大鼠肝再生中脂肪滴蛋白质组的差异分析
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摘要
肝脏的再生机制是肝脏研究中一个关键而复杂的问题。对于这一涉及复杂调控网络的多阶段生理过程,全面系统的展示其分子基础至关重要。近几年,比较蛋白质组学技术的发展为研究肝再生分子机制提供了技术平台,迅速推进了肝再生相关效应因子、信号通路的数据积累。而针对亚细胞蛋白质组的研究策略能提供蛋白质的亚细胞定位信息,从而有助于研究者阐释其作用机理,更进一步理解其在肝再生整体调控网络中所处的位置。
脂肪滴(Lipid Droplets)作为脂质代谢调控的中心细胞器,广泛参与多种生理过程。研究显示脂肪滴与其他细胞器存在普遍的相互作用和分子交流,并能通过调节细胞内的脂质平衡,辅助细胞应对特定时期和状况下的细胞环境。近年有研究报道肝脏细胞内的脂肪滴在肝再生早、中期大量积累,与肝再生进程密切关联,但其中机制尚未明确,有待探讨。
本研究以2/3肝切除的大鼠为肝再生模型,首次应用八标iTRAQ (8-plex isobaric Tags for Relative and Absolute Quantitation)技术结合2D LC-MS/MS以正常大鼠肝组织和假手术大鼠肝组织为对照,全面分析了再生早期(6h,12h)、中期(24h,48h)和晚期(72h,120h)肝脏细胞内脂肪滴蛋白质的表达差异,以期为深入探讨脂肪滴调控肝再生的机制做好铺垫。目前,课题主要获得如下结果。
第一、大鼠肝再生各时间点的脂肪滴蛋白质的质谱鉴定结果通过IPI数据库检索共得到248种蛋白质,对应到200个基因,与以往的脂肪滴蛋白质组研究结果比较,其中158个为本研究首次鉴定。利用GO(Gene Ontology)对这些基因的分子功能、生物过程和细胞组分的注释进行分类分析,结果提示脂肪滴蛋白质可能与细胞增殖状态相关,参与调节肝再生进程中脂肪合成、积累和分解代谢,以保持细胞能量供给稳定,提供生物膜合成底物,并调节胞内的氧化水平,防止胞浆中脂质过量形成的细胞毒性等。
第二、利用Protein Pilot软件给出的蛋白质定量信息,对各时间点的脂肪滴蛋白质的整体表达模式进行分层聚类分析,结果显示术后脂肪滴蛋白质组的表达变化与肝再生进程紧密相关：肝再生早期6h和12h的表达模式相似,而24h的与二者相近；随后48h和72h的表达谱相似,而120h的与二者相近；在这6个时间点中,肝再生晚期120h的蛋白表达变化最接近于假手术组。这些结果与我们的理论预期相符,也显示了八标iTRAQ技术是平行分析多组蛋白质样品的有效工具。
第三、本研究共发现29种蛋白质在大鼠肝再生过程中呈现差异表达,其中14种主体上呈上调表达,15种呈下调表达。如脂肪滴表面17-βHSD 11/13、ACSL的水平在术后24h显著上调,而脂肪结合蛋白L-FABP在肝再生24h-72h期间出现显著下调,这些蛋白质可能通过调节细胞内脂质的运输、合成和代谢,参与肝再生中损伤应激和细胞增殖等生物进程。
总结：本研究首次建立了脂肪滴蛋白质在肝再生中的动态变化图谱,发现了潜在的差异分子,并系统探讨了脂肪滴及其表面蛋白参与调节肝再生进程的可能途径和方式：1)参与细胞应激,调节胞内氧化水平;2)储存脂类,降低脂质过量产生的细胞毒性；3)保持脂质代谢平衡,供给细胞增殖所需能量和生物膜合成底物；4)募集和转运蛋白因子,与其它细胞器相互交流；5)暂存胞内大量合成的蛋白,收容失活异常蛋白辅助降解,维持细胞内环境平衡等。本研究为进一步研究脂肪滴功能和肝再生机理提供了有价值的线索。
The mechanism of liver's unique regenerative capacity is a hotspot as well as a complicated issue in liver research. Determining the molecular base is crucial to understand this multi-stage pathophysiological process involving complex networks. The comparative proteomic approach is a powerful tool to investigate molecular changes and has been employed to elucidate crucial information about liver regeneration in recent years. Among them, subcellular proteomics is a valuable addition to the commonly used proteomic approaches in liver regeneration for providing subcellular compartments information which facilitates understand proteins' functional roles in the regulating system of regeneration.
Lipd droplets (LDs), the central organelles regulating lipid synthesis and metabolism, are volved in multiple physiological processes. Studies revealed universal interaction between LDs and other organelles, as well as LDs' roles in regulating lipid homeostasis that function in specific cellular condition. A recent work reported LDs accumulated in hepatocytes in early and middle stages of liver regeneration, and correlated with regeneration process. Wheras the mechanism remains undetermined.
In the present study, comparative proteomic analysis in rat liver regeneration after 2/3 hepatectomy (PH) was performed using 8-plex iTRAQ technology coupled with 2D LC-MS/MS for the first time. Differentially expressed proteins in the early stage (6h,12h), middle stage (24h,48h) and late stage (72h,120h) were profiled, using normal and sham operated liver as control.4 main results were achieved as follows.
Firstly, the data from 2D LC-MS/MS were analyzed against IPI database and 248 distinct proteins, matching 200 genes, were identified in total.158 out of 200 genes were newly detected on LDs. The outcome from functional analysis with GO annotation system suggested LDs proteins might regulate intracellular lipid homeostasis, through which LDs function as stable energy supplier for cell proliferation and repository for substrates of membrane synthesis. LDs, together with their surface proteins, could further adjust intracellular oxidative stress and prevent cytotoxicity caused by excessive lipid.
Secondly, hierarchical clustering analysis of the proteins expression patterns demonstrated correlation between LDs proteome changes and liver regeneration progress:proteins expression at 6h and 12h after PH hold a like pattern, which of 24h was similar to those of 6h and 12h, that of 48h similar to 72h, and that of 120h was more close to sham than any other time points.
Thirdly, we employed significance analysis using ProteinPilot, and found 29 proteins with notable changes (p<0.05) at one time point or more, of which 14 were up-regulated and 15 were down-regulated. Some of these proteins, for instance L-FABP,17-βHSD 11/13 and ACSL, might be directly linked to cell proliferation and other reponses following stimulus by modulating intracellular lipid transport, synthesis and metabolism.
In conclusion, this study figured out a dynamically quantitative expression pattern of LD proteins during liver regeneration for the first time, and detected some protential key factors. These findings indicated the possible roles of LDs in liver regeneration as follows:1. regulating intracellular oxidation level responding to the stimulus; 2. storing lipid and buffering cells from the toxic effects of excessive amounts of lipid,3. regulating lipid homestasis, providing energy for cell proliferation and building blocks for biological membranes, such as phospholipids and sterols; 4. recruiting and delivering proteins, crosstalking with the other organelles; 5. sequestrating abnormal proteins before degradation, maintaining intracellular microenviroment. Our results provided useful clues for further research on this issue.
脂肪滴(Lipid Droplets)作为脂质代谢调控的中心细胞器,广泛参与多种生理过程。研究显示脂肪滴与其他细胞器存在普遍的相互作用和分子交流,并能通过调节细胞内的脂质平衡,辅助细胞应对特定时期和状况下的细胞环境。近年有研究报道肝脏细胞内的脂肪滴在肝再生早、中期大量积累,与肝再生进程密切关联,但其中机制尚未明确,有待探讨。
本研究以2/3肝切除的大鼠为肝再生模型,首次应用八标iTRAQ (8-plex isobaric Tags for Relative and Absolute Quantitation)技术结合2D LC-MS/MS以正常大鼠肝组织和假手术大鼠肝组织为对照,全面分析了再生早期(6h,12h)、中期(24h,48h)和晚期(72h,120h)肝脏细胞内脂肪滴蛋白质的表达差异,以期为深入探讨脂肪滴调控肝再生的机制做好铺垫。目前,课题主要获得如下结果。
第一、大鼠肝再生各时间点的脂肪滴蛋白质的质谱鉴定结果通过IPI数据库检索共得到248种蛋白质,对应到200个基因,与以往的脂肪滴蛋白质组研究结果比较,其中158个为本研究首次鉴定。利用GO(Gene Ontology)对这些基因的分子功能、生物过程和细胞组分的注释进行分类分析,结果提示脂肪滴蛋白质可能与细胞增殖状态相关,参与调节肝再生进程中脂肪合成、积累和分解代谢,以保持细胞能量供给稳定,提供生物膜合成底物,并调节胞内的氧化水平,防止胞浆中脂质过量形成的细胞毒性等。
第二、利用Protein Pilot软件给出的蛋白质定量信息,对各时间点的脂肪滴蛋白质的整体表达模式进行分层聚类分析,结果显示术后脂肪滴蛋白质组的表达变化与肝再生进程紧密相关：肝再生早期6h和12h的表达模式相似,而24h的与二者相近；随后48h和72h的表达谱相似,而120h的与二者相近；在这6个时间点中,肝再生晚期120h的蛋白表达变化最接近于假手术组。这些结果与我们的理论预期相符,也显示了八标iTRAQ技术是平行分析多组蛋白质样品的有效工具。
第三、本研究共发现29种蛋白质在大鼠肝再生过程中呈现差异表达,其中14种主体上呈上调表达,15种呈下调表达。如脂肪滴表面17-βHSD 11/13、ACSL的水平在术后24h显著上调,而脂肪结合蛋白L-FABP在肝再生24h-72h期间出现显著下调,这些蛋白质可能通过调节细胞内脂质的运输、合成和代谢,参与肝再生中损伤应激和细胞增殖等生物进程。
总结：本研究首次建立了脂肪滴蛋白质在肝再生中的动态变化图谱,发现了潜在的差异分子,并系统探讨了脂肪滴及其表面蛋白参与调节肝再生进程的可能途径和方式：1)参与细胞应激,调节胞内氧化水平;2)储存脂类,降低脂质过量产生的细胞毒性；3)保持脂质代谢平衡,供给细胞增殖所需能量和生物膜合成底物；4)募集和转运蛋白因子,与其它细胞器相互交流；5)暂存胞内大量合成的蛋白,收容失活异常蛋白辅助降解,维持细胞内环境平衡等。本研究为进一步研究脂肪滴功能和肝再生机理提供了有价值的线索。
The mechanism of liver's unique regenerative capacity is a hotspot as well as a complicated issue in liver research. Determining the molecular base is crucial to understand this multi-stage pathophysiological process involving complex networks. The comparative proteomic approach is a powerful tool to investigate molecular changes and has been employed to elucidate crucial information about liver regeneration in recent years. Among them, subcellular proteomics is a valuable addition to the commonly used proteomic approaches in liver regeneration for providing subcellular compartments information which facilitates understand proteins' functional roles in the regulating system of regeneration.
Lipd droplets (LDs), the central organelles regulating lipid synthesis and metabolism, are volved in multiple physiological processes. Studies revealed universal interaction between LDs and other organelles, as well as LDs' roles in regulating lipid homeostasis that function in specific cellular condition. A recent work reported LDs accumulated in hepatocytes in early and middle stages of liver regeneration, and correlated with regeneration process. Wheras the mechanism remains undetermined.
In the present study, comparative proteomic analysis in rat liver regeneration after 2/3 hepatectomy (PH) was performed using 8-plex iTRAQ technology coupled with 2D LC-MS/MS for the first time. Differentially expressed proteins in the early stage (6h,12h), middle stage (24h,48h) and late stage (72h,120h) were profiled, using normal and sham operated liver as control.4 main results were achieved as follows.
Firstly, the data from 2D LC-MS/MS were analyzed against IPI database and 248 distinct proteins, matching 200 genes, were identified in total.158 out of 200 genes were newly detected on LDs. The outcome from functional analysis with GO annotation system suggested LDs proteins might regulate intracellular lipid homeostasis, through which LDs function as stable energy supplier for cell proliferation and repository for substrates of membrane synthesis. LDs, together with their surface proteins, could further adjust intracellular oxidative stress and prevent cytotoxicity caused by excessive lipid.
Secondly, hierarchical clustering analysis of the proteins expression patterns demonstrated correlation between LDs proteome changes and liver regeneration progress:proteins expression at 6h and 12h after PH hold a like pattern, which of 24h was similar to those of 6h and 12h, that of 48h similar to 72h, and that of 120h was more close to sham than any other time points.
Thirdly, we employed significance analysis using ProteinPilot, and found 29 proteins with notable changes (p<0.05) at one time point or more, of which 14 were up-regulated and 15 were down-regulated. Some of these proteins, for instance L-FABP,17-βHSD 11/13 and ACSL, might be directly linked to cell proliferation and other reponses following stimulus by modulating intracellular lipid transport, synthesis and metabolism.
In conclusion, this study figured out a dynamically quantitative expression pattern of LD proteins during liver regeneration for the first time, and detected some protential key factors. These findings indicated the possible roles of LDs in liver regeneration as follows:1. regulating intracellular oxidation level responding to the stimulus; 2. storing lipid and buffering cells from the toxic effects of excessive amounts of lipid,3. regulating lipid homestasis, providing energy for cell proliferation and building blocks for biological membranes, such as phospholipids and sterols; 4. recruiting and delivering proteins, crosstalking with the other organelles; 5. sequestrating abnormal proteins before degradation, maintaining intracellular microenviroment. Our results provided useful clues for further research on this issue.
引文
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