大鼠足细胞的原代培养及其Nephrin的特异表达

发布时间：2018-03-12 15:20

本文选题：足细胞　切入点：细胞培养技术　出处：《广东医学》2017年06期 　论文类型：期刊论文

【摘要】：目的建立简便的大鼠足细胞原代培养体系,检测其去氧肾上腺素(Nephrin)mRNA及蛋白的表达水平。方法无菌取肾,分离肾皮质,胶原酶消化法分离肾小球,培养9~10 d,胰蛋白酶差异消化法传代,过筛去除肾小球继续培养5~7 d:形态学观察和间接免疫荧光法进行足细胞鉴定;流式细胞仪分析足细胞纯度;实时定量PCR(qRT-PCR)、间接免疫荧光法和Western blot法比较前三代足细胞中Nephrin mRNA及蛋白的表达水平。结果接种5 d后大部分肾小球贴壁,肾小球周围有细胞爬出,其最外缘有树枝状细胞分布,形态学及特异性抗体Nephrin、WT-1鉴定为足细胞;流式细胞仪分析足细胞纯度为94.7%;间接免疫荧光和qRT-PCR均显示:与第二、三代足细胞相比,第一代足细胞中Nephrin mRNA和蛋白表达均较高(均P0.05);Western blot示体外培养大鼠足细胞表达两种分子量的Nephrin,且第一代足细胞Nephrin灰度值高于第二、三代足细胞(P0.05)。结论酶消化法结合差异过筛法接种可促进肾小球贴壁,有利于培养纯度高的足细胞;大鼠足细胞体外培养条件下能特异性表达Nephrin结构及功能蛋白。
[Abstract]:Objective to establish a simple primary culture system of rat podocytes and to detect the expression of Nephrinn mRNA and protein in rat podocytes. After culture for 9 ~ 10 days, trypsin differential digestion method was passed through and the glomeruli were removed from the screen for 5 ~ 7 days. Morphological observation and indirect immunofluorescence assay were used to identify the podocytes, flow cytometry was used to analyze the purity of podocytes. The expression levels of Nephrin mRNA and protein in the first three generations of podocytes were compared with indirect immunofluorescence assay and Western blot method. Results after 5 days of inoculation, most of the glomeruli adhered to the wall, the cells around the glomeruli crawled out, and the dendritic cells were distributed in the outer edge of the cells. Morphology and specific antibody Nephrinn WT-1 were identified as podocytes. Flow cytometry showed that the purity of podocytes was 94.7. Indirect immunofluorescence and qRT-PCR showed that compared with the second and third generation podocytes, The expression of Nephrin mRNA and protein in the first generation of podocytes was higher than that in the first generation of podocytes (both P0.05 blot showed that the rat podocytes expressed two molecular weights of Nephrinin, and the grayscale value of Nephrin in the first generation of podocytes was higher than that in the second generation. Conclusion Enzymatic digestion combined with differential sieve method can promote glomerular adhesion and promote the culture of podocytes with high purity, and rat podocytes can specifically express Nephrin structure and functional proteins in vitro.
【作者单位】：石河子大学医学院;石河子大学医学院第一附属医院肾内科;
【基金】：国家自然科学基金资助项目(编号:81160090)
【分类号】：R692

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