摘要
体外33℃许可条件下培养由H-2Kb-tsA58转基因小鼠所建立的未分化足细胞系,并在37℃非许可条件下诱导其分化。观察足细胞分化后形态学改变;MTT法测定细胞的生长曲线;红色荧光染料PKH-26标记足细胞,追踪其在子代细胞中的分布,检测细胞增殖能力;流式细胞仪检测细胞周期的改变;Western印迹检测足细胞相关蛋白CD2AP、α-actinin和足细胞分化相关蛋白nephrin的表达;免疫荧光结合激光共聚焦方法检测CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白的表达变化。结果显示:与未分化足细胞相比,分化足细胞形态发生改变,生长速度减慢,增殖能力下降;细胞周期表现为G0/G1期细胞比例的增多和S期及G2/M期的细胞比例下降;CD2AP、nephrin和α-actinin的表达明显增高;CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白在表达分布上均发生明显的改变。以上结果表明,足细胞分化后生物学性状明显发生改变,细胞骨架重新分布;CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白均在足细胞的分化过程中发挥重要作用。
Conditionally immortalized murine podocyte cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃ permissive conditions and shifted to 37℃ non- permissive conditions to induce differentiation. Podocytes morphology changes, growth curve, proliferation ability and cell cycle were measured respectively in undifferentiated and differentiated podocytes. The expressions of CD2AP, α-actinin, nephrin and cytoskeletal proteins （i.e. F-actin and tubulin） were detected by laser scanning confocal microscope. The results demonstrated that in differentiated podocytes, cell morphology changed along with secondary foot process formation and reproductive activity descended. Cells in G0/G1 period accumulated and in S, G2/M period decreased. The expressions of CD2AP, nephrin and α-actinin were elevated in differentiated podocytes. The distribution profiles of podocyte associated proteins and cytoskeletal proteins apparently altered. In conclusion, differentiated podocytes biological characters changed and cytoskeleton rearranged. CD2AP, nephrin, α-actinin, F-actin and tubulin may play a crucial role in podocytes differentiation.
出处
《细胞生物学杂志》
CAS
CSCD
2008年第1期82-88,共7页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30500245)
湖北省卫生厅科研基金(No.NX200510)资助项目~~
关键词
足细胞
分化
足细胞相关蛋白
细胞骨架
CD2相关蛋白
podocytes
differentiation
podocytes associated protein
cytoskeleton
CD2-associated protein
