绵羊气管上皮细胞的分离培养及绵羊肺炎支原体对其的吸附

Isolation and culture of sheep tracheal epithelium and establishment of adherence of Mycoplasma ovipneumoniae to host cells

为建立简便、高效、稳定的绵羊气管上皮细胞的体外培养及鉴定的方法,并在所培养的细胞上研究绵羊肺炎支原体侵染模型,本研究利用链蛋白酶冷消化法对绵羊气管上皮细胞进行体外分离,通过差速贴壁法纯化气管上皮细胞并进行传代后用液氮保存,细胞复苏后通过测定生长曲线、上皮细胞骨架蛋白角蛋白8和18以及对染色体与微生物的检测等对所培养细胞进行鉴定,并将鉴定纯化的气管上皮细胞用绵羊肺炎支原体进行侵染。结果显示,成功培养出绵羊气管上皮细胞,纯化的细胞在显微镜下排列紧密,形态均一,呈鹅卵石铺路样。细胞生长曲线呈"S"型,分子生物学手段和增菌试验未检测到气管上皮细胞中含有微生物污染,通过细胞免疫组化检测到了细胞角蛋白8、18,染色体核型数目为2n=54。绵羊肺炎支原体能黏附于分离纯化的绵羊支气管上皮细胞,为进一步研究支原体侵染细胞的机制等奠定了基础。

It was to set up a simple,efficient and stable method to culture and identify sheep trachea epitheliumin vitro,and also to study adhesion to host cells.Sheep trachea epithelium cells were isolated by sterile sheep tracheal.Pronase XIV was poured into tracheal and digested.The process of cultivation using differential velocity adherents could purify tracheal epithelium cells,and then were cultured up to passages.After cryopreservation,the growth curve,keratin 8,keratin 18,karyotype,microorganism of recoverd cells were identified.With observed by microscope,the purified cells displayed tightly arranging,a typical cobblestone appearance,homogeneous cell morphology.The cell growth curve was＂S＂.keratin 8and keratin 18 were positively expressed in the cells by immunofluorescence,microorganism were not found in the cells.The karyotype result was the same as sheep.Mycoplasma ovipneumoniae adhered to sheep tracheal epithelium was observed.This research provided a fundation on the adherence mechanism of Mycoplasma ovipneumoniae.