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小鼠肾小球分离方法比较及原代足细胞鉴定
Comparison of two methods for glomerular separation and identification of primary podocytes in mice
目的 通过比较小鼠肾小球分离技术,明确小鼠原代足细胞培养的最优方法。方法 分别采用差异过筛法和免疫磁珠法分离C57/BL6J小鼠肾小球,用Ⅳ型胶原酶消化去除包曼囊,用相差显微镜观察肾小球纯度和形态结构,采用免疫荧光染色和聚合酶链反应法(polymerase chain reaction,PCR)鉴定足细胞的标志蛋白Podocin的分布和表达。结果 差异过筛法获得肾小球纯度达90.2%±1.6%,一只C57/BL6J小鼠可以得到(10 421±2421)个肾小球;视野中肾小球结构完整,可见肾小管碎片。免疫磁珠法获得的肾小球纯度达96.7%±1.2%,一只C57/BL6J小鼠可以得到(16 112±2651)个肾小球;视野中肾小球结构完整,基本无肾小管。免疫磁珠法获得肾小球纯度和数目均高于差异过筛法,差异均有统计学意义(P＜0.05)。7～10 d可见肾小球周围大量多边形细胞爬出,呈铺路石样外观,符合足细胞特征;免疫荧光染色和PCR法均证实有足细胞特异性蛋白Podocin表达。结论 C57/BL6J小鼠肾小球的分离,免疫磁珠法明显优于差异过筛法,可以更简便、高效地培养出原代足细胞。
Objective To find the optimal method of culturing primary podocytes in mice by comparing different glomerular separation techniques.Methods The renal glomeruli of C57/BL6J mice were obtained by sieving and with Dynabeads,while Bowman’s capsules were removed by digesting the glomeruli in type IV collagenase. Furthermore,the inverted phase contrast microscope was used to observe the structure of isolated renal glomeruli,expressions of podocin,while DAPI was observed by double-labeling immunofluorencence. Finally,the levels of podocin mRNA were measured by PCR.Results By using the conventional sieving method, the number of glomeruli isolated from one C57/BL6J mouse was 10 421±2421 with a purity of 90.2%±1.6%.The glomerular structure was complete and renal tubule fragments could be seen. However, with the Dynabeads method,the number of glomeruli isolated from one C57/BL6J mouse was 16112±2651 with a purity of 96.7%±1.2%,the glomerular structure was complete and there were few renal tubules. The purity and number of glomeruli obtained with Dynabeads were higher or larger than those obtained with the differential screening method,and the difference was statistically significant.A larger number of cobblestone-like cells that outgrew around nearly each glomerulus from the 7th to 10th day were observed with immunofluorescent staining, which was the characteristic of podocytes. The PCR results showed that the podocytes expressed podocin.Conclusions The test result has offered more proof that Dynabeads method is superior to the differential sieving method in isolating glomeruli from C57/BL6J mice because it is simple and highly effective for culturing primary glomerular podocytes.
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国家自然科学基金(81600571)
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