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目的研究足细胞裂孔隔膜(SD)复合体分子nephrin、podoein和CD2AP,以及足细胞骨架蛋白α-辅肌动蛋白(actinin)-4的作用及分子间反应。方法针对nephrin、podocin、CD2AP和α-aetinin-4mRNA序列分别设计并构建2个特异RNA干扰质粒-psiRNA-hH1GFPzeo,分别导入小鼠足细胞系MPC5以“敲低”其表达。免疫荧光染色观察其分布方式。半定量RT-PCR和免疫蛋白印迹检测其mRNA和蛋白表达。结果(1)podocin敲低组(siPod966和siPod54):未检测到podocin及nephrinmRNA,其蛋白分别下降了92%、79%及82%、67%。而CD2APmRNA和蛋白分别增加了62%、42%及71%、46%。α-actinin-4无变化。(2)nephrin敲低组(siNep492):未检测到nephrinmRNA和蛋白。而CD2APmRNA和蛋白分别增加了35%、48%。podocin和α-actinin-4无变化。(3)CD2AP敲低组(siCda744和siCda21):未检测到CD2APmRNA,其蛋白分别下降了92%和83%。nephrinmRNA和蛋白分别下降了60%、48%及76%、72%;而podocinmRNA和蛋白分别增加了38%、22%及56%、44%。α-actinin-4无变化。(4)α-actinin-4敲低组(siAct1790和siAct319):α-actinin-4和nephrin的mRNA分别下降了69%、58%及64%、49%:蛋白分别下降了81%和55%以及71%、64%。而podocin以及CD2APmRNA分别增加了50%、34%及45%、28%;蛋白分别增加了64%、46%及65%、42%。(5)敲低nephrin、podocin和CD2AP后,这些表达量降低的分子的分布发生了明显改变,即以核周为主;而相应分子敲低后引起的podocin和CD2AP表达增加,其分布亦主要以核周染色增强为主。α-actinin-4即使表达降低,分布亦无变化,仍呈细丝状分布于胞质及足细胞伸出的突起中。结论(1)在SD复合体分子中,nephrin可能具有相对独立的作用。(2)a-actinin-4对nephrin、podocin和CD2AP有直接或间接的作用。(3)足细胞分子间的作用和联系不总是“一致的”,可能是“单向的”、也可能是“双向的”。(4)nephrin、podocin、CD2AP和α-actinin-4在足细胞的分布有赖于其表达量的正常及正常的分子间反应。
Objective To investigate the effects of nephrin, podoein and CD2AP and actinin - 4, a podocyte cytoskeleton protein, on the cellular compartments of podocyte and the intermolecular reaction. Methods Two specific RNA interference plasmids-psiRNA-hH1GFPzeo were designed and constructed respectively for nephrin, podocin, CD2AP and α-aetinin-4 mRNA sequences and were respectively introduced into mouse podocyte line MPC5 to knock down the expression. Immunofluorescence staining to observe the distribution. Semi-quantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression. Results (1) podocin knockdown group (siPod966 and siPod54): no detectable podocin and nephrin mRNA, the protein decreased by 92%, 79% and 82%, 67%. While CD2AP mRNA and protein increased by 62%, 42% and 71%, 46% respectively. α-actinin-4 no change. (2) nephrin knockdown group (siNep492): Nephrin mRNA and protein were not detected. While CD2AP mRNA and protein increased by 35% and 48% respectively. No change in podocin and α-actinin-4. (3) CD2AP knockdown group (siCda744 and siCda21): CD2AP mRNA was not detected, the protein decreased by 92% and 83% respectively. nephrin mRNA and protein decreased by 60%, 48% and 76% and 72% respectively, while podocin mRNA and protein increased by 38%, 22% and 56% and 44% respectively. α-actinin-4 no change. (4) The mRNA levels of α-actinin-4 and nephrin decreased by 69%, 58% and 64%, and 49% respectively in the α-actinin-4 knockdown group (siAct1790 and siAct319) And 71%, 64%. While podocin and CD2AP mRNA increased by 50%, 34% and 45%, 28% respectively; protein increased by 64%, 46% and 65% and 42% respectively. (5) After knockdown of nephrin, podocin and CD2AP, the distributions of these molecules whose expression decreased were significantly changed, that is, the perinuclear cycle was predominant; while the expression of podocin and CD2AP was increased after knockdown of corresponding molecules Perinuclear staining enhanced mainly. α-actinin-4 showed no change in distribution even though the expression was decreased, and remained filamentous distribution in the protuberances protruding from the cytoplasm and podocyte. Conclusion (1) nephrin may play a relatively independent role in the SD complex molecule. (2) a-actinin-4 has a direct or indirect effect on nephrin, podocin and CD2AP. (3) The roles and connections among podocyte molecules are not always “consistent” and may be “one-way” or “two-way”. (4) The distribution of nephrin, podocin, CD2AP and α-actinin-4 in podocytes depends on the normal and normal intermolecular reaction of their expression level.