the isolated sheep trophoblast cells expressed IFN-τnormally.In addition,the results of treatment with P_(4),E_(2) and IFN-τshowed that the expression levels of embryo implantation-related genes ISG15,CXCL10,RSAD2,CTSL,SPP1,and MUC1 were up-regulated.In conclusion,high-purity trophoblast cells can be successfully isolated from Kazakh sheep by trypsin digestion+tissue block culture with differential trypsin digestion time.This laid foundation for future research on the regulation mechanism related to Kazakh sheep embryo implantation.

关 键 词：哈萨克羊 滋养层细胞 胰酶+组织块培养 

分 类 号：S826.9[农业科学—畜牧学]