發(fā)布時(shí)間：2018-06-22 17:47

  本文選題：鎘 + 細(xì)胞自噬��； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鎘(Cd)是生態(tài)環(huán)境中一種常見的重金屬,在環(huán)境中具有穩(wěn)定、積累和不易清除的特點(diǎn),環(huán)境中的Cd主要經(jīng)呼吸道和消化道進(jìn)入體內(nèi)。鎘對哺乳動(dòng)物的毒性具有多器官性和多組織性,已被國際癌癥研究機(jī)構(gòu)歸類為人類致癌物質(zhì)。腎臟是鎘蓄積的主要部位,是急性或慢性鎘暴露的主要靶器官,而且近曲小管是鎘腎毒性的主要損傷部位。自噬是一種高度動(dòng)態(tài)的多步驟生物學(xué)過程,其負(fù)責(zé)將溶酶體中損傷的細(xì)胞器、錯(cuò)誤折疊的蛋白質(zhì)和長壽命大分子的降解和再循環(huán),其作為細(xì)胞保護(hù)機(jī)制來維持細(xì)胞穩(wěn)態(tài)和適應(yīng)不利條件。自噬是一種細(xì)胞防御過程,其中胞質(zhì)成分、細(xì)胞器和入侵細(xì)菌由自噬體轉(zhuǎn)運(yùn)到溶酶體以進(jìn)行降解。自噬的失調(diào)已經(jīng)涉及腎臟疾病的發(fā)病機(jī)制,如:急性腎損傷和慢性腎臟疾病等。mTOR是一類進(jìn)化上非常保守的絲氨酸/蘇氨酸蛋白激酶,屬于磷脂酰肌醇激酶相關(guān)激酶家族成員�？墒苌L因子、營養(yǎng)物質(zhì)等多種因素的影響,通過使其下游的靶蛋白磷酸化,參與基因轉(zhuǎn)錄和蛋白的表達(dá),進(jìn)而影響自噬、凋亡等一系列生物活動(dòng)。在哺乳動(dòng)物細(xì)胞中,mTOR至少具有兩種復(fù)合物mTORC1和mTORC2,具有不同的底物特異性。mTORC1調(diào)節(jié)p70S6激酶1(S6K1)和真核起始因子4E(eIF4E)結(jié)合蛋白1(4E-BP1)的磷酸化。mTORC2調(diào)節(jié)Akt磷酸化或活性。此外,mTOR是啟動(dòng)自噬的主要調(diào)節(jié)因子。實(shí)驗(yàn)研究表明,Cd染毒能夠抑制自噬,但是否通過mTOR的活化對Cd染毒所致的大鼠腎小管上皮細(xì)胞的自噬起抑制作用尚不明確。本實(shí)驗(yàn)以原代培養(yǎng)大鼠腎小管上皮細(xì)胞為模型,進(jìn)行鎘染毒以及mTOR抑制劑共孵育處理12h。通過蛋白質(zhì)免疫印跡技術(shù)(Western Blot)分析Cd染毒對mTOR及其底物的蛋白水平的表達(dá)。采用激光共聚焦顯微技術(shù)和蛋白質(zhì)免疫印跡技術(shù)檢測mTOR活化對大鼠腎小管上皮細(xì)胞自噬流的影響。同時(shí),又檢測了mTOR活化對溶酶體功能及V-ATP酶的影響。結(jié)果表明:Cd染毒能夠激活大鼠腎小管上皮細(xì)胞mTOR,mTORC1和mTORC2兩條通路都發(fā)揮作用。而且mTOR主要是通過mTOR的磷酸化起作用,對mTOR總蛋白沒有影響。與此同時(shí),mTOR活化對大鼠腎小管上皮細(xì)胞的自噬流具有抑制作用,阻礙了自噬體與溶酶體的融合。此外,mTOR活化影響了大鼠腎小管上皮細(xì)胞溶酶體的功能,具體表現(xiàn)在抑制了溶酶體V-ATP酶的表達(dá),使溶酶體酸性環(huán)境減弱。總之,Cd染毒所致的大鼠腎小管上皮細(xì)胞mTOR的活化,可通過移位到溶酶體膜上,來抑制溶酶體中V-ATP酶的活性,從而減弱溶酶體的酸性環(huán)境,進(jìn)而損壞溶酶體的功能,最后抑制了溶酶體與自噬體的融合,在Cd所致的大鼠腎小管上皮細(xì)胞中發(fā)揮自噬抑制作用。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S859.8

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