线虫CPL-1介导细胞自噬参与饮食诱导脂肪沉积的作用研究
发布时间：2018-11-08 19:24
【摘要】：肥胖及相关代谢疾病正极大地危害着人类健康,而脂肪过量积累是引起肥胖的关键的诱因。组织蛋白酶L(Cathepsin L,CPL)通过降解胞外基质而参与饮食诱导的脂肪沉积的过程,但是其是否介导细胞自噬参与脂质代谢的分子机制还不清楚。CPL作为溶酶体半胱氨酸蛋白酶主要是在溶酶体中发挥作用,溶酶体是细胞内酸性细胞器,溶酶体与自噬小体融合形成自噬溶酶体,从而参与细胞自噬过程。最近研究表明,细胞自噬在生物体脂质重构中发挥着重要的作用,但是CPL通过调控细胞自噬参与脂肪积累过程的作用还有待确认。为了在模式动物秀丽隐杆线虫(C.elegans)中研究这一作用,本论文首先扩增了线虫Cpl-1(Cathepsin L-like proteases,CPL-1,Cathepsin L在线虫中同源基因)基因,构建重组原核表达质粒pET28a::CPL-1,并将其转化到大肠杆菌BL21中诱导表达,纯化了CPL-1融合蛋白,制备了相应的多克隆抗体,经ELISA和western blot检测该抗体具有良好的效价和专一性。在此基础上,本研究进一步探讨了CPL-1介导细胞自噬参与了饮食诱导的线虫脂肪沉积过程的作用。利用LGG-1::GFP线虫模式,我们发现在葡萄糖诱导脂质沉积过程中,细胞自噬相关基因表达都显著升高。其中,4mM葡萄糖诱导脂肪沉积诱导自噬水平最高。而且,CPL-1与细胞自噬水平有着很强的相关性。相应地,在高糖饮食诱导下的线虫cpl-1突变体中,自噬相关基因表达显著降低。最终,通过RNA干扰降低线虫自噬相关基因bec-1及lgg-1的表达,也能降低高糖饮食诱导的线虫脂肪沉积。这些结果表明,CPL-1所介导的细胞自噬参与了线虫中的饮食诱导的脂肪沉积过程。综上所述,本研究制备了线虫CPL-1的多克隆抗体,并发现了CPL-1介导细胞自噬参与线虫饮食诱导脂肪沉积过程中的作用,提出了组织蛋白酶调控脂肪沉积的新机制。
[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.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2
本文编号：2319431
[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.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589.2
【参考文献】
相关期刊论文 前1条
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
，本文编号：2319431
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