山羊乳中乳腺上皮细胞的体外分离培养及鉴定

Isolation,culture and identification of goat mammary epithelial cells in goat milk in vitro

为了探索获得乳腺上皮细胞(MECs)的新途径,试验采用乳汁分离法分离培养山羊乳腺上皮细胞(GMECs),并以山羊耳缘成纤维细胞(GEFs)为对照,通过形态学观察、免疫荧光染色观察细胞形态,油红O染色检测脂滴形成情况;实时荧光定量PCR检测特异性基因;Western-blot检测乳腺分泌的特异性蛋白;此外,对GMECs和GEFs进行转录组测序(RNA-seq)分析及GO功能注释和KEGG信号通路富集分析。结果表明:从山羊奶中分离出来的细胞表现出MECs的结构特征;与GEFs对照相比,GMECs中乳腺上皮特异性基因CDH1、LTF、CSN2、CSN3、EpCAM、KRT18、ELF3和ELF5的相对表达量在第8代(GMEC8)极显著增加(P<0.01),GMEC8可以表达CDH1、EpCAM、CSN2和LTF等蛋白;共获得4481个明显差异表达的基因,其中上调基因1975个,下调基因2506个;上皮的形态发生和上皮发育为显著富集的GO条目;乳腺发育相关的信号通路如WNT、PI3K-Akt和MAPK信号通路明显富集。说明从山羊奶中可以分离出GMECs。

To explore a novel approach to obtain the mammary epithelial cells(MECs),goat mammary epithelial cells(GMECs)were isolated and cultured by milk separation and compared with goat ear border fibroblasts(GEFs).Morphological observation and immunofluorescence staining were used to observe cell morphology,and oil red O staining was used to detect lipid droplet formation.Real-time fluorescent quantitative PCR was used to detect specific genes.Western-Blot was used to detect protein secreted in mammary epithelial cells.In addition,transcriptome sequencing(RNA-seq)analysis and GO functional annotation and KEGG signaling pathway enrichment were performed on GMECs and GEFs.The results showed that the cells isolated from goat milk showed structural characteristics of MECs.The expression of mammary epithelial-specific genes CDH1,LTF,CSN2,CSN3,EpCAM,KRT18,ELF3,and ELF5 in GMECs was highly significantly increased(P<0.01)in the 8th generation(GMEC8)compared to the GEFs control,and the GMEC8 could express proteins such as CDH1,EpCAM,CSN2,and LTF;a total of 4481 significantly expressed genes,including 1975 up-regulated genes and 2506 down-regulated genes were obtained;epithelial morphogenesis and epithelial development were significantly enriched GO entries;mammary gland development-related signaling pathways such as WNT,PI3K-Akt and MAPK signaling pathways were significantly enriched.It indicated that GMECs could be isolated from goat milk.