鸡胚小肠上皮细胞的体外分离培养和鉴定

Culture and Identification of the Chicken Intestinal Epithelial Cells Isolated from Chick Embryo in Vitro

本试验旨在提供鸡小肠上皮细胞的体外分离方法,为进一步研究鸡肠道疾病的致病机理、鸡肠道营养物质转运吸收与免疫机理提供模型。本试验采用组织块培养法来分离培养鸡小肠上皮细胞。运用刮除法、酶消化法、有限稀释法纯化鸡小肠上皮细胞。利用噻唑蓝(MTT)法来鉴定鸡小肠上皮细胞生长情况。通过实时荧光定量PCR(RT-PCR)检测鸡小肠上皮细胞特异性基因的表达来鉴定鸡小肠上皮细胞。结果表明:1)采用组织块细胞培养法能够成功获得鸡小肠上皮细胞系。2)接种组织块8h内鸡小肠上皮细胞开始大量增殖。3)鸡小肠上皮细胞的生长曲线为"S"形,符合细胞增殖规律。4)传至第5代的鸡小肠上皮细胞体积开始变大,细胞形态模糊并开始衰老凋亡;细胞传代时进行接种,大量的细胞未贴壁。综上所述,采用在6孔板内接种鸡小肠组织快,能够获得具有稳定性的鸡小肠上皮细胞,为鸡肠道疾病致病机理、鸡肠道营养物质转运吸收和永生化鸡小肠上皮细胞提供细胞素材。

This experiment was conducted to provide a stable model of chicken intestinal epithelial cell line for the study the pathogenesis of chicken intestinal diseases, chicken intestinal absorption of nutrients transport and immune mechanism. Our research applied the chicken intestinal tissue culture to successfully isolate and culture the chicken intestinal epithelial cell. We applied curettage, enzyme digestion, limiting dilution method to purify the chicken intestinal epithelial cell. MTT method was used to identify the growth curve of the chicken intestinal epithelial cell. The specific genes of chicken intestinal epithelial cells were detected by quantitative real-time PCR （RT-PCR）. The results showed as follow： 1） the chicken intestinal epithelial cells could be successfully cultured by tissue culture cells. 2 ） The chicken intestinal epithelial cells started the proliferation 8 h after seeding the tissue. 3） The＂ S＂ shape of the growth curve of the chicken intestinal epithelial cells was accordance with the law of cell proliferation. 4） The chicken intestinal epithelial cells were swollen and started senescence and apoptosis after 5 passages. And many cells were not adherent to the bottom of the plate after seeded by passage. In conclusion, seeding the chicken intestinal tissue in 6-well plate, we are able to obtain a stable chicken intestinal epithelial cells line, which provides a stable cell model to study the pathogenesis of chicken intestinal disease, the absorption of chicken intestinal nutrients transport, immortalized chicken intestinal epithelial cells line.