Ad-IL-24对人胶质瘤细胞生长抑制效应的体外实验

研究携带人白介素24(IL-24)的腺病毒表达载体(Ad-IL-24)对人U251胶质瘤细胞生长的影响和抗肿瘤分子机制。将不同MOI Ad-IL-24感染U251人胶质瘤细胞后, MTT法检测Ad-IL-24对U251细胞生长的抑制作用, 流式细胞仪和Hochest 染色法检测细胞的凋亡率。RT-PCR检测bcl-2、bax、ICE、C-myc、HIF-1a和p53等基因的转录表达水平, Western blotting检测Cleaved Caspase-3的表达。结果表明100 MOI Ad-IL-24感染U251细胞后能明显抑制细胞生长, 并能明显诱导细胞凋亡, 感染72 h后细胞凋亡率可达42%, 感染4 d后细胞生长抑制率可达50%。RT-PCR检测发现Ad-IL-24能引起与细胞凋亡和血管形成相关基因bax/bcl-2、ICE、C-myc、p53的上调和HIF-1a的下调, 并促进Caspase-3的活化。本研究结果显示Ad-IL-24能明显抑制人胶质瘤细胞U251生长和诱导细胞凋亡, 其抗肿瘤机制可能与通过bax/ bcl-2、ICE、c-myc、p53的上调和HIF-1a的下调, 进而导致Caspase-3的活化而诱导肿瘤细胞发生凋亡有关。     

Ad-IL-24对SGC-7901胃癌细胞生长抑制的体外实验

旨在研究携带人IL-24基因的腺病毒表达载体(Ad-IL-24)对SGC-7901人胃癌细胞的生长抑制作用并分析其分子机制。以不同MOI(感染复数)的Ad空载体腺病毒感染SGC-7901人胃癌细胞,筛选出最佳感染剂量;Ad-IL-24以最佳感染剂量感染SGC-7901胃癌细胞,RT-PCR法和Western blotting法检测腺病毒介导的IL-24基因在SGC-7901胃癌细胞中的转录;MTT法检测Ad-IL-24对SGC-7901胃癌细胞的生长抑制作用,流式细胞仪检测其诱导SGC-7901人胃癌细胞凋亡和细胞周期改变的效应,Hoechst33258染色荧光显微镜检测其诱导胃癌细胞凋亡的核形态改变;RT-PCR半定量法进一步检测SGC-7901胃癌细胞中凋亡相关基因的转录。结果显示,100MOI为感染SGC-7901胃癌细胞的腺病毒最佳感染剂量;Ad-IL-24能成功介导IL-24基因在SGC-7901胃癌细胞中转录性表达;Ad-IL-24感染SGC-7901胃癌细胞后,能明显抑制胃癌细胞生长和诱导凋亡;Ad-IL-24能显著上调SGC-7901胃癌细胞中bax、caspase-3和p53的表达和下调bc...     

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.     

Ad-ING4-IL-24 双基因共表达对人肺腺癌化疗的增敏效应

研究ING4 (肿瘤生长抑制因子4) 和IL-24 (人白细胞介素24) 双基因共表达腺病毒载体 (Ad-ING4-IL-24) 对肺腺癌的化疗增敏效应及分子机制。将Ad-ING4-IL-24感染A549肺癌细胞及联合顺铂 (DDP) 化疗药物作用,RT-PCR和Western blotting检测ING4和IL-24基因在A549肺癌细胞中的转录和表达,MTT法、流式细胞仪 (FCM) 和 Hoechst 染色法检测Ad-ING4-IL-24联合DDP (联合组) 对A549肺癌细胞的生长抑制和凋亡效应。采用A549细胞株建立人肺腺癌裸鼠模型,然后瘤体局部注射干预用药,动态测量肿瘤体积,并计算瘤重抑瘤率,免疫组化检测ING4、IL-24、bax、bcl-2、VEGF等基因的表达。结果表明,Ad-ING4-IL-24感染A549肺癌细胞后可获得成功转录和表达,体外联合组能明显抑制A549肺癌细胞生长和诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。体内联合组同样能显著抑制肿瘤生长,瘤重抑瘤率达52.81％,免疫组化结果显示联合组能上调bax基因表达,同时下调bcl-2、VEGF等基因的表达。以上结果表明Ad-ING4-IL-24具有化疗增敏的作用,该作用机制可能与促进肿瘤细胞凋亡和抑制血管形成有关。     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

腺病毒介导IL-24-E1A双基因载体的构建及其体外抑制SMMC-7721肝癌细胞生长作用初探

构建IL-24和E1A双基因腺病毒载体,获得Ad-IL-24-E1A重组腺病毒子,分析其体外抑瘤作用。PCR及BglⅡ和SalⅠ酶切获得IL-24,与空载体构建成pAdTrack-IL-24-IRES。XhoI和EcoRV酶切获得E1A片段,与pAdTrack-IL-24-IRES连接后成功构建pAdTrack-IL-24-IRES-E1A,同源重组、包装和扩增获得Ad-IL-24-E1A重组病毒子。用50MOI重组腺病毒感染SMMC-7721肝癌细胞,MTT法测定Ad-IL-24-E1A的细胞生长抑制作用;流式细胞仪分析细胞凋亡情况。本研究成功获得Ad-IL-24-E1A重组病毒子。与其他组相比,Ad-IL-24-E1A明显抑制肿瘤细胞生长,感染48h后凋亡率达52%,而抑制正常细胞作用不明显。研究提示Ad-IL-24-E1A双基因重组载体明显抑制SMMC-7721肝癌细胞生长。     

爪哇伪枝藻胞外多糖诱导皮肤癌细胞(A431)凋亡的研究

为探讨爪哇伪枝藻胞外多糖(Extracellular polymeric substances of Scytonema javanicum, EPS)诱导人表皮癌A431细胞凋亡及其对凋亡相关基因caspase-3、bcl-2和bax表达的影响,本实验利用MTT法检测细胞生长抑制情况;HE染色法及透射电镜进行形态学观察;单细胞凝胶电泳法(SCGE/彗星电泳)分析DNA受损情况;免疫组织化学法检测细胞内caspase-3、bcl-2和bax表达水平。结果显示EPS能显著抑制A431细胞增殖,并呈时间和剂量依赖性,作用96h的半数抑制浓度IC50为4.25mg/mL,并出现细胞凋亡的形态学改变;彗星电泳结果与对照相比6mg/mL EPS作用48h能引起A431细胞DNA严重损伤;免疫组织化学检测发现6mg/mL EPS作用72h能显著上调A431细胞内凋亡相关基因caspase-3和bax的表达,而下调bcl-2的表达。     

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.     

NALP1 基因与骨肉瘤细胞的相关研究

目的:探讨NALP1基因在骨肉瘤细胞株MG-63、U-2OS中的表达,以及高表达NALP1基因对于骨肉瘤细胞体外凋亡的影响。方法:使用RT-PCR、Western-blot法检测骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平并与人成骨细胞株hFOB1.19比较。将NALP1基因转染质粒PcDNA3.1,将重组质粒转染骨肉瘤细胞,分成高表达基因组、空质粒组及对照组,加入抗肿瘤药物顺铂及甲氨蝶呤促使肿瘤细胞凋亡,使用流式细胞仪测定各组肿瘤细胞凋亡率。结果:通过统计分析,骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平均低于人成骨细胞株hFOB1.19(P<0.05),NALP1高表达组的肿瘤细胞凋亡率明显高于空白质粒组及对照组。结论:上调骨肉瘤细胞株MG-63、U-2OS中的NALP1的表达量可以促进肿瘤细胞凋亡。     

腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。     

Myricetin inhibits the induction of anti-Fas IgM-, tumor necrosis factor-alpha- and interleukin-1beta-mediated apoptosis by Fas pathway inhibition in human osteoblastic cell line MG-63

The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients with inflamed synovium, such as in rheumatoid arthritis (RA). By means of alkaline phosphatase (ALP) activity and osteocalcin ELISA assay, I have shown that myricetin exhibits a significant induction of differentiation in the human osteoblast-like cell line MG-63. In addition, I also assessed whether myricetin affects inflammatory cytokines-mediated apoptosis in osteoblast cells. TNF-alpha or IL-1beta enhances apoptotic DNA fragmentation in anti-Fas IgM-treated MG-63 cells by increasing Fas receptor expression. However, TNF-alpha or IL-1beta treatment alone does not induce apoptosis. Treatment of MG-63 cells with myricetin not only inhibited anti-Fas IgM-induced apoptosis, but also blocked the synergetic effect of anti-Fas IgM with TNF-alpha or IL-1beta on cell death. The apoptotic inhibition of myricetin is associated with inhibition of TNF-alpha and IL-1beta-mediated Fas expression and enhancement of FLIP expression, resulting in a decrease of caspase-8 and caspase-3 activation. These results indicate a potential use of myricetin in preventing osteoporosis by inhibiting inflammatory cytokines-mediated apoptosis in osteoblast cells.     

不同浓度白花丹素对骨肉瘤细胞MG-63凋亡迁移、基质金属蛋白酶及Bcl-2、Bax、Ezrin蛋白表达的影响

目的:研究不同浓度白花丹素对骨肉瘤细胞MG-63凋亡迁移、基质金属蛋白酶(MMP)及Bcl-2、Bax、Ezrin蛋白表达的影响。方法:取对数生长期的骨肉瘤MG-63细胞,传代培养成细胞株后以随机法分成对照组、低剂量组、中剂量组、高剂量组。其中对照组加入到0.1%浓度的DMSO完全培养基中培养,低剂量组、中剂量组、高剂量组分别加入到浓度为5、10、20μmol/L的白花丹素的有关培养基中培养。培养24 h后,采用Transwell法检测MG-63细胞迁移率、Hoechst33342染色法检测MG-63细胞凋亡率、Western blot法检测四组MG-63细胞的MMP-2、MMP-9、Bcl-2、Bax、Ezrin蛋白表达水平。结果:培养24 h后,低剂量组、中剂量组、高剂量组的骨肉瘤细胞MG-63凋亡率及Bax蛋白表达水平均较对照组升高(P<0.05),且随白花丹素浓度的增加而升高(P<0.05);骨肉瘤细胞MG-63的细胞迁移率、MMP-2、MMP-9、Bcl-2及Ezrin蛋白表达水平较对照组降低(P<0.05),且随白花丹素浓度的增加而降低(P<0.05)。结论:白花丹素对骨肉瘤细胞MG-63凋亡的促进作用以及迁移的抑制作用明显,其作用机制可能与抑制骨肉瘤细胞MG-63中的MMP-2、MMP-9、Bcl-2、Ezrin蛋白表达及促进Bax蛋白表达有关,且浓度越高,抑制或促进作用越明显。     

TNF-α is upregulated in T2DM patients with fracture and promotes the apoptosis of osteoblast cells in vitro in the presence of high glucose

Fracture healing is regulated by proinflammatory mediators such as tumor necrosis factor-α (TNF-α), which poses influence on the balance between bone formation and remodeling. And the diabetes is thought to contribute to the delayed diabetic fracture healing. In the present study, we examined the promotion to proinflammatory cytokines and chemokines in type 2 diabetes mellitus (T2DM) patients with bone fractures, and then evaluated the promotion to TNF-α by the high glucose treatment in human osteoblast-like MG-63 cells and the regulatory role of the promoted TNF-α on the MG-63 cell apoptosis. It was demonstrated that there were significantly-upregulated high-sensitivity C-reactive protein (hsCRP) TNF-α, IL-1β, IL-6, IFN-γ-inducible protein 10 (IP-10) and RANTES in T2DM patients with bone fracture. And the promotion to TNF-α and IL-1β was confirmed in vitro in both mRNA and protein levels in high glucose-treated MG-63 cells. And either TNF-α or high glucose reduced the viability of MG-63 cells, promoted apoptosis and upregulated apoptosis-associated markers, such as released cytochrome c, cleaved caspase 3 and lyzed PARP. Moreover, there was a synergistic effect between TNF-α and high glucose. The viability reduction and the apoptosis induction of MG-63 cells were significantly higher in the group with both TNF-α and high glucose treatments, than in the group with singular TNF-α treatment. In conclusion, our study demonstrated that proinflammatory cytokines and chemokines were promoted in T2DM patients with bone fracture or in osteoblasts by the high glucose stimulation. TNF-α and high glucose synergistically reduced the viability and induced the apoptosis in the osteoblast-like MG-63 cells in vitro. It implies the significant regulatory role of TNF-α in the delayed fracture healing in T2DM.     

基于JAK2/STAT3信号通路探讨白藜芦醇对骨肉瘤MG-63细胞凋亡及迁移的影响

摘要 目的：研究白藜芦醇(RES)通过蛋白酪氨酸激酶2/信号转导子与激活子3（JAK2/STAT3）信号通路对人骨肉瘤体外细胞株MG-63细胞凋亡、侵袭和迁移的影响。方法：体外培养MG-63细胞,以不同浓度的RES作用于MG-63细胞。Annexin V-FITC/PI双染流式细胞术检测不同时间和不同浓度的RES对MG-63细胞凋亡的影响。划痕实验和Transwell实验检测不同时间和不同浓度的RES对MG-63细胞侵袭和迁移能力的影响。免疫印迹实验检测不同时间和不同浓度的RES对MG-63细胞磷酸化蛋白酪氨酸激酶2（p-JAK2）、磷酸化信号转导子与激活子3（p-STAT3）、凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2家族促凋亡蛋白（Bax）及基质金属蛋白酶（MMP）-2、MMP-9表达的影响。结果：RES浓度越高,时间越久,MG-63细胞凋亡率越高（P<0.05）。RES浓度越高,MG-63细胞迁移和侵袭能力越弱(P<0.05)。RES处理MG-63细胞后其p-JAK2、p-STAT3、Bcl-2以及MMP-2、MMP-9的表达明显降低,而Bax蛋白表达明显升高,且p-JAK2、p-STAT3、Bax、Bcl-2以及MMP-2、MMP-9的表达水平变化具有RES浓度依赖性（P<0.05）。结论：RES可能通过调控JAK2/STAT3信号通路促使人骨肉瘤MG-63细胞凋亡,并抑制MG-63细胞侵袭和迁移。     

Dieckol exerts anticancer activity in human osteosarcoma (MG-63) cells through the inhibition of PI3K/AKT/mTOR signaling pathway

BackgroundOsteosarcoma (OS) is the most common malignant bone cancer with more metastasis and increased occurrence in children and teen-agers and being responsible for more number of morbidity and mortality worldwide.ObjectiveThe current exploration was planned study the in vitro anticancer actions of dieckol against human OS MG-63 cells via PI3K/AKT/mTOR signaling inhibition.MethodologyThe cytotoxicity of dieckol was scrutinized by MTT assay. Effects of dieckol on the ROS accumulation, apoptotic cell death, and MMP level in the MG-63 cells were studied by respective fluorescence staining assays. The levels of proliferative, inflammatory, and apoptotic markers in the dieckol treated MG-63 cells were scrutinized by marker specific kits. The expressions of PI3K, AKT, and mTOR was assayed by RT-PCR.ResultsThe MTT assay revealed that the dieckol dose dependently prevented MG-63 cells viability and the IC50 was found at 15 µM. Dieckol treatment effectively reduced the MMP level and improved the ROS generation and apoptosis in MG-63 cells. Dieckol also regulated the proliferative (cyclin D1), inflammatory (COX-2, IL-6, TNF-α, and NF-κB), and apoptotic (caspase-3, Bax, Bcl-2) markers in the MG-63 cells. The PI3K/AKT/mTOR signaling in the MG-63 cells were effectively inhibited by the dieckol treatment.ConclusionIn conclusion, our findings from this study recommends that the dieckol could be a talented anticancer candidate for the OS management in the future.     

Regulation of integrin-type cell adhesion receptors by cytokines.

Integrin heterodimers which share a common beta 1 subunit are the major cellular receptors for many extracellular matrix proteins. Here, we show that two inflammatory mediators, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), can regulate the expression of the alpha 1 beta 1 integrin heterodimer, known to be a laminin and collagen receptor. In human skin fibroblasts 10 units/ml IL-1 beta increase the biosynthesis of the alpha 1 integrin subunit an average of 4.5-fold. Furthermore, IL-1 beta can turn on alpha 1 subunit expression in MG-63 human osteosarcoma cells even in conditions where the untreated MG-63 cells do not express it in detectable amounts. The effect of TNF-alpha on alpha 1 subunit expression is similar. Both IL-1 beta and TNF-alpha increased MG-63 cell adhesion on laminin. The effect of transforming growth factor-beta 1 (TGF-beta 1) on integrin expression in MG-63 cells has been previously described (Heino, J., and Massagué, J. (1989) J. Biol. Chem. 264, 21806-21811). TGF-beta 1 decreases the biosynthesis of alpha 3 subunit but increases the production of alpha 2 subunit. IL-1 beta potentiates the effects of TGF-beta 1. Furthermore, in the presence of TGF-beta 1 the increase in the expression of alpha 1 subunit by IL-1 beta is even larger. Thus, IL-1 beta and TGF-beta 1, which usually have antagonistic functions in connective tissue, can regulate integrin expression in a synergistic way.     

Effects of Survivin on cell proliferation and apoptosis in MG-63 cells in vitro

Osteosarcoma is the most frequent primary malignant tumor of the skeleton and occurs mainly in children and adolescent. The prognosis of osteosarcoma is very poor due to its aggressive and no effective treatment. This study is the first to investigate the anti-cancer effects of antisense pEGFP-C1-Survivin on human osteosarcoma cells. It was shown in our results that Survivin blockaded could significantly induce apoptosis and inhibit the invasive of osteosarcoma cells line MG-63. The effects were probably produced by the decreased expression of Survivin induced by antisense pEGFP-C1-Survivin which was examined by RT-PCR and western blotting. All these suggested that Survivin should be very important in the development of osteosarcoma and Survivin blockaded by using antisense pEGFP-C1-Survivin could markedly inhibit the proliferation and invasion of osteosarcoma cells line MG-63, partially reversed their malignant phenotype. Targeting Survivin might be a promising option in the treatment of osteosarcoma.     

Interleukin-1 regulates FGF-2 mRNA and localization of FGF-2 protein in human osteoblasts

Interleukin-1 (IL-1) and basic fibroblast growth factor (FGF-2) are potent stimulators of osteoclast formation. However, the role of FGF-2 in the responses to IL-1 in bone has not been reported. We examined the effect of IL-1 on FGF-2 mRNA and protein expression in human osteosarcoma MG-63 osteoblasts, normal human osteoblasts (NHOB), and osteoblasts from osteoarthritic patients (F2 and F13). IL-1 increased FGF-2 mRNA expression in osteoblasts within 1.5 to 3 h. Multiple FGF-2 protein isoforms were expressed in human osteoblasts. Twenty-four hours of treatment of MG-63 and NHOB cells with IL-1 increased the high-molecular-weight(HMW, 22/24 kDa) and low-molecular-weight (LMW, 18 kDa) FGF-2 proteins intracellularly. In contrast, IL-1 preferentially increased the LMW protein signal intracellularly as well as on the cell surface of F2 and F13 osteoblasts. We conclude that IL-1 is a major stimulator of FGF-2 expression in human osteoblasts. Furthermore, selective increases in the exportable LMW protein in osteoblasts from osteoarthritic patients may be of clinical relevance.