新疆军垦细毛羊肾小管上皮细胞的原代培养及鉴定

【目的】建立一种高效、实用的新疆军垦细毛羊肾小管上皮细胞原代培养方案,为肾脏损伤引起的疾病研究奠定基础。【方法】取5～8个月大的出栏细毛羊肾脏皮质,剪成1～2 mm3大小组块,分别采用传统组织块法和改良组织块法,在含15%～20%新生小牛血清的DMEM的培养基中进行原代培养,无需研磨或使用酶和化学试剂,根据上皮细胞自身的生长特性,首次结合刮除法,相差消化法和差速贴壁法,分离纯化上皮细胞,再选用0.037 mm孔径网筛过滤除去肾小球上皮细胞,分离纯化培养肾小管上皮细胞,细胞免疫组化进行鉴定。【结果】改良方法与传统方法相比,细胞贴壁早,生长快,处理时间短,减少了污染机率,提高了细胞培养效率,分离培养的肾小管上皮细胞,纯度达90%以上,细胞呈多角形,鹅卵石状,折光性强,连接紧密,可传至第9代,免疫细胞化学(CK-18)染色鉴定为阳性。【结论】改良的组织块培养与传统的组织块培养以及常用的酶消化培养相比,经济、简单、不易污染,分离培养的肾小管上皮细胞,活性高,纯度高,是一种高效、实用的细毛羊肾小管上皮细胞培养方法。

Primary Culture and Identification of Renal Tubular Epithelial Cells of Xinjiang Fine-wool Sheep

【Objective】 The projected aimed to explore an effective and practical method for primary culture of Xinjiang fine-wool sheep renal tubular epithelial cells,so as to establish the basis for the study of diseases caused by kidney damage.【Method】Renal cortex of fine-wool sheep at 5-8 months old were chosen and cut into pieces of 1-2 mm3 Traditional and modified tissue culture methods were used,and cultured in DMEM supplemented with 15%-20%newborn calf serum,without grinding or using enzymes and chemicals.All this was done based on the characteristics of epithelial cell.The renal tubular epithelial cells were separated and purified by combining scraping with the different time for cell digestion and attachment,and then filtered by 0.037 mm aperture sieve to remove glomeruli epithelial cells.The isolated cells were identified by immunohistochemistry.【Result】Compared with the traditional one,the modified method could lead to this result: cells adhered early and grew fast,low pollution was produced and high efficiency of cell culture could be reached.Renal tubular epithelial cells were successfully cultivated and isolated,and the purity reached as high as 90%.The cultured cells displayed polygon,cobblestone layout and strong refraction,tight connection,and they were successively propagated nine times.They were identified as cells of epithelial origin by immunohistochemistry(CK-18).【Conclusion】Compared with traditional tissue culture and enzymatic digestion culture,this economic,simple and pollution-free culture system is an effective one to cultivate renal tubular epithelial cells with high activity and purity.