上海交通大学学报（医学版）

›› 2010, Vol. 30 ›› Issue (7): 747-.

• 论著（基础研究） • 上一篇    下一篇

CD2AP突变对肾小球足细胞骨架蛋白F-actin 分布的影响

赵彩霞, 潘晓霞, 冯晓蓓, 隆 琦, 王伟铭, 王朝晖, 陈 楠   

  1. 上海交通大学 医学院瑞金医院肾脏内科, 上海 200025
  • 出版日期:2010-07-25 发布日期:2010-07-26
  • 通讯作者: 陈 楠, 电子信箱: chen-nan@medmail.com.cn。
  • 作者简介:赵彩霞（1983—）, 女, 硕士生；电子信箱: zhaocaixia2010@yahoo.cn。
  • 基金资助:

    国家自然科学基金（30871001）；上海市重点学科建设项目(T0201)；上海市科委重点项目(08D21900502)

Effects of CD2AP mutation on distribution of cytoskeletal protein F-actin in podocytes

ZHAO Cai-xia, PAN Xiao-xia, FENG Xiao-bei, LONG Qi, WANG Wei-ming, WANG Zhao-hui, CHEN Nan   

  1. Department of Nephrology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2010-07-25 Published:2010-07-26
  • Supported by:

    National Natural Science Foundation of China, 30871001；Shanghai Key Discipline Foundation, T0201；Shanghai Science and Technology Committee Foundation, 08D21900502

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摘要：

目的 构建突变型CD2相关蛋白（CD2AP）荧光表达载体并转染肾小球足细胞，观察细胞内骨架蛋白F-actin分布的变化。方法 定点诱变真核表达质粒pcDNA6-V5-CD2AP致CD2AP第2外显子160G>A杂合突变，测序鉴定。利用野生型和突变型质粒和pEGFP-C1荧光表达载体，分别构建野生型和突变型重组质粒pEGFP-C1-CD2AP荧光表达载体并转染小鼠肾小球足细胞，荧光显微镜观察处于不同细胞周期的足细胞内F-actin分布情况。结果 测序结果显示，pcDNA6-V5-CD2AP真核表达载体CD2AP的2号外显子第160位碱基G诱变为A，而其他碱基未发生改变。在细胞分裂间期，未转染足细胞内F-actin平行排列成纤维束；野生型重组质粒转染足细胞内F-actin在细胞核周围呈点、块状浓聚；而突变型重组质粒转染足细胞内F-actin主要在细胞膜上勾出轮廓。在细胞分裂中期，野生型重组质粒转染足细胞内F-acin纤维丝呈粗纤维状，环细胞浓聚；突变型重组质粒转染足细胞则仅见胞质内F-acin纤维丝增多，部分区域浓聚。结论 成功构建野生型和突变型重组CD2AP荧光表达载体。CD2AP基因160G>A杂合突变使肾小球足细胞分裂间期和分裂中期的F-actin纤维骨架发生改变。

关键词: CD2相关蛋白, F-actin, 表达载体, 肾小球足细胞, 突变

Abstract:

Objective To construct mutant CD2-associated protein (CD2AP) fluorescence expression vector and transfect into podocytes, and investigate the distribution of F-actin in podocytes. Methods Eukaryotic expression plasmid pcDNA6-V5-CD2AP site-directed mutagenesis for 160G>A heterozygous mutation in exon 2 of CD2AP was conducted, and identification was performed by sequencing. Wild-type and mutant recombinant plasmid pEGFP-C1-CD2AP fluorescence expression vectors were constructed with wild-type and mutant plasmid and pEGFP-C1 fluorescence expression vectors, respectively, and mouse podocytes were transfected. The distributions of F-actin in podocytes at different cell cycles were observed by fluorescence microscopy. Results Sequencing analysis revealed that sitedirect mutagenesis of 160 G in exon 2 of pcDNA6-V5-CD2AP eukaryotic expression vector CD2AP to A was performed, while there was no changes in the other bases. In the interphase of cell division, F-actin in untransfected podocytes lined in parallel as fiber bundle. F-actin in podocytes transfected with wild-type recombinant plasmid displayed thick, short dot-like structures mainly around the nuclei, while F-actin in podocytes transfected with mutant recombinant plasmid expressed along the cytoplasm membrane. In the metaphase of cell division, F-actin filaments transfected with wild-type recombinant plasmid aggregated encircling podocytes, while those transfected with mutant recombinant plasmid only emerged as a number increase in cytoplasm with some regional aggregation. Conclusion Wild-type and mutant recombinant CD2AP fluorescence expression vectors are successfully constructed. CD2AP gene 160G>A heterozygous mutation may lead to the cytoskeletal changes of F-actin in the interphase and metaphase of cell division of podocytes.

Key words: CD2-associated protein, F-actin, expression vector, podocytes, mutation