Nephrin,podocin及α-actinin在小鼠肾小球足细胞系的表达与分布

Distribution and expression of nephrin,podocin and α-actinin in a conditionally immortalized mouse podocyte cell line

目的 :探讨nephrin ,podocin及α actinin在小鼠肾小球足细胞系 (MPC5)的表达与分布 ,为进一步研究上述分子间的相互作用建立稳定的实验平台。 方法 :培养小鼠MPC5,以γ 干扰素诱导在 33℃传代增生 ,继而在37℃培养两周使细胞分化成熟以用于实验研究。相差显微镜观察足细胞形态 ;免疫荧光染色观察nephrin ,podocin ,α actinin及WT1在足细胞的分布 ;RT PCR检测足细胞nephrin ,podocin及α actinin 4的mRNA ;免疫蛋白印迹检测nephrin ,podocin ,α actinin及WT1蛋白。 结果 :成熟足细胞呈星形且有突起形成。免疫荧光及免疫蛋白印迹显示足细胞特异性的表达WT1分子。免疫荧光染色可见nephrin和podocin在足细胞内均呈线状分布于胞膜表面 ,α actinin呈细丝状分布于胞质内及伸出的突起中。在mRNA水平及蛋白水平均检测到nephrin ,podocin和α actinin 4表达 ,其蛋白大小分别为 180KDa,4 5KDa和 10 0KDa。 结论 :首次在mRNA水平及蛋白水平证实了小鼠MPC5能够表达nephrin ,podocin及α actinin ,为进一步研究这些分子间的相互作用奠定了基础。

Objective: To analyze the distribution and expression of nephrin, podocin and a-actinin in a conditionally immortalized mouse podocyte cell line (MPC 5), which will be in vitro model for studying the relationship among podocyte molecules. Methodology: MPC 5 were induced to propagate and passage by γ-inferon at 33℃, which were subsequently cultured at 37℃ for differentiation for two weeks. The podocyte morphology was observed by phase-contrast microscope. The distribution of nephrin, podocin and α-actinin was revealed by immunofluorescence (IF) staining. Results: ① In the light microscopy, differentiated cells presented with the polygoneal shape and with the formation of processes. ② IF the staining of nephrin and podcin was mainly distributed on the surface of cellular membrane in a filamentous pattern. The staining of α-actinin was specifically localized to the nuclear. ③ Expressions of nephrin, podocin and α-actinin mRNAs in MPC 5 were detected by RT-PCR. ④ Expression of nephrin, podocin, α-actinin and WT1 in MPC 5 were shown by Western blotting with the size of approximately 180 KDα, 45 kdα, 100 KDα and 52 KDα, respectively. Conclusion: For the first time, we demonstrated the expression of nephrin, podocin and α-actinin both at mRNA level and at protein level in mouse MPC 5.