鸡肠上皮细胞原代培养与鉴定

Primary culture and identification of chicken intestinal epithelial cells

为了探讨鸡肠上皮细胞体外分离培养方法,进一步研究鸡源志贺菌Ipa C毒力蛋白在鸡肠上皮细胞上的受体蛋白分子,试验分别无菌取14,16,18日龄鸡胚肠组织,用300 U/m LⅪ型胶原酶和0.1 mg/m LⅠ型中性蛋白酶混合搅拌消化25 min,用壁差法纯化鸡肠上皮细胞,贴壁时间差为3 h,分别种植于20%FBS包被过的和未包被过的一次性塑料细胞培养瓶中,在倒置显微镜下观察细胞生长状态,透射电镜下对培养细胞进行鉴定。结果表明:采用16日龄鸡胚肠组织用Ⅺ型胶原酶和Ⅰ型中性蛋白酶联合消化25 min,可获得较多的肠隐窝单位,肠上皮细胞在20%FBS包被过的一次性细胞培养瓶中贴壁效果良好,细胞生长状态良好;透射电镜下可见鸡肠上皮细胞的微绒毛,鉴定为鸡肠上皮细胞。

To investigate a method of the in vitro isolation and culture of chicken intestinal epithelial cells, the receptor protein molecule of IpaC virulence protein from chicken Shigella was further studied. The intestinal tissues were taken aseptically from chicken embryos at 14, 16, and 18 days of age separately, and then mixed, stirred and digested with 300 U/mL XI collagenase and 0.1 mg/mL type I neutral protease for 25 min. A differential adhesion method was used to purify chicken intestinal epithelial cells and the adherence time difference was 3 h. The intestinal crypt unit was planted in the 20% FBS - coated and non - coated disposable plastic cell culture flasks, respectively. The growth state of the cells were observed under an inverted microscope, and the identification of the intestinal epithelial cells was conducted under transmission electron microscope. The results showed that more intestinal crypt units could be obtained using the intestinal tissues from 16 - day - old chicken embryos with mixed 300 U/mL Ⅺ collagenase and 0.1 mg/mL type Ⅰ neutral protease to digest for 25 rain. The intestinal epithelial cells had a better adherence result in the 20% FBS - coated disposable plastic cell culture flasks. Microvillus on the chicken intestinal epitbdial cells could be observed with transmission electron microscope, and the cultured cells were identified as chicken intestinal epithdial ceils.