【摘要】：肥胖及相關(guān)代謝疾病正極大地危害著人類(lèi)健康,而脂肪過(guò)量積累是引起肥胖的關(guān)鍵的誘因。組織蛋白酶L(Cathepsin L,CPL)通過(guò)降解胞外基質(zhì)而參與飲食誘導(dǎo)的脂肪沉積的過(guò)程,但是其是否介導(dǎo)細(xì)胞自噬參與脂質(zhì)代謝的分子機(jī)制還不清楚。CPL作為溶酶體半胱氨酸蛋白酶主要是在溶酶體中發(fā)揮作用,溶酶體是細(xì)胞內(nèi)酸性細(xì)胞器,溶酶體與自噬小體融合形成自噬溶酶體,從而參與細(xì)胞自噬過(guò)程。最近研究表明,細(xì)胞自噬在生物體脂質(zhì)重構(gòu)中發(fā)揮著重要的作用,但是CPL通過(guò)調(diào)控細(xì)胞自噬參與脂肪積累過(guò)程的作用還有待確認(rèn)。為了在模式動(dòng)物秀麗隱桿線蟲(chóng)(C.elegans)中研究這一作用,本論文首先擴(kuò)增了線蟲(chóng)Cpl-1(Cathepsin L-like proteases,CPL-1,Cathepsin L在線蟲(chóng)中同源基因)基因,構(gòu)建重組原核表達(dá)質(zhì)粒pET28a::CPL-1,并將其轉(zhuǎn)化到大腸桿菌BL21中誘導(dǎo)表達(dá),純化了CPL-1融合蛋白,制備了相應(yīng)的多克隆抗體,經(jīng)ELISA和western blot檢測(cè)該抗體具有良好的效價(jià)和專一性。在此基礎(chǔ)上,本研究進(jìn)一步探討了CPL-1介導(dǎo)細(xì)胞自噬參與了飲食誘導(dǎo)的線蟲(chóng)脂肪沉積過(guò)程的作用。利用LGG-1::GFP線蟲(chóng)模式,我們發(fā)現(xiàn)在葡萄糖誘導(dǎo)脂質(zhì)沉積過(guò)程中,細(xì)胞自噬相關(guān)基因表達(dá)都顯著升高。其中,4mM葡萄糖誘導(dǎo)脂肪沉積誘導(dǎo)自噬水平最高。而且,CPL-1與細(xì)胞自噬水平有著很強(qiáng)的相關(guān)性。相應(yīng)地,在高糖飲食誘導(dǎo)下的線蟲(chóng)cpl-1突變體中,自噬相關(guān)基因表達(dá)顯著降低。最終,通過(guò)RNA干擾降低線蟲(chóng)自噬相關(guān)基因bec-1及l(fā)gg-1的表達(dá),也能降低高糖飲食誘導(dǎo)的線蟲(chóng)脂肪沉積。這些結(jié)果表明,CPL-1所介導(dǎo)的細(xì)胞自噬參與了線蟲(chóng)中的飲食誘導(dǎo)的脂肪沉積過(guò)程。綜上所述,本研究制備了線蟲(chóng)CPL-1的多克隆抗體,并發(fā)現(xiàn)了CPL-1介導(dǎo)細(xì)胞自噬參與線蟲(chóng)飲食誘導(dǎo)脂肪沉積過(guò)程中的作用,提出了組織蛋白酶調(diào)控脂肪沉積的新機(jī)制。
[Abstract]:Obesity and related metabolic diseases are harmful to human health, and fat accumulation is the key cause of obesity. Cathepsin L (Cathepsin is involved in the process of fat deposition induced by diet by degrading extracellular matrix. However, it is not clear whether or not it mediates the molecular mechanism of autophagy involved in lipid metabolism. As a lysosomal cysteine protease, CPL plays an important role in lysosome, which is the intracellular acidic organelle. Lysosomes fused with autophagy to form autophagic lysosomes, thus participating in the process of autophagy. Recent studies have shown that autophagy plays an important role in lipid remodeling, but the role of CPL in lipid accumulation by regulating autophagy has yet to be confirmed. In order to study this role in model animal C.elegans, the gene of Cpl-1 (homologous gene in Cathepsin L-like proteases,CPL-1,Cathepsin L online worm) was first amplified. The recombinant prokaryotic expression plasmid pET28a::CPL-1, was constructed and transformed into Escherichia coli BL21 to induce expression. The fusion protein of CPL-1 was purified and the corresponding polyclonal antibody was prepared. The antibody was tested by ELISA and western blot with good titer and specificity. On this basis, we further investigated the role of CPL-1 mediated autophagy in the diet-induced fat deposition of nematodes. Using the LGG-1::GFP nematode model, we found that the expression of autophagy related genes increased significantly during glucose induced lipid deposition. Among them, 4mM glucose-induced fat deposition induced autophagy was the highest. Moreover, there is a strong correlation between CPL-1 and autophagy level. Accordingly, the expression of autophagy related genes decreased significantly in the cpl-1 mutants of nematode induced by high glucose diet. Finally, the expression of bec-1 and lgg-1 in nematode autophagy was reduced by RNA interference, and the fat deposition of nematode induced by high glucose diet was also decreased. These results suggest that CPL-1 mediated autophagy is involved in diet-induced fat deposition in nematodes. In conclusion, the polyclonal antibodies against nematode CPL-1 were prepared, and the role of CPL-1 mediated autophagy in the process of nematode diet induced fat deposition was found, and a new mechanism of cathepsin regulating fat deposition was proposed.
【學(xué)位授予單位】：合肥工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R589.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Marton Siklos;Manel Ben Aissa;Gregory R.J.Thatcher;;Cysteine proteases as therapeutic targets:does selectivity matter? A systematic review of calpain and cathepsin inhibitors[J];Acta Pharmaceutica Sinica B;2015年06期

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本文編號(hào)：2319431