人参皂苷Rg1组合诱导人成骨肉瘤MG-63细胞分化过程中Prohibitin的表达与定位变化

选择性抽提经人参皂苷Rg1组合（RCT）诱导处理前后的人成骨肉瘤MG-63细胞核基质,对prohibitin在核基质中的存在、分布及其与相关基因产物在RCT处理前后MG-63细胞中的共定位关系进行观察研究.蛋白质组学分析结果显示,prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在RCT处理后细胞核基质中表达下调;蛋白质印迹杂交确证了prohibitin在MG-63细胞核基质中的存在及其在RCT处理后下调变化;免疫荧光显微镜观察进一步证实prohibitin定位在核基质上,经RCT处理后出现分布位置与表达水平变化;激光共聚焦显微镜观察可见prohibitin与c-Fos、c-Myc、p53和Rb基因产物均存在共定位关系,并在RCT处理后共定位分布区域出现变化.本研究证实了prohibitin是一种新发现的核基质蛋白,其在核基质上的定位与表达在RCT诱导分化前后发生显著变化,并与相关癌基因、抑癌基因产物存在共定位关系.实验表明RCT处理引起的prohibitin的变化与人成骨肉瘤MG-63细胞的诱导分化与调控具有密切关系,为深入揭示RCT等中药有效成分诱导肿瘤细胞分化的机理提供了重要科学依据和深入探索的新方向.     

人参皂甙Rg1、肉桂酸和丹参酮IIA组合对成骨肉瘤MG-63细胞增殖与相关基因表达的影响

研究中药有效成分人参皂甙Rg1、肉桂酸和丹参酮IIA组合对人成骨肉瘤MG-63细胞增殖抑制和相关基因表达影响,探索其对肿瘤细胞的生物学效应.以33 μg/ml人参皂甙Rg1、296.32 μgml肉桂酸和0.3 μg/ml丹参酮IIA的组合(简称RCT)处理人成骨肉瘤MG-63细胞,以肿瘤细胞分化诱导物HMBA处理MG-63细胞为平行对照,用流式细胞仪、免疫细胞化学检测及光镜观察系统研究RCT组合对MG-63细胞的作用.生长曲线及细胞周期检测显示RCT组合可显著抑制MG-63细胞的增殖,细胞生长抑制率达72.37%,细胞周期阻滞于G0/G1期;免疫细胞化学检测显示RCT组合处理后MG-63细胞的癌基因c-fos、c-myc表达下调,抑癌基因p27、Rb表达上调.RCT组合对MG-63细胞增殖及相关基因表达的影响与分化诱导物六亚甲基双乙酰胺(HMBA)处理组相似.     

HMBA诱导人成骨肉瘤MG-63细胞分化过程中prohibitin的表达与定位变化

本文以HMBA诱导处理前后的人成骨肉瘤MG-63细胞为对象,对prohibitin在核基质中存在、分布及其与相关基因产物在HMBA处理前后MG-63细胞中的共定位关系进行观察研究.蛋白印迹杂交结果确证prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调,免疫荧光显微镜观察显示prohibitin定位在核基质上,经HMBA处理后出现分布位置与表达水平的变化.激光共聚焦显微镜观察可见prohibitin与MG-63细胞中c-fos、c-myc、p53和rb基因产物均存在共定位关系,但在HMBA处理后细胞中其共定位分布区域出现变化.研究结果证实prohibitin是一种核基质蛋白,定位于核基质上,prohibitin在人成骨肉瘤MG-63细胞诱导分化过程中的表达分布及其与相关癌基因、抑癌基因产物的共定位现象值得进一步探索和研究.     

姜黄素诱导人成骨肉瘤MG-63细胞凋亡过程中hnRNP A2/B1表达与定位

姜黄素(curcumin)诱导处理的人成骨肉瘤MG-63细胞,在光镜和电镜观察细胞凋亡的基础上,对hnRNP A2/B1在核基质中存在、分布及其与凋亡相关基因产物在MG-63细胞中的共定位关系进行了研究.经姜黄素处理后,细胞出现染色质凝聚、细胞核固缩、凋亡小体等典型的细胞凋亡形态特征;双向凝胶电泳和质谱鉴定结果显示,hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,在姜黄素处理后细胞核基质蛋白中表达下调.蛋白质印迹杂交结果,证实hnRNP A2/B1在姜黄素处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化.免疫荧光显微镜观察显示,hnRNP A2/B1定位于MG-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平变化.激光扫描共聚焦显微镜的观察结果显示,hnRNP A2/B1在MG-63细胞凋亡过程中与Bax、Bcl-2、Fas和p53等基因产物具有共定位关系,且其共定位区域发生了变化.研究结果证实了hnRNP A2/B1定位于核基质纤维上,是一种核基质蛋白,在姜黄素诱导人成骨肉瘤MG-63凋亡过程中的表达与分布变化及其与凋亡相关基因的关系显然对MG-63细胞凋亡具有重要影响,这为深入揭示肿瘤细胞凋亡的机制提供了重要科学依据和深入探索的新方向.     

人成骨肉瘤MG-63细胞在HMBA诱导分化后核基质蛋白表达的变化

HMBA诱导处理人成骨肉瘤MG-63细胞分化后,选择性抽提核基质蛋白,经双向凝胶电泳技术分析．共有12个蛋白点表达发生变化,经肽指纹图谱分析鉴定了9个蛋白质,其中,MHCⅡ类抗原、IFN刺激的基因因子3α蛋白、DKFZp434M2221．1蛋白、8-羟基-鸟嘌呤糖基化酶同功酶oggl和波形蛋白表达上调,hnRNP A2/B1和肌动蛋白表达下调,60S核糖体蛋白L21和ST2蛋白仅在分化的MG-63细胞中表达。研究结果表明肿瘤细胞增殖分化过程中伴随核基质蛋白表达的变化,并为深入揭示核基质蛋白与细胞癌变和逆转的关系以及阐明细胞增殖分化的基因表达调控原理提供了依据。     

姜黄素诱导人成骨肉瘤MG-63细胞凋亡过程中核基质蛋白表达的变化

应用选择性抽提、整装透射电镜观察和双向聚丙烯酰胺凝胶电泳与质谱鉴定技术研究细胞凋亡诱导物姜黄素处理前后人成骨肉瘤MG-63细胞核基质-中间纤维系统的构型变化．及其核基质蛋白表达的差异。经姜黄素处理后,MG-63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MG-63细胞凋亡前后存在27个差异表达的核基质蛋白．经质谱鉴定了其中的21个蛋白．在凋亡的MG-63细胞中表达上调的蛋白鉴定为：DNA聚合酶zeta等7种蛋白：表达下调的蛋白质为：Prohibitin等14种蛋白。其中首次在核基质中发现的蛋白质有17个。因此．在MG-63细胞凋亡过程中不仅其核基质-中间纤维系统构型产生了典型的的凋亡特征性变化．而且伴有核基质蛋白表达的差异。由此证实了与肿瘤细胞凋亡诱导相关特异核基质蛋白的存在及其对肿瘤细胞凋亡诱导的调控作用．从而对揭示核基质构型及其蛋白组成与细胞凋亡的关系和阐明细胞凋亡过程中的基因表达调控机理．均具有重要意义。     

HMBA诱导人肝癌SMMC-7721 细胞分化过程中 Nucleophosmin 的表达与定位变化

通过选择性抽提经环六亚甲基双乙酰胺（hexamethylene bisacetamide,HMBA）诱导处理前后的人肝癌SMMC-7721细胞核基质,并运用亚细胞蛋白质组学等分析技术,研究nucleophosmin (NPM)在核基质上的表达和定位变化,及其与相关基因产物的共定位关系,观察研究了nucleophosmin 在诱导分化前后人肝癌SMMC-7721细胞核基质中的存在、分布及其与相关基因产物的共定位关系.双向凝胶电泳和质谱鉴定结果显示,nucleophosmin 存在于 SMMC-7721 细胞核基质蛋白组分中,在 HMBA 处理后细胞核基质中表达下调.蛋白质印迹杂交实验结果确证了 nucleophosmin 在核基质中的存在及其在诱导处理后细胞核基质中表达下调的变化.免疫荧光显微镜观察显示,nucleophosmin 定位在 SMMC-7721细胞核基质上,经 HMBA处理后出现分布位置与表达水平的变化.激光扫描共聚焦显微镜观察结果显示,SMMC-7721细胞中,nucleophosmin与 c-fos、c-myc、rb、p53 等基因产物具有共定位关系,但在诱导处理后细胞内的共定位区域发生了改变.研究结果证实,nucleophosmin 是一种核基质蛋白,定位于核基质纤维上,nucleophosmin 在人肝癌 SMMC-7721 细胞诱导分化过程中的表达分布,及其与相关癌基因、抑癌基因产物的关系对 SMMC-7721 细胞分化具有重要影响.     

丹参酮ⅡA对人成骨肉瘤MG-63细胞形态结构和终末分化指标的影响

目的:观察中药有效成分丹参酮ⅡA(Tan ⅡA)对人成骨肉瘤MG-63细胞形态与超微结构和相关终末分化指标的影响,以鉴定其对肿瘤细胞终末分化的诱导作用.方法:0.5μg/mL丹参酮ⅡA处理MG-63细胞,光镜、电镜观察和免疫细胞化学检测系统研究MG-63细胞处理前后细胞形态、超微结构变化和成骨细胞相关终末分化蛋白的表达变化.结果:光镜与电镜观察结果显示经Tan ⅡA处理细胞产生了核质比例减小、异染色质减少、常染色质增多、细胞器丰富发达、细胞表面微绒毛减少等显著变化;免疫细胞化学检测显示处理后MG-63细胞I型胶原蛋白、骨粘素和骨钙蛋白的表达呈阳性,并观察到钙化糖原颗粒增多和典型骨结节的形成.结论:丹参酮ⅡA能显著改变MG-63细胞形态与超微结构恶性特征,并促进与成骨细胞相关的终末分化指标的表达变化,从而对人成骨肉瘤细胞的终末分化具有明显的诱导作用.     

三氧化二砷下调MG-63骨肉瘤细胞IEX-1基因表达的转录调控机制研究

三氧化二砷(arsenic trioxide, As₂O₃)是中国传统中药砒霜的主要有效成分,最早应用于血液系统肿瘤的治疗,随后研究表明其对实体瘤也具有抑制细胞增殖并诱导凋亡的作用．早期研究发现,1.0 μmol/L的As₂O₃可以体外诱导骨肉瘤细胞系MG-63细胞凋亡,进一步的cDNA芯片分析、RT-PCR、RNA印迹证实细胞凋亡与As2O3干预后IEX-1基因表达下调有关．IEX-1为早期诱导应答基因,调节细胞生长和凋亡．通过荧光素酶分析,EMSA、蛋白质印迹等实验,发现As₂O₃干预骨肉瘤细胞系MG-63后能诱导p53蛋白表达上调,增加的p53蛋白通过与IEX-1的启动子结合,转录抑制IEX-1的转录,导致IEX-1基因表达下调．进一步证实了IEX-1与骨肉瘤的重要关系,同时也阐明As₂O₃诱导IEX-1基因表达下调的转录调控机制．     

Oxymatrine Extracted from Sophora flavescens Inhibited Cell Growth and Induced Apoptosis in Human Osteosarcoma MG-63 Cells In Vitro

Oxymatrine, one of the most active components of the ethanol extracts from Sophora flavescens, is known for its potent antitumor activity both in vitro and in vivo. However, the mechanism of its action in mediating the cell apoptosis remains elusive. In this study, we investigated the proliferation inhibitory and apoptotic activities of oxymatrine against human osteosarcoma MG-63 cells. The compound was found to markedly and dose-dependently inhibit the cell proliferation determined by 5-bromo-2-deoxyuridine incorporation. Oxymatrine also induced the cell apoptosis in a dose- and time-dependent manner as showed by the annexin V-FITC/PI double staining and TUNEL assay. Furthermore, a disruption of mitochondrial membrane potential and an up-regulation of cleaved caspases-3, and-9 and downregulation of Bax/Bcl-2 was evidenced in the oxymatrine-treated cells. These proteins have been known to play a pivotal role in the regulation of apoptosis. In conclusion, these observations indicate of the oxymatrine potential as an effective antitumor agent against osteosarcoma. Moreover, the compound appears to exert its anti-tumor action by stimulating the caspase-triggered signaling pathway.     

维甲酸对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响

目的:研究维甲酸对人成骨肉瘤MG-63细胞增殖和相关基因表达的影响,以探索其对成骨肉瘤细胞的生物学效应.方法:以1μmol/L维甲酸处理人成骨肉瘤MG-63细胞,生长曲线测定,流式细胞仪分析、光镜观察和免疫细胞化学检测等研究维甲酸对MG-63细胞的生长曲线、细胞周期和相关癌基因、抑癌基因表达的影响,并对其作用机理进行初步分析.结果:维甲酸处理7天后,MG-63细胞生长抑制率达到42.2%,G0/G1期比例达到61.8%,细胞形态铺展,排列趋于规则,癌基因c-myc、c-fos的表达降低.而抑癌基因Rb、p27表达上调.结论:1μmol/L维甲酸可以有效抑制细胞的增殖活动,改变细胞恶性形态特征,下调癌基因c-myc、c-fos和上调抑癌基因Rb、p27的表达,从而对人成骨肉瘤细胞分化具有诱导作用.     

姜黄素对人成骨肉瘤MG-63细胞增殖抑制和凋亡相关基因表达的影响

目的:探讨姜黄素对人成骨肉瘤MG-63细胞增殖抑制及凋亡相关基因表达的影响.方法:用姜黄素处理MG-63细胞,细胞计数法检测细胞增殖抑制的效果;荧光染色观察细胞的凋亡;流式细胞仪(FCM)进行细胞周期时相分析;免疫细胞化学法和western blotting免疫法检测细胞凋亡相关基因的表达水平.结果:随着姜黄素浓度的增加及其作用时间延长,对细胞的增殖抑制作用增强,最高抑制率可达89.07%;光镜下可观察到细胞发生染色质浓缩,细胞核凝聚和碎裂等典型的凋亡形态学改变;FCM检测结果显示细胞经姜黄素处理后出现明显的凋亡峰;免疫反应结果显示,凋亡相关基因bcl-2和P53表达水平降低,而bax和Fas表达水平升高.结论:姜黄素能显著抑制MG-63细胞增殖并可有效诱导其凋亡,其作用机制可能与bcl-2和bax二者的比值发生变化从而接受了凋亡刺激信号有关.     

Biological Characteristics of the MG-63 Human Osteosarcoma Cells on Composite Tantalum Carbide/Amorphous Carbon Films

Tantalum (Ta) is a promising metal for biomedical implants or implant coating for orthopedic and dental applications because of its excellent corrosion resistance, fracture toughness, and biocompatibility. This study synthesizes biocompatible tantalum carbide (TaC) and TaC/amorphous carbon (a-C) coatings with different carbon contents by using a twin-gun magnetron sputtering system to improve their biological properties and explore potential surgical implant or device applications. The carbon content in the deposited coatings was regulated by controlling the magnetron power ratio of the pure graphite and Ta cathodes. The deposited TaC and TaC/a-C coatings exhibited better cell viability of human osteosarcoma cell line MG-63 than the uncoated Ti and Ta-coated samples. Inverted optical and confocal imaging was used to demonstrate the cell adhesion, distribution, and proliferation of each sample at different time points during the whole culture period. The results show that the TaC/a-C coating, which contained two metastable phases (TaC and a-C), was more biocompatible with MG-63 cells compared to the pure Ta coating. This suggests that the TaC/a-C coatings exhibit a better biocompatible performance for MG-63 cells, and they may improve implant osseointegration in clinics.     

Dendropanoxide Induces Autophagy through ERK1/2 Activation in MG-63 Human Osteosarcoma Cells and Autophagy Inhibition Enhances Dendropanoxide-Induced Apoptosis

Anticancer effects of dendropanoxide (DP) newly isolated from leaves and stem of Dendropanax morbifera Leveille were firstly investigated in this study. DP inhibited cell proliferation and induced apoptosis in dose- and time-dependent manner in MG-63 human osteosarcoma cells, which was dependent on the release of cytochrome c to the cytosol and the activation of caspases. Moreover, the DP-treated cells exhibited autophagy, as characterized by the punctuate patterns of microtubule-associated protein 1 light chain 3 (LC3) by confocal microscopy and the appearance of autophagic vacuoles by MDC staining. The expression levels of ATG7, Beclin-1 and LC3-II were also increased by DP treatment. Inhibition of autophagy by 3-methyladenine (3-MA) and wortmannin (Wort) significantly enhanced DP-induced apoptosis. DP treatment also caused a time-dependent increase in protein levels of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased DP-induced autophagy that was accompanied by an increased apoptosis and a decreased cell viability. These results indicate a cytoprotective function of autophagy against DP-induced apoptosis and suggest that the combination of DP treatment with autophagy inhibition may be a promising strategy for human osteosarcoma control. Taken together, this study demonstrated for the first time that DP could induce autophagy through ERK1/2 activation in human osteosarcoma cells and autophagy inhibition enhanced DP-induced apoptosis.     

人参皂苷Rg1促进人牙周膜干细胞增殖及骨向分化

人参皂苷Rg1 (GS Rg1) 是人参的主要药理活性成分. GS Rg1有刺激造血干细胞的形成和促进骨髓间充质干细胞增殖和分化的作用.而人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）具有自我更新和多向分化的干细胞特性.但目前关于GS Rg1能否促进牙周膜干细胞增殖和分化的研究尚不多见.本研究证明,1×10-5 mol/L GS Rg1作用于hPDLSCs后,能明显促进牙周膜干细胞的增殖与分化.MTT检测细胞增殖显示,培养液中加入了GS Rg1实验组在第2,3,4,5 d增殖情况明显高于对照组,提示GS Rg1成分促进了牙周膜干细胞的增殖. 检测ALP表达量,RUNX2,Collagen I,OPN,OCN表达水平,加药实验组表达水平均高于未加药对照组,它们的表达量的增高提示成骨分化的增强. 牙周膜干细胞与纳米羟基磷灰石支架结合的电镜图片及其支架植入小鼠体内后的免疫组化检测显示,含有GS Rg1成分的细胞支架能促进形成更多的骨样组织.因此,GS Rg1能促进hPDLSCs体内体外的增殖及骨向分化,并在替代传统生长因子应用于牙周组织工程方面有较好前景.     

The Beneficial Effect of Ginsenoside Rg1 on Schwann Cells Subjected to Hydrogen Peroxide Induced Oxidative Injury

Ginsenoside Rg1 (GRg1) has been considered to have therapeutic potential in promoting peripheral nerve regeneration and functional recovery after sciatic nerve injuries. However, the mechanism underlying the beneficial effect of GRg1 on peripheral nerve regeneration is currently unclear. The possible effect of GRg1 on Schwann cells (SCs), which were subjected to oxidative injury after nerve injury, might contribute to the beneficial effect of GRg1 on nerve regeneration. The present study was designed to investigate the potential beneficial effect of GRg1 on SCs exposed to oxidative injury. The oxidative injury to SCs was induced by hydrogen peroxide. The effect of GRg1 (50 μM) on SCs exposed to oxidative injury was measured by the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) in SCs. The cell number and cell viability of SCs were evaluated through fluorescence observation and MTT assay. The apoptosis of SCs induced by oxidative injury was evaluated by an apoptosis assay. The expression and secretion of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were evaluated using RT-PCR, Western blotting, and an ELISA method. We found that GRg1 significantly up-regulated the level of SOD, GSH and CAT, and decreased the level of MDA in SCs treated with hydrogen peroxide. In addition, GRg1 has been shown to be able to inhibit the proapoptotic effect of hydrogen peroxide, as well as inhibit the detrimental effect of hydrogen peroxide on cell number and cell viability. Furthermore, GRg1 also increased the mRNA levels, protein levels and secretion of NGF and BDNF in SCs after incubation of hydrogen peroxide. Further study showed that preincubation with H89 (a PKA inhibitor) significantly inhibited the effects induced by hydrogen peroxide, indicating that the PKA pathway might be involved in the antioxidant effect and neurotrophic factors (NTFs) promoting effect of GRg1. In addition, a short-term in vivo study was performed to confirm and validate the antioxidant effect and nerve regeneration-promoting effect of GRg1 in a sciatic crush injury model in rats. We found that GRg1 significantly increased SOD, CAT and GSH, decreased MDA, as well as promoted nerve regeneration after crush injury. In conclusion, the present study showed that GRg1 is capable of helping SCs recover from the oxidative insult induced by hydrogen peroxide, which might account, at least in part, for the beneficial effect of GRg1 on nerve regeneration.