新生猪肾近曲小管上皮细胞的体外培养与鉴定

Culture and Identification of Newborn Pig's Renal Tubular Epithelial Cells in vitro

目的 分离新生猪肾小管上皮细胞进行体外培养 ,为研究围生期肾小管上皮细胞生物学行为特征提供理论基础。方法 采用机械分散结合 型胶原酶 (0 .5 g/ L)消化的方法纯化肾小管片段 ,选择含 10 %的 FCS低糖DMEM培养基 ,置于 5 %CO2 ,37℃孵箱培养。 0 .2 5 %胰蛋白酶用于肾小管上皮细胞纯化和传代培养。应用细胞形态学 (光镜和电镜 )、免疫细胞化学 (Pan CK和 CK18单抗 )和酶组织化学 (酸性磷酸酶和碱性磷酸酶 )方法进行肾近曲小管上皮细胞鉴定 ;用流式细胞术做细胞纯度鉴定。结果 出生 12～ 2 4小时的新生猪肾小球直径为 5 0～ 70 μm,出生5～ 6天的肾小球直径为 70～ 98μm。新生猪肾近曲小管上皮细胞体外培养对葡萄糖的需求量不高。培养成功并传 6～8代 ,肾小管上皮细胞纯度达 95 %～ 98%。综合形态学、免疫细胞化学和酶组织化学结果 ,支持细胞来源于肾单位的近曲小管。结论 采用机械分散 -酶消化纯化肾小管 ,0 .2 5 %胰蛋白酶纯化肾小管上皮细胞及低糖 DMEM培养新生猪肾近曲小管的方法是可行的。

Objective Establish a method for the isolation and culture of newborn pig's renal tubular epithelial cells in vitro so as to provide a basis for researches on the biologic characteristics of perinatal renal tubular epithelial cells. Methods Mechanical-isolation and digesting with collagenase Ⅴ were used in purification of renal tubular fragment. 0.25% trypsase was used in purification of renal tubular epithelial cells and sub-culture. Morphological analysis ( electron microscope, light microscope), immunocytochemistry (PanCK, CK-18) and enzyme histochemistry (acid phosphatase, alkaline phosphatase) were used in identification of proximal tubular epithelial cells. Purity of tubular epithelial cells was determined by flow cytometry. Results The size of glomeruli is about 50-70μm in piglets one day old and 70-98μm in piglets 5-6 days old. Low concentration of glucose in the media may be more propitious to culture of neonatal pig's proximal tubular epithelial cells.The primary and secondary culture of proximal tubular epithelial cells were successful(the passage number could reach 6-8 times). The purity of renal tubular epithelial cells reached 95%-98%. According to the results of morphological, immunocytochemistry and enzymehistochemistry, the proximal tubules of the nephron were defined as the site of origin of the cells. Conclusion The method for isolation of renal tubular fragment with machinery-enzyme, for purification of renal tubular epithelial cells with 0.25% trypsase and for culture in media of low glucose is doable.