摘要
本研究以人参皂甙Rg1、肉桂酸、丹参酮ⅡA等中药有效成分组合(RCT)处理人成骨肉瘤MG-63细胞,鉴定中药有效成分对人成骨肉瘤细胞终末分化的诱导效果。在此基础上,综合应用亚细胞蛋白质组学及免疫细胞化学分析等技术,对MG-63细胞诱导分化过程中的核基质蛋白变化进行了系统研究。分析鉴定和确证与肿瘤细胞增殖分化相关的特异核基质蛋白,并研究特异核基质蛋白与人成骨肉瘤细胞相关癌基因、抑癌基因在细胞内的共定位关系与变化,探索它们在肿瘤细胞诱导分化过程中的调控作用。以期能够在较为整体水平上阐明中药有效成分诱导肿瘤细胞分化的作用机制并进一步认识细胞癌变与逆转调控机理。
实验结果显示,33μg/mL人参皂甙Rg1+2mmol/L肉桂酸+0.3μg/mL丹参酮ⅡA的组合能有效抑制人成骨肉瘤MG-63细胞的增殖活动,细胞增殖抑制率达72.37%,G0/G1期的细胞比例由47.5%上升为72.5%,S期细胞比例由20%下降为7.3%,细胞周期被阻滞于G0/G1期;光镜和透射电镜观察结果显示,经RCT诱导处理后的MG-63细胞出现了细胞形态规则、核质比例减小、细胞核形状规则、核内异染色质减少、常染色质增多、线粒体和粗糙型内质网增多、高尔基体结构较为典型、细胞表面微绒毛减少等变化;人成骨肉瘤细胞终末分化标志物Ⅰ型胶原、骨钙蛋白、钙化糖原颗粒增多并形成典型的骨节结结构;免疫细胞化学检测结果显示,癌基因c-fos和c-myc表达产物减少,抑癌基因Rb和p27的表达产物增多。选择性抽提整装光镜和电镜观察显示,经RCT组合处理的MG-63细胞的核基质纤维和中间纤维丰富,分布均匀,单丝成份增多,并分别与核纤层密切连系,形成贯穿核质区域的较为规则的网络结构。双向凝胶电泳及MALDI—TOF质谱分析共鉴定出17个在MG-63细胞分化前后存在差异表达的核基质蛋白,其中表达上调的主要有Msh3、Annexin A1、Vimentin、Cep290等7种蛋白,表达下调的有Prohibitin、Nucleophosmin、hnRNP A2/B1、Actin、TRIP-1、Lamin-B1、Lamin-A/C、Mitosin等10种蛋白。蛋白印迹杂交与免疫荧光显微镜观察确证了Vimentin、Actin、hnRNP、Nucleophosmin和Prohibitin等在MG—63细胞分化过程中的表达变化。免疫荧光激光共聚焦显微镜观察结果显示Prohibitin、Nucleophosmin与癌基因c-myc、c-fos和抑癌基因p53、Rb表达产物有共定位关系,它们的表达水平和细胞定位在诱导分化前后发生了变化。
研究表明,RCT组合能有效抑制人成骨肉瘤MG-63细胞增殖活动,改变MG-63细胞形态与超微结构特征,下调癌基因c-fos、c-myc和上调抑癌基因p27和Rb等的表达,并增强Ⅰ型胶原、骨钙蛋白、骨粘素的表达和钙化糖原颗粒及典型骨结节的形成,从而对人成骨肉瘤细胞的终末分化具有显著诱导作用。在RCT组合诱导MG-63细胞分化过程中,其核基质-中间纤维系统构型特征产生了恢复性改变,并出现了多种核基质蛋白表达水平的显著变化。其中Nucleophosmin、Prohibitin、hnRNP、Vimentin和Actin为MG-63细胞分化的共同差异核基质蛋白。而Prohibitin和Nucleophosmin分别与c-fos、c-myc、p53、Rb均存在共定位关系并在MG-63细胞分化过程中出现分布和位移变化现象。由此证实了RCT组合能够干预MG-63细胞相关癌基因、抑癌基因的活性,改变一些与细胞DNA复制、RNA转录加工与运输、基因表达和信号转导相关的重要核基质蛋白的表达,进而阻滞细胞周期,促使人成骨肉瘤细胞分化。从而为在较为整体的水平上深入研究中药有效成分诱导肿瘤分化过程中相关癌基因和抑癌基因表达、细胞信号转导和细胞周期调控及其相互作用等一系列细胞生命活动过程的相互关系提供了科学依据与探索研究的新方向,并为肿瘤的病理诊断和抗癌药物研究等提供新的靶向性蛋白。
In this study, the combination of effective ingredients from Chinese materia medica (33μg/mL ginsenoside Rg1, 2mmol/L cinnamic acid and 0.3μg/mL tanshinone II A, shortened form RCT in this paper) was used to induce the osteosarcoma MG-63 cells into terminal differentiation, and its effects were investigated by cellular biology and immunocytochemistry. The differentially expressed nuclear matrix proteins were analyzed by subcellular proteomics methods and laser Confocal scanning microscope in order to find out a new way to explore the molecular mechanisms of carcinogenesis and malignant phenotypic reversion in a systematic level.
The results revealed that the proliferation of MG-63 cells were inhibited, as the percentage of cells increased from 47.5% to 72.5% in G0/G1 phase while decreased from 20.0% to 7.3% in S phase. The malignant morphological and ultrastructural characteristics were reversed towards the normal related cells, e.g., the morphology of the cells were regular and inclined to the same volume, the nucleo-cytoplasm ratio lessened, the heterochromatin decreased while euchromatin increased, the organelle increased and their structure appeared well-developed ,typical and polarized, microvilli were rare on the cellular surface. The expression level of the phenotype markers of osteoblast such as collagen, osteocalcin and osteonectin, was upregulated and the typical mineralized bone nodules could be observed under light microscopy in the treated cells. Immunocytochemistry assay revealed that the expression level of oncogene including c-myc, c-fos was downregulated, while the expression level of tumor suppressor genes including p27, Rb was upregulated. The configuration of nuclear matrix-intermediate filament was altered towards the normal related cells as the filament was more abundant, the distribution was regular and the filaments of nuclear matrix, nuclear lamina and intermediatefilment connected tightly into a compact meshwork. Nuclear matrix proteins, selectively extracted from MG-63 cells treated with or without RCT, were subjected to Proteomic analysis. Seventeen differentially expressed proteins were identified, including up-regulated proteins Msh3, Annexin A1, Vimentin, mago nashi homolog, Cep290 and Pseudouridylate synthase 7 homolog ; down-regulated proteins Prohibitin, Nucleophosmin, hnRNP A2/B1, Actin, TRIP-1, Lamin-B1, Lamin-A/C and Mitosin. Most of them are related to DNA replication and repair, cell cycle regulation, gene expression and regulation. Some proteins were confirmed by western blot and immunofluorescence analysis. Prohibitin and nucleophosmin, two of the specific nuclear matrix proteins, were colocalized with oncogene c-fos and c-myc products, as well as tumor suppressor gene p53 and Rb products. The expression level and location of them were changed following the proliferation and differentiation of MG-63 cells.
Our study showed that the osteosarcoma MG-63 cells were induced into terminal differentiation after treatment with RCT, as the proliferation of MG-63 cells were inhibited, the cell cycle were arrested in G0/G1, the malignant morphological and ultrastructural characteristics were reversed, the configuration of nuclear matrix-intermediate filament was altered, the expression of the osteoblat- like phenotype markers were highly increased. Some nuclear matrix proteins which play an important role in DNA replication and repair, cell cycle regulation, gene expression and regulation, have changed in expression level and location during the differentiation. The colocalization of prohibitin and nucleophosmin with products of osteosarcoma associated oncogenes c-myc, c-fos and tumor suppressor genes Rb, p53, suggested the pathway and mechanisms in which specific nuclear matrix proteins regulated the proliferation and differentiation of the human osteosarcoma MG-63 cells. These results indicated that RCT can induce MG-63 cells into terminal differentiation by regulating the activities of some oncogenes and antioncongenes, altering the expression of some key nuclear matrix proteins, arresting cell cycle, and facilitating the expression of osteoblat-like phenotype markers. Our study provides the proofs and a new way to explore the mechanisms and the relationships of a series of life activities during the differentiation in tumor cells induced by effective ingredients from Chinese materia medica, e.g., the expression of oncogenes and tumor suppressor genes, signal transduction, and cell cycle regulation. Besides, this study provides several potential target proteins for cancer diagnosis and cancer therapy.
引文
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3.《中医药创新发展规划纲要(2006-2020年)》,科技部、卫生部、国家中医药管理局、国家食品药品监督管理局等国务院16个部门3月21日联合发布.
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