PPARα介导共轭亚油酸(CLA)降低草鱼肝脂积累的分子机制研究
结题报告
批准号:
31672666
项目类别:
面上项目
资助金额:
62.0 万元
负责人:
董桂芳
依托单位:
学科分类:
C1905.水产动物营养与饲料学
结题年份:
2020
批准年份:
2016
项目状态:
已结题
项目参与者:
余登航、付书林、赵秀举、邹琦、郭超、常家智、孔龙
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中文摘要
营养性脂肪肝是制约水产养殖业可持续发展的瓶颈,其防治措施尚不多见。本课题组证实,CLA可显著降低草鱼肝脂积累,且CLA适宜添加组和未添加CLA组间肝脏中PPARα基因表达存在显著差异,提示PPARα可能介导了CLA降低草鱼肝脂积累过程,但其分子调控机制尚不清楚。因此,本项目以草鱼肝细胞系和活体为研究对象,首先通过PPARα激动剂或抑制剂与细胞共培养/过表达和沉默PPARα基因/PPARα挽救实验,结合细胞脂肪蓄积表型,鉴定PPARα的功能;继而运用iTRAQ定量蛋白质组学技术,筛选细胞中沉默PPARα基因后的下游差异表达蛋白,构建PPARα-靶蛋白互作网络,鉴定核心PPARα-靶蛋白;最后在细胞和活体水平验证核心PPARα-靶蛋白功能并厘清PPARα与PPARα-靶蛋白互作调控机制,最终阐明PPARα介导CLA降低草鱼肝脂积累的分子机制,为草鱼营养性脂肪肝防治和抗病新品种选育提供理论依据。
英文摘要
Nutrition-associated liver disease (NALD) is one of the bottlenecks for a sustainable development of aquaculture. However, it is rare in effectively treatment for NALD. It has been revealed that dietary CLA can significantly decrease liver lipid concentration in grass carp, and there are significant differences in the mRNA and protein expressions of PPARα in the livers between the CLAopt and CLA-free diets of the fish in our previous experiment. It is possible that the liver lipid-lowering effects of conjugated linoleic acid in grass carp could be mediated by PPARα. However, it is unclear the molecular mechanism of the liver lipid-lowering effects of conjugated linoleic acid in grass carp mediated by PPARα. Therefore, in the present study, grass carp and it’s liver cell lines will be used. In the present study, three strategies combined with the phenotype of lipid deposition in the cell lines will be used for the gene function identification of PPARα: PPARα agonist and inhibitor co-cultured with liver cell line, over-expression of the mRNA of PPARα and silencing of PPARα gene, and rescue experiment for PPARα. The quantitative proteomics using iTRAQ will be used to screen for differentially expressed proteins when the PPARα gene will have been silenced, and then the protein network of PPARα-targeted protein interactions is constructed to screen the PPARα-targeted protein, and the core PPARα-targeted proteins will be identified. Moreover, the gene function identification of the core PPARα-targeted proteins will be identified at both the cell and whole-body levels, and the interaction regulation will be clarified between the PPARα and PPARα-targeted proteins. The results of the present study will elucidate the molecular mechanism of the liver lipid-lowering effects of conjugated linoleic acid in grass carp mediated by PPARα, and it will help for the targeted prevention and controling of the NALD, and for breeding of the new disease-resistant species.
营养性脂肪肝是制约水产养殖业可持续发展的瓶颈,其防治措施尚不多见。我们的前期研究证实,共轭亚油酸(CLA)可显著降低草鱼肝脂积累,且PPARα可能介导了CLA降低草鱼肝脂积累过程,但其分子调控机制尚不清楚。基于上述研究基础,本项目以草鱼肝细胞系L8824和活体为研究对象,首先通过不同浓度的油酸(OA)与L8824共培养诱导其高脂模型并确定其适宜OA诱导浓度(高脂模型组,OAopt);确定0.5 mM OA组为OAopt。随后采用不同浓度的CLA与OAopt共培养,确定CLA在OAopt中发挥降脂功效的适宜浓度(CLAopt)为50 μM t10, c12 CLA, 继而采用PPARα-siRNA、PPARα激动剂或抑制剂与OAopt或CLAopt组细胞共培养,结合细胞脂肪代谢相关基因表达和脂肪蓄积表型,进一步鉴定到PPARα介导了CLA降低草鱼肝脂积累。而后运用iTRAQ定量蛋白质组学技术,筛选细胞中siRNA介导沉默PPARα基因后的下游差异表达蛋白,鉴定到核心PPARα-靶蛋白-Perilipin-2,确定CLA-PPARα- Perilipin-2调控通路;最后在细胞和活体水平验证CLA-PPARα-Perilipin-2调控通路,初步阐明PPARα介导CLA降低草鱼肝脂积累的分子机制,为草鱼营养性脂肪肝防治和抗病新品种选育提供理论依据。
期刊论文列表
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科研奖励列表
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专利列表
DOI:10.7541/2019.084
发表时间:2019
期刊:水生生物学报
影响因子:--
作者:孔龙;邹琦;程时焰;杨严鸥;余登航;黄峰;董桂芳
通讯作者:董桂芳
Effects of dietary conjugated linoleic acid on growth performance, tissue adipocytokine levels and lipid metabolism of grass carp
日粮共轭亚油酸对草鱼生长性能、组织脂肪细胞因子水平及脂质代谢的影响
DOI:10.1111/anu.12815
发表时间:2018-08
期刊:Aquaculture Nutrition
影响因子:3.5
作者:Qi Zou;Yan‐ou Yang;Bang‐Hong Wei;Deng‐hang Yu;Lu Chen;Teng Zhou;Feng Huang;Gui‐Fang Dong
通讯作者:Gui‐Fang Dong
DOI:10.1016/j.fsi.2018.11.071
发表时间:2019-03-01
期刊:FISH & SHELLFISH IMMUNOLOGY
影响因子:4.7
作者:Kong, Long;Cheng, Shi-yan;Dong, Gui-Fang
通讯作者:Dong, Gui-Fang
共轭亚油酸降低草鱼肝脏脂肪积累关键调控因子的筛选与鉴定
  • 批准号:
    31302195
  • 项目类别:
    青年科学基金项目
  • 资助金额:
    24.0万元
  • 批准年份:
    2013
  • 负责人:
    董桂芳
  • 依托单位:
国内基金
海外基金