mTOR活化在镉所诱导大鼠肾小管上皮细胞自噬抑制中的作用

发布时间：2018-06-22 17:47

本文选题：镉 + 细胞自噬　；参考：《山东农业大学》2017年硕士论文

【摘要】：镉(Cd)是生态环境中一种常见的重金属,在环境中具有稳定、积累和不易清除的特点,环境中的Cd主要经呼吸道和消化道进入体内。镉对哺乳动物的毒性具有多器官性和多组织性,已被国际癌症研究机构归类为人类致癌物质。肾脏是镉蓄积的主要部位,是急性或慢性镉暴露的主要靶器官,而且近曲小管是镉肾毒性的主要损伤部位。自噬是一种高度动态的多步骤生物学过程,其负责将溶酶体中损伤的细胞器、错误折叠的蛋白质和长寿命大分子的降解和再循环,其作为细胞保护机制来维持细胞稳态和适应不利条件。自噬是一种细胞防御过程,其中胞质成分、细胞器和入侵细菌由自噬体转运到溶酶体以进行降解。自噬的失调已经涉及肾脏疾病的发病机制,如:急性肾损伤和慢性肾脏疾病等。mTOR是一类进化上非常保守的丝氨酸/苏氨酸蛋白激酶,属于磷脂酰肌醇激酶相关激酶家族成员。可受生长因子、营养物质等多种因素的影响,通过使其下游的靶蛋白磷酸化,参与基因转录和蛋白的表达,进而影响自噬、凋亡等一系列生物活动。在哺乳动物细胞中,mTOR至少具有两种复合物mTORC1和mTORC2,具有不同的底物特异性。mTORC1调节p70S6激酶1(S6K1)和真核起始因子4E(eIF4E)结合蛋白1(4E-BP1)的磷酸化。mTORC2调节Akt磷酸化或活性。此外,mTOR是启动自噬的主要调节因子。实验研究表明,Cd染毒能够抑制自噬,但是否通过mTOR的活化对Cd染毒所致的大鼠肾小管上皮细胞的自噬起抑制作用尚不明确。本实验以原代培养大鼠肾小管上皮细胞为模型,进行镉染毒以及mTOR抑制剂共孵育处理12h。通过蛋白质免疫印迹技术(Western Blot)分析Cd染毒对mTOR及其底物的蛋白水平的表达。采用激光共聚焦显微技术和蛋白质免疫印迹技术检测mTOR活化对大鼠肾小管上皮细胞自噬流的影响。同时,又检测了mTOR活化对溶酶体功能及V-ATP酶的影响。结果表明:Cd染毒能够激活大鼠肾小管上皮细胞mTOR,mTORC1和mTORC2两条通路都发挥作用。而且mTOR主要是通过mTOR的磷酸化起作用,对mTOR总蛋白没有影响。与此同时,mTOR活化对大鼠肾小管上皮细胞的自噬流具有抑制作用,阻碍了自噬体与溶酶体的融合。此外,mTOR活化影响了大鼠肾小管上皮细胞溶酶体的功能,具体表现在抑制了溶酶体V-ATP酶的表达,使溶酶体酸性环境减弱。总之,Cd染毒所致的大鼠肾小管上皮细胞mTOR的活化,可通过移位到溶酶体膜上,来抑制溶酶体中V-ATP酶的活性,从而减弱溶酶体的酸性环境,进而损坏溶酶体的功能,最后抑制了溶酶体与自噬体的融合,在Cd所致的大鼠肾小管上皮细胞中发挥自噬抑制作用。
[Abstract]:Cadmium (CD) is a common heavy metal in the ecological environment. It has the characteristics of stable accumulation and difficult to remove in the environment. CD in the environment mainly enters the body through the respiratory tract and digestive tract. Cadmium has been classified as a human carcinogen by the International Agency for Cancer Research (IARC) for its multi-organ and multi-tissue toxicity to mammals. The kidney is the main site of cadmium accumulation, the main target organ of acute or chronic cadmium exposure, and the proximal convoluted tubule is the main injury site of cadmium nephrotoxicity. Autophagy is a highly dynamic multistep biological process responsible for the degradation and recycling of damaged organelles, misfolded proteins and long-lived macromolecules in lysosomes. It acts as a cell protection mechanism to maintain cell homeostasis and adapt to adverse conditions. Autophagy is a cell defense process in which cytoplasmic components organelles and invading bacteria are transported from autophagy to lysosome for degradation. Autophagy disorder has been involved in the pathogenesis of renal diseases, such as acute renal injury and chronic renal disease. MTOR is an evolutionarily conserved serine / threonine protein kinase, which belongs to the phosphatidylinositol kinase-associated kinase family. By phosphorylation of target protein downstream, gene transcription and protein expression are involved, and a series of biological activities such as autophagy and apoptosis can be affected by growth factor, nutrition and other factors. In mammalian cells, mTOR has at least two complexes, mTORC1 and mTORC2, and has different substrate specificity. MTORC1 regulates the phosphorylation or activity of p70S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In addition, mTOR is the main regulatory factor to initiate autophagy. The experimental results showed that CD could inhibit autophagy, but it was not clear whether it could inhibit autophagy of renal tubular epithelial cells induced by CD by activation of mTOR. The primary cultured rat renal tubular epithelial cells were treated with cadmium and mTOR inhibitor for 12 h. The expression of mTOR and its substrates was analyzed by Western blot. The effects of mTOR activation on autophagy of renal tubular epithelial cells in rats were studied by confocal laser microscopy and Western blot. At the same time, the effects of mTOR activation on lysosomal function and V-ATPase were examined. The results showed that two pathways, mTORmTORC1 and mTORC2, were activated in rat renal tubular epithelial cells. Moreover, mTOR is mainly mediated by phosphorylation of mTOR and has no effect on the total protein of mTOR. At the same time, the activation of mTOR inhibited the autophagy of renal tubular epithelial cells and blocked the fusion of autophagy and lysosome. In addition, the activation of mTOR affected the function of lysosome in rat renal tubular epithelial cells, which was shown by inhibiting the expression of lysosomal V-ATPase and weakening the acidic environment of lysosome. In short, the activation of mTOR in rat renal tubular epithelial cells induced by CD exposure can inhibit the activity of V-ATPase in lysosomes by translocation to lysosomal membranes, thereby weakening the acidic environment of lysosomes and further damaging the function of lysosomes. Finally, the fusion of lysosome and autophagy was inhibited in CD-induced renal tubular epithelial cells.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8

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