核基质蛋白在姜黄素诱导人永生化表皮HaCaT细胞凋亡过程中的变化研究
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摘要
本文在鉴定姜黄素(curcumin,Cur)诱导人永生化表皮HaCaT细胞凋亡的基础上,应用选择性抽提整装透射电镜和蛋白质组学分析技术,对HaCaT细胞诱导凋亡过程中核基质-中间纤维系统的构型变化和核基质蛋白组成的变化进行系统研究。分析鉴定与表皮细胞凋亡相关的特异核基质蛋白,并探索它们在表皮细胞凋亡过程中的调控作用,以期能够在较为整体的水平上进一步认识表皮细胞凋亡及其机理,从而找出表皮细胞凋亡研究的新方向。
实验结果显示,经7.5μg/mL姜黄素处理之后,人永生化表皮HaCaT细胞的增殖受到明显抑制,细胞生长抑制率达83.03%;细胞周期检测出现亚二倍体(亚G_1期)细胞峰值,细胞凋亡率达13.1%,并发生G_2/M期阻滞;琼脂糖凝胶电泳出现细胞凋亡典型的DNA“梯状”条带:光镜和透射电镜观察结果显示,经姜黄素诱导处理后的HaCaT细胞出现了细胞体积缩小、核质比例减小、细胞核固缩、核内染色质凝聚、线粒体肿胀、内质网网腔扩大、出现凋亡小体等显著的凋亡特征;而且与细胞凋亡相关的癌基因bcl-2表达下调,抑癌基因p53、fas、bax等表达上调。选择性抽提整装光镜和电镜观察显示,经姜黄素处理的HaCaT细胞的核基质和中间纤维比对照组明显稀疏,且分布更加不均匀,并分别与核纤层连系,形成趋于断裂但相对还较为完整的网状结构;双向凝胶电泳分析显示在姜黄素诱导HaCaT细胞凋亡前后存在27个差异表达的核基质蛋白,经质谱分析,鉴定了其中的21个蛋白,其中在凋亡的HaCaT细胞中表达上调的9种蛋白鉴定为:Chaperonin containing TCP1、RNA binding motif、Hypothetical proteinAt4g18230、serine proteinase inhibitor、SUMO-1-specific protease、nucleoporinNUP107、coagulation factor V precursor、Apoptosis-inducing factor、Caspase 3;表达下调的6种蛋白质为LMNA protein、Vimentin、Glycosyl transferase,family 2、Actin、MAP/microtubule affinity-regulating kinase、translation initiation factor IF-2。诱导凋亡处理后新出现的6种蛋白质为Alcohol dehydrogenase[NADP~+]、锌指蛋白ZNF483、MHC classⅠantigen、M-phase phosphoprotein-1、Heat shock 90kDaprotein、Protein kinase D3。鉴定出的21种核基质蛋白中除vimentin、actin,LMNAprotein,NUP107,HSP90,SUMO-4蛋白酶,ZNF483 protein,MHC classⅠantigen,Chaperonin containing TCP1外,其余均为首次在核基质中发现的蛋白质。
研究结果表明,7.5μg/mL姜黄素对人永生化表皮HaCaT细胞的凋亡具有显著诱导作用,在HaCaT细胞凋亡过程中不仅其核基质-中间纤维系统构型产生了明显的凋亡特征性变化,而且其核基质蛋白组成也相应发生了明显改变。由此证实了与表皮细胞凋亡诱导相关特异核基质蛋白的存在及其对表皮细胞凋亡诱导的调控作用。
In this study, the curcumin(Cur) was used to induce the immortalized human epithelial cell line HaCaT cells into apoptosis, and its effects were investigated by selective extraction-whole mount optic and transmission electron microscopy and techniques of proteomic. The differentially expressed nuclear matrix proteins were analyzed in order to explore the molecular mechanisms in a system level.
The results revealed that the HaCaT cells were induced into apoptosis after treated with 7.5μg/mL curcumin as the proliferation of HaCaT cells were inhibited, and the inhibitory rate is 83.03%. The results of flow cytometry analysis showed that curcumin could induce the emergence of the phase of apoptosis, and the rate is 13.1% ,the cell cycle were arrested in G_2/M phase. Agarose gel electrophoresis revealed that cell DNA fragment exhibited characteristic " DNA ladder " . Light microscope and electron microscope showed that the morphology of the cells treated with curcumin appeared shrinked, cell nucleus concerntrated,chromatin agglutination, mitochondria swelling, and apoptosis body forming. Immunocytochemistry revealed that the expression of wt p53, Bax, Fas were upregulated significatntly while the products of Bcl-2 proteins were downregulated significantly in the cells treated with Cur. Selective extraction, whole-mount optic and transmission electron microscopy showed that after treated with 7.5μg/mL curcumin the configuration of nuclear matrix-intermediate filament in HaCaT cells was remarkably changed as the filament was few and scattered, not well distributed and arranged irregularly.and connected to lamina respectively, and the filaments of nuclear matrix,nuclear lamin and intermediatefilment connected not very tightly and the formation but tends to fracture but also relatively more complete network structure.There were 27 spots changed remarkably during the apoptosis induced by curcumin,21 of which were identified. the nine up-regulated proteins including nucleoporin NUP107, six down-regulated proteins including LMNA protein and six new proteins were identified.
In conclusion, the configuration and composition of nuclear matrix were changed following the HaCaT cell apoptosis.It is confirmed that specific nuclear matrix proteins take part in the regulation of cell proliferation and apoptosis, and the associated signal transduction pathways are suggested. It provides proofs and a new way to study the machnisms of epithelial cell apoptosis and it is possible to get a system understanding of the molecular mechanisms from the studies of nuclear matrix, as well as to reveal the relationship of a series of life activities, such as DNA replication, gene expression, signal transduction, and cell cycle regulation.
实验结果显示,经7.5μg/mL姜黄素处理之后,人永生化表皮HaCaT细胞的增殖受到明显抑制,细胞生长抑制率达83.03%;细胞周期检测出现亚二倍体(亚G_1期)细胞峰值,细胞凋亡率达13.1%,并发生G_2/M期阻滞;琼脂糖凝胶电泳出现细胞凋亡典型的DNA“梯状”条带:光镜和透射电镜观察结果显示,经姜黄素诱导处理后的HaCaT细胞出现了细胞体积缩小、核质比例减小、细胞核固缩、核内染色质凝聚、线粒体肿胀、内质网网腔扩大、出现凋亡小体等显著的凋亡特征;而且与细胞凋亡相关的癌基因bcl-2表达下调,抑癌基因p53、fas、bax等表达上调。选择性抽提整装光镜和电镜观察显示,经姜黄素处理的HaCaT细胞的核基质和中间纤维比对照组明显稀疏,且分布更加不均匀,并分别与核纤层连系,形成趋于断裂但相对还较为完整的网状结构;双向凝胶电泳分析显示在姜黄素诱导HaCaT细胞凋亡前后存在27个差异表达的核基质蛋白,经质谱分析,鉴定了其中的21个蛋白,其中在凋亡的HaCaT细胞中表达上调的9种蛋白鉴定为:Chaperonin containing TCP1、RNA binding motif、Hypothetical proteinAt4g18230、serine proteinase inhibitor、SUMO-1-specific protease、nucleoporinNUP107、coagulation factor V precursor、Apoptosis-inducing factor、Caspase 3;表达下调的6种蛋白质为LMNA protein、Vimentin、Glycosyl transferase,family 2、Actin、MAP/microtubule affinity-regulating kinase、translation initiation factor IF-2。诱导凋亡处理后新出现的6种蛋白质为Alcohol dehydrogenase[NADP~+]、锌指蛋白ZNF483、MHC classⅠantigen、M-phase phosphoprotein-1、Heat shock 90kDaprotein、Protein kinase D3。鉴定出的21种核基质蛋白中除vimentin、actin,LMNAprotein,NUP107,HSP90,SUMO-4蛋白酶,ZNF483 protein,MHC classⅠantigen,Chaperonin containing TCP1外,其余均为首次在核基质中发现的蛋白质。
研究结果表明,7.5μg/mL姜黄素对人永生化表皮HaCaT细胞的凋亡具有显著诱导作用,在HaCaT细胞凋亡过程中不仅其核基质-中间纤维系统构型产生了明显的凋亡特征性变化,而且其核基质蛋白组成也相应发生了明显改变。由此证实了与表皮细胞凋亡诱导相关特异核基质蛋白的存在及其对表皮细胞凋亡诱导的调控作用。
In this study, the curcumin(Cur) was used to induce the immortalized human epithelial cell line HaCaT cells into apoptosis, and its effects were investigated by selective extraction-whole mount optic and transmission electron microscopy and techniques of proteomic. The differentially expressed nuclear matrix proteins were analyzed in order to explore the molecular mechanisms in a system level.
The results revealed that the HaCaT cells were induced into apoptosis after treated with 7.5μg/mL curcumin as the proliferation of HaCaT cells were inhibited, and the inhibitory rate is 83.03%. The results of flow cytometry analysis showed that curcumin could induce the emergence of the phase of apoptosis, and the rate is 13.1% ,the cell cycle were arrested in G_2/M phase. Agarose gel electrophoresis revealed that cell DNA fragment exhibited characteristic " DNA ladder " . Light microscope and electron microscope showed that the morphology of the cells treated with curcumin appeared shrinked, cell nucleus concerntrated,chromatin agglutination, mitochondria swelling, and apoptosis body forming. Immunocytochemistry revealed that the expression of wt p53, Bax, Fas were upregulated significatntly while the products of Bcl-2 proteins were downregulated significantly in the cells treated with Cur. Selective extraction, whole-mount optic and transmission electron microscopy showed that after treated with 7.5μg/mL curcumin the configuration of nuclear matrix-intermediate filament in HaCaT cells was remarkably changed as the filament was few and scattered, not well distributed and arranged irregularly.and connected to lamina respectively, and the filaments of nuclear matrix,nuclear lamin and intermediatefilment connected not very tightly and the formation but tends to fracture but also relatively more complete network structure.There were 27 spots changed remarkably during the apoptosis induced by curcumin,21 of which were identified. the nine up-regulated proteins including nucleoporin NUP107, six down-regulated proteins including LMNA protein and six new proteins were identified.
In conclusion, the configuration and composition of nuclear matrix were changed following the HaCaT cell apoptosis.It is confirmed that specific nuclear matrix proteins take part in the regulation of cell proliferation and apoptosis, and the associated signal transduction pathways are suggested. It provides proofs and a new way to study the machnisms of epithelial cell apoptosis and it is possible to get a system understanding of the molecular mechanisms from the studies of nuclear matrix, as well as to reveal the relationship of a series of life activities, such as DNA replication, gene expression, signal transduction, and cell cycle regulation.
引文
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