绵羊胎盘滋养层细胞的体外培养与鉴定

Culture and identification of sheep trophoblast cells in vitro

为建立一种稳定、方便地获得较高纯度的绵羊绒毛膜滋养层细胞的方法,为后续的细胞转染和RNA干扰试验提供细胞学基础,采用组织块法培养滋养层细胞,消化差速法纯化细胞,相差显微镜观察滋养层细胞的形态学特点,应用常规HE染色和免疫组织化学染色、透射电镜及PCR技术鉴定细胞来源。结果显示,倒置相差显微镜下,可见细胞为上皮样细胞形态,呈片状铺展生长。免疫组织化学染色显示,细胞角蛋白染色阳性细胞占90%以上,波形蛋白染色呈阴性;透射电镜可观察到滋养层细胞所特有的结构;台盼蓝排斥试验检测细胞活力,存活率超过95%,细胞活力良好。常规RT-PCR技术可扩增出滋养层细胞所分泌的特有的干扰素蛋白-1基因片段。证实该方法可以有效获得较高纯度的、具有生物学活性的绵羊绒毛膜滋养层细胞,为本实验室后续的体外研究提供试验基础。

To establish a stable and convenient method for preparation of highly purified trophoblast cells from sheep placenta and to provide the cellular basis for the study of cell transfection and RNA interference test,the sheep trophoblast cells were cultured by explant culture technique,and purified by digestion differential method.Phase-contrast microscope was used to observe the morphological characteristics of trophoblast cells,and general HE staining,immunohistochemical staining,electron microscopy and RT-PCR technology were used to identify the cell source.Inverted phase contrast microscope observation showed that the cells were the epithelial-like cell morphology,paving-like growth.Keratin staining showed that the purity of the cultured cells exceeded 90%,vimentin staining was negative.The unique structure of trophoblast cells could be observed by transmission electron microscopy.The cell viability evaluated by trypan blue exclusion test was greater than 95%.Conventional RT-PCR technique can detect trophoblast cells secreted interferon specific protein-1 gene.The results showed that this primary culture method can provide highly purified sheep trophoblast cells with good cellular viability.The present study provides an experimental basis for our laboratory in vitro studies.