microRNA沉默TRPC6对肾小球足细胞自噬调控的研究

Research found that glomerular podocyte autophagy activity level is relevant to podocyte damage and proteinuria development. But the mechanism is not clearly.Our previous research demonstrated that puromycin (PAN) induced podocytes injury can cause more expression and abnormal distribution of Transient Receptor Potential Cation Channel 6(TRPC6).Therefore, we assume that if PAN damage glomerular podocyte, podocyte autophagy levels are enhanced,which damages SD molecules,induces proteinuria. Knocking down TRPC6, stabling the expression of autophagy, will maintain normal biological function of podocyte. In order to prove this hypothesis, we use PAN induced podocytes, silence TRPC6 initiation factor by microRNA, study the morphology and number of podocytes and the formation of autophagosome; Detect the change of mRNA and protein expression of LC3 and Beclin-1,which have close relationship with autophagy. Explore that stable autophagy activity level can play a role of podocyte protection or not; Use autophagy specific blockers,which named 3-methyl adenine (3-MA),to restrain autophagy process, can accelerate podocyte damagination or not.The experiment may demonstrate the molecular mechanism of TRPC6 participate in autophagy regulation of podocytes, which will reveal that the regulation of autophagy will be a new target for preventing proteinuria.

研究发现肾小球足细胞的自噬活性水平与足细胞的损伤和蛋白尿的发生密切相关，其机制尚未明确。我们的前期研究发现嘌呤霉素（PAN）损伤体外培养的肾小球足细胞使TRPC6分子表达增强及分布异常。因此，我们推测PAN损伤足细胞时，足细胞自噬水平异常增强，进而损伤足细胞裂孔隔膜（SD）分子，导致蛋白尿的发生；通过敲低TRPC6，稳定自噬体的表达，进而维持足细胞正常的生物功能状态。为证实这一假说，我们建立足细胞损伤模型，应用microRNA干扰技术沉默TRPC6的起始因子，观察足细胞形态、数目和自噬体的形成；检测足细胞自噬相关基因LC3、Beclin-1的mRNA、蛋白的表达和分布的变化；探讨稳定自噬活性水平是否发挥保护足细胞的作用，应用自噬特异性阻断剂3-甲基腺嘌呤（3-MA）抑制自噬过程是否加速足细胞的损伤；阐明TRPC6参与足细胞自噬的分子机制，揭示自噬的调控可能成为防治蛋白尿的新靶点。

研究发现肾病足细胞损伤和蛋白尿的发生与肾小球足细胞的自噬活性水平密切相关，其机制尚未明确。我们的前期研究发现嘌呤霉素（PAN）损伤体外培养的肾小球足细胞使TRPC6分子表达增强及分布异常,造成足细胞CD2AP的异常表达，抑制PI3K/AKT信号通路，进而导致足细胞凋亡。因此，我们推测PAN损伤足细胞时，足细胞自噬水平异常增强，进而损伤足细胞裂孔隔膜（SD）分子，导致蛋白尿的发生。为证实这一设想，我们建立足细胞损伤模型，应用microRNA干扰技术沉默TRPC6的起始因子，观察足细胞形态、数目和自噬体的形成；应用地塞米松(DEX)处理细胞，作用于PI3K/AKT信号通路.检测足细胞自噬相关基因LC3、Beclin-1的mRNA、蛋白的表达和分布的变化；探讨他克莫司在足细胞损伤中的保护作用及TRPC6在足细胞损伤修复中的相关性，应用自噬特异性阻断剂3-甲基腺嘌呤（3-MA）抑制自噬过程是否加速足细胞的损伤；研究阐明了TRPC6参与足细胞自噬的分子机制，揭示自噬的调控可能成为防治蛋白尿的新靶点。