H-2K^b-tsA58转基因小鼠肾小球内皮和足细胞株的分离培养与鉴定

Isolation,Cultivation and Characterization of Glomerular Endothelial,Podocyte Cell Lines from H-2K^b-tsA58 Transgenic Mice

目的：利用H-2K^b-tsA58转基因小鼠的条件永生性,从肾脏中分离、纯化和培养肾小球内皮细胞株和足细胞株。方法：筛网法分离肾小球,利用内皮细胞表面特异性抗原CD31的表达,采用免疫磁珠初步分离肾小球内皮细胞,结合限制性稀释法获取均一细胞来源的细胞克隆后持续传代培养。间接免疫荧光法鉴定分离纯化后的细胞特性。结果：分离纯化后的细胞克隆在33℃和γ-干扰素诱导下具有无限增殖能力,37℃无γ-干扰素条件下细胞趋于分化成熟。所获得的内皮细胞株特异性表达CDl31,并形成管腔样结构；所获得的足细胞分化成熟,形成大量足突结构并特异性表达成熟足细胞的标志性抗原nephrin和synaptopodin。结论：本研究成功地建立了获取具有成熟足细胞特征的条件永生性小鼠足细胞株和肾小球内皮细胞株的技术方法,为体外研究这两种细胞的功能及其在疾病中的作用提供了条件。

Objective：Establish glomerular endothelial and podocyte cell lines from conditionally immortal mice. Methods：Glomeruli were isolated with sieving method. Glomerular endothelial cells grown from glomeruli were isolated from magnetic beads loaded with CD31. Different cell clones were obtained by limiting dilution and characterized by indirect immunofluorescence. Resulls：Clonal cell lines proliferated infinitely at 33C with induction ofγ-interferon, while differentiated at 37℃ in the absence ofγ-interferon. Glomerular endothelial clone formed tube-like structures and was positive for CD31 at 37℃. In same conditions, differentiated podocyte clone with foot processes formation expressed both nephrin and synaptopodin which were exclusively expressed on podocytes in kidney in vivo. Conclusion： The glomerular endothelial and podocyte cell lines of conditionally immortal mice were obtained, which provides an important tool in vitro to understand their functions and roles in diseases.