绵羊原代Cajal间质细胞的分离培养与鉴定

Isolation,culture and identification of primary interstitial cells of Cajal derived from sheep

采集美利奴绵羊的十二指肠,在解剖显微镜下剥去黏膜层及黏膜下层,剪碎后采用Ⅱ型胶原酶和胰酶消化并分离获得绵羊十二指肠原代Cajal间质细胞(ICC);传代培养后,通过倒置显微镜观察不同时间的细胞形态,使用反转录PCR和免疫荧光染色检测ICC特异性标记物c-Kit。结果显示,成功分离了ICC,且细胞在培养24 h后贴壁,呈现出其固有的形态。RT-PCR检测c-kit基因为阳性,免疫荧光染色鉴定ICC表达c-Kit蛋白。本试验成功分离ICC且传代后细胞稳定增殖生长,为后期开展病原微生物导致ICC改变造成胃肠道炎性病变提供材料。

The duodenum of Merino sheep was collected,the mucosa and submucosa were peeled off under the anatomical microscope,and the primary interslitial cells of Cajal(ICC)of duodenum of sheep was obtained by digestion with collagenaseⅡand pancreatin after being cut.After subculture,the cell morphology at different times was observed by inverted microscope,and reverse transcription PCR and immunofluorescence staining were used to detect ICC specific marker c-Kit.The results indicated that ICC was successfully isolated,and the cells adhered to the wall after 24 hours of culture,showing its inherent morphology.RT-PCR showed that c-kit gene was positive,and immunofluorescence staining showed that ICC expressed c-Kit protein.In this study,ICC was successfully isolated and stably proliferated after subculture,providing materials for the later development of pathogenic microorganisms leading to ICC changes and gastrointestinal inflammatory diseases.