摘要
本文旨在研究肾脏足细胞的分化特点及CD2相关蛋白(CD2-associated protein,CD2AP)在足细胞分化过程中的作用。用RPMI1640培养基在33oC许可条件下培养永生化小鼠足细胞系(未分化组),转染针对CD2AP的小分子干扰RNA(small interfering RNA,siRNA)后置于37oC非许可条件下培养(转染组),并将非许可条件下未转染组作为对照组。用MTT法检测足细胞的生长速度;用RT-PCR方法检测CD2AP、WT1、synaptopodin和nephrin mRNA表达;用Westernblot检测CD2AP、WT1和nephrin蛋白表达;用免疫荧光结合激光共聚焦方法检测CD2AP、nephrin、F-actin和tubulin在分化及未分化足细胞中的分布及其共定位情况。结果显示,CD2AP、WT1和nephrin在分化及未分化足细胞中均可稳定表达,而synaptopodin仅表达于已分化足细胞,在未分化足细胞无表达。在足细胞分化过程中,CD2AP和nephrin的表达上调(P<0.05);CD2AP、tubulin和F-actin在细胞内的分布发生改变,CD2AP与nephrin及F-actin在未分化足细胞中存在共定位关系。转染特异性siRNA下调CD2AP表达,细胞生长速度明显减慢,synaptopodin mRNA表达下调(P<0.05),细胞分化迟滞。结果表明,足细胞分化过程中伴随细胞骨架的重新分布和细胞形态的改变;CD2AP可能作为足细胞裂孔隔膜分子与细胞骨架的连接蛋白,在足细胞分化过程中发挥重要作用。
To study the cellular changes and the potential role of CD2-associated protein （CD2AP） in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33℃. After transfection with CD2AP small interfering RNA （siRNA） the cells were shifted to non-permissive condition at 37℃. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferen- tiated podocytes was detected by laser scanning confocal microscopy. The results showed： （1） CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes, （2） CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation （P〈0.05）. （3） CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. （4） After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.
出处
《生理学报》
CAS
CSCD
北大核心
2008年第1期135-142,共8页
Acta Physiologica Sinica
基金
supported by the National Natural Science Foundation of China (No. 30500245)
the Science Research Founda-tion of Health Department of Hubei Province (No. NX200510)
