SD大鼠肾小管上皮细胞的原代培养与鉴定.docVIP

SD大鼠肾小管上皮细胞的原代培养和鉴定 （作者:___________单位: ___________邮编: ___________） 作者：何锦园,曾国华,吴文起,钟文,桂志明,麦赞林 【摘要】 目的：探讨大鼠肾小管上皮细胞的体外培养及鉴定方法，为肾结石病的研究提供实验平台。方法：采用机械研磨、Ⅰ型胶原酶消化法分离出肾小管节段，在含10%胎牛血清和1%上皮细胞生长因子的上皮细胞培养基中培养，0.25%胰蛋白酶消化后传代培养，并用原代和传1代细胞做免疫细胞化学染色鉴定。结果：细胞培养至第3天完全贴壁，4～7 d处于对数生长期，为多边鹅卵石样;免疫细胞化学染色显示cytokeratin 18表达阳性。结论:机械研磨、Ⅰ型胶原酶消化法结合使用上皮细胞培养基可以培养得到较纯的肾小管，是大鼠肾小管上皮细胞原代培养的理想方法,为进一步研究泌尿系结石病因和机制提供了实验基础。 【关键词】 肾小管上皮细胞; 原代培养; CK18; 大鼠 　　[Abstract] Objective： To discuss the method of the primary culture and identification of SD rats renal tubular epithelial cells, thus to supply an experimental platform for the study of renal calculi.Methods： The renal tubular segments were isolated by mechanical grinding and collagenase Ⅰdigestion. The collected cells were cultured with EpiCM,which contained 10% FBS and 1% EpicGs, in incubation with 5% CO2 at 37 ℃, and subcultured with 0.25% trypsin.The cultured cells were identified by immunocytochemistry. Results: The cultured cells were completely adhered after 3 days, the logarithmic growth phase was beween 4 and 7 days. The cells were large, displaying the typical cobblestone appearance.Immunocytochemistry suggested that the expressions of cytokeratin 18 in renal tubular epithelial cells were positive. Conclusion: Mechanical grinding and collagenase Ⅰdigestion combined with EpiCM is an ideal method to collect and culture the renal tubular epithelial cells,and it provides the experimental basis for further study of the etiology and mechanism of urinary tract stones. 　　[Key words] renal tubular epithelial cells; primary culture; CK18; rats 　　尿石症是泌尿外科最常见的疾病之一，主要病因包括遗传、环境、饮食、代谢等。一般认为,它的形成是一系列化学的、生化的、生理的及分子调节等因素综合作用的结果[1]，其早期病变是微小晶体黏附在肾小管上皮细胞表面并引起损伤。可见，肾小管上皮细胞可以作为进一步研究尿石症病因及发病机制的一个实验平台。目前，国外已建立部分种属的肾小管上皮细胞株，但价格昂贵且不易获得。本实验旨在探讨一种理想的肾小管上皮细胞原代培养方法。 　　1 材料和方法 　　1.1 材料 　　实验动物：SD大鼠2只，一雄一雌，体重分别为220 g和200 g，由广州中医药大学动物实验中心提供[许可证号为SCXK(粤)20080020粤监证字2008A002]。主要试剂：胎牛血清(Gibco公司)、上皮细胞培养基EpiCM(美国ScienCell公司)、Ⅰ型胶原酶(Sigma公司)、胰蛋白酶(Gibco公司)、青/链霉素(Sigma公司)、cyt