牡蛎低分子活性肽BPO-L对人肺腺癌A549细胞周期和相关癌基因、抑癌基因表达的调控作用

采用酸抽提、凝胶柱层析等方法,从僧帽牡蛎体内分离提取到牡蛎低分子活性多肽组分BPO-L,以HMBA处理组为平行对照,流式细胞仪检测细胞周期变化及以免疫细胞化学方法检测相关癌基因、抑癌基因表达变化.研究BPO-L对人肺腺癌A549细胞分化的生物学效应,探索其对肺癌细胞的作用机理.实验结果显示,BPO-L能有效抑制A549细胞增殖活动,促使细胞阻滞于G0/G1期.在此过程中,A549细胞c-myc,MTp53等癌基因蛋白表达减弱,p21WAF1/CIP1和Rb等抑癌基因蛋白表达活性的增强.本研究证实BPO-L对肺癌细胞具有显著的诱导分化作用.其诱导癌细胞分化机理与其调节和干预c-myc、MTp53等癌基因与p21WAF1/CIP1和Rb等抑癌基因的表达有关.     

丹参酮ⅡA对人成骨肉瘤MG-63细胞增殖的抑制作用

应用细胞培养、台盼蓝拒染计数、光学显微镜观察、流式细胞仪检测及免疫细胞化学等技术研究中药有效成分丹参酮ⅡA对人成骨肉瘤MG-63细胞增殖抑制作用.实验结果显示,丹参酮ⅡA处理MG-63细胞后, 细胞增殖活动受到抑制,抑制率为52.10%,细胞倍增时间由对照组的48 h延长至65 h;细胞发生G0/G1期阻滞,G0/G1期细胞比例由对照组的47.5%上升到59.5%,S期细胞比例则由对照组的20.0%下降至9.0%;并出现细胞形态规则、大小趋于一致、细胞体积增大、核质比例减小等变化;MG-63细胞增殖分化调控相关的癌基因c-fos和c-myc表达活性降低.结果表明,丹参酮ⅡA对人成骨肉瘤MG-63细胞增殖有显著抑制作用,其作用可能与干预c-fos和c-myc等癌基因表达从而调控细胞周期有关.     

丹参酮ⅡA对人成骨肉瘤MG-63细胞增殖的抑制作用

应用细胞培养、台盼蓝拒染计数、光学显微镜观察、流式细胞仪检测及免疫细胞化学等技术研究中药有效成分丹参酮ⅡA对人成骨肉瘤MG-63细胞增殖抑制作用．实验结果显示，丹参酮ⅡA处理MG-63细胞后，细胞增殖活动受到抑制，抑制率为52．10％，细胞倍增时间由对照组的48h延长至65h；细胞发生G0／G1期阻滞，G0／G1期细胞比例由对照组的47．5％上升到59，5％，S期细胞比例则由对照组的20．0％下降至9．0％；并出现细胞形态规则、大小趋于一致、细胞体积增大、核质比例减小等变化；MG-63细胞增殖分化调控相关的癌基因c-fos和c-myc表达活性降低．结果表明，丹参酮ⅡA对人成骨肉瘤MG-63细胞增殖有显著抑制作用，其作用可能与干预c-fos和c-myc等癌基因表达从而调控细胞周期有关．     

熊去氧胆酸和维甲酸对人成骨肉瘤细胞MG-63细胞周期与分化的影响

以癌细胞诱导分化物维甲酸为平行对照,应用中药有效成份熊去氧胆酸处理人成骨肉瘤MG-63细胞,观察比较熊去氧胆酸及其与维甲酸的组合对MG-63细胞形态、细胞周期及分化的影响.实验结果显示,经熊去氧胆酸、维甲酸及其组合联合处理之后,MG-63细胞体积增大,核质比例减少,形态趋于规则.细胞周期发生G0/G1期阻滞,G0/G1期细胞比例显著增加,而S期和G2/M期细胞比例则明显下降.细胞终末分化标志物I型胶原表达明显增强,骨结节形成数目增多,且结节结构更为典型.其中,熊去氧胆酸和维甲酸联合组对MG-63细胞周期的阻滞及细胞终末分化指标I型胶原的表达、骨结节形成的诱导均强于单独处理组.结果表明,熊去氧胆酸具有与维甲酸相近似的诱导人成骨肉瘤MG-63细胞分化的作用,并且两者组合具有协同诱导成骨肉瘤细胞分化的效果.     

沉默PPARγ对骨肉瘤MG-63细胞增殖的影响及相关机制

目的:探讨过氧化物酶体增殖物激活受体γ(PPARγ)基因沉默对骨肉瘤MG-63细胞增殖的影响及其机制.方法:将PPARγshRNA(干扰质粒),scramble shRNA(阴性对照质粒)转染入骨肉瘤MG-63细胞中,Western blot检测PPARγshRNA对PPARγ蛋白的抑制作用;MTT法观察PPARγ沉默对骨肉瘤MG-63细胞增殖的影响,克隆形成实验与检测PPARγ沉默对骨肉瘤MG-63细胞克隆形成能力的影响;Western Blot检测PPARγ沉默后对骨肉瘤MG-63细胞中细胞增殖相关蛋白p-AKT、p-GSK-3β、CyclinD1的蛋白表达水平的影响.结果:PPARγ在骨肉瘤MG-63细胞中沉默成功;MTT及克隆形成实验结果显示沉默PPARγ可促进骨肉瘤MG-63细胞的增殖;Western Blot检测结果显示PPARγ沉默上调骨肉瘤MG-63细胞p-AKT、p-GSK-3β、CyclinD1蛋白的表达.结论:沉默PPARγ可能通过调控Akt/GSK-3β/CyclinD1信号通路促进骨肉瘤MG-63细胞的增殖.     

人参皂甙Rg1对人成骨肉瘤MG-63细胞形态结构和终末分化指标的影响

为了研究人参皂甙Rg1对人成骨肉瘤MG-63细胞(以下简称"MG-63细胞")形态与超微结构及终末分化指标的影响,鉴定其对MG-63细胞的诱导分化作用,以50 μg/mL人参皂甙Rg1处理MG-63细胞,光学显微镜与电子显微镜观察MG-63细胞形态、超微结构变化,免疫细胞化学方法检测成骨细胞相关终末分化蛋白的表达变化,并同步以HMBA处理MG-63细胞作为阳性对照.光学显微镜与电子显微镜观察结果显示细胞形态与超微结构产生了细胞形态规则、大小一致、细胞铺展体积增大,核质比例减小、核内核仁数目减少、细胞器丰富发达等与正常细胞相似的恢复性变化.观察到MG-63细胞终末分化指标I型胶原、骨粘素、骨钙蛋白的阳性表达及钙化糖原颗粒的增多与典型骨节结的形成,其变化结果与HMBA处理细胞类似.本研究证实人参皂甙Rg1能显著改变MG-63细胞形态与超微结构恶性特征,并增强成骨细胞相关的终末分化指标的表达,从而对MG-63细胞的终末分化具有一定的诱导作用.     

HMBA对人成骨肉瘤MG-63细胞形态结构和终末分化指标表达的影响

应用环六亚甲基双乙酰胺处理人成骨肉瘤MG-63细胞,观察MG-63细胞处理前后形态与超微结构及其相关终末分化指标的表达变化.实验结果显示,经5 mmol/L HMBA处理后,MG-63细胞体积增大,趋于扁平铺展状态,细胞大小较为一致,排列较为规则,细胞核形态规则,核质比例减小,核仁减少,核内异染色质减少,常染色质增多,细胞核内的线粒体和高尔基体较为发达,内质网数量增多,细胞表面的微绒毛减少,在成熟细胞中可见钙化糖原颗粒,细胞表面出现钙化小泡沉积,并且形成典型的骨结节.常规细胞化学和免疫细胞化学检测显示,HMBA处理后的细胞中Ⅰ型胶原纤维、骨钙素、骨粘素的表达显著增加,实验结果表明HMBA能够有效诱导人成骨肉瘤细胞的分化,并促进其终末分化指标的表达.     

白花蛇舌草中多糖抑制骨肉瘤的作用机制

为探讨白花蛇舌草(Hedyotis diffusa)活性成分对骨肉瘤的作用及其机制,对白花蛇舌草中分别提取的黄酮类、三萜酸类、香豆素类和多糖类化合物,用噻唑蓝(MTT)法检测不同提取物对MG-63细胞活性的影响;同时建立裸鼠骨肉瘤移植瘤模型,观察不同提取物对移植瘤生长的抑制作用;并通过流式细胞仪检测细胞凋亡及周期变化,蛋白质免疫印记(Western blot)检测Bax及Bcl-2蛋白表达水平.结果显示:4种提取物都对MG-63细胞的增殖具有明显抑制作用,并呈现明显的剂量依赖性,最优药物质量浓度为80μg/mL;在此质量浓度下4种提取物对裸鼠移植骨肉瘤均具有抑制作用,其中多糖类对MG-63细胞活性和移植瘤生长的抑制作用最强,明显促进MG-63细胞凋亡,阻滞细胞周期;Bax表达量在48h内随着白花蛇舌草多糖类提取物作用时间延长而增加,而Bcl-2表达量则降低.由上述结果可知,白花蛇舌草活性成分中多糖类的抗肿瘤活性最强,其作用机制可能是通过抑制骨肉瘤细胞增殖、促进细胞凋亡和阻滞细胞周期来达到抑瘤效果.     

Cyclin D1与p21WAF1在鼻咽癌中的表达

目的探讨鼻咽上皮癌变过程中周期蛋白CyclinD1与抑癌基因p21（wild—type p53 activated fragment 1,p21wAF1）蛋白的表达及其意义。方法应用免疫组化方法检测47例鼻咽癌组织、21例癌旁上皮组织和14例正常鼻咽粘膜中Cyclin D1与p21WAF1蛋白表达。结果从鼻咽正常粘膜、癌旁粘膜上皮组织到癌组织,Cyclin D1表达阳性率逐渐增加,并且差异有显著意义（均为P〈0．05）。p21WAF1蛋白在以上组织中阳性率较高,且各组间差异无显著性。结论Cyclin D1、p21WAF1蛋白与鼻咽部上皮细胞恶性增生密切相关,p21wA兀蛋白表达可能与细胞周期调控的反馈机制有关。     

ING4及IL-24双基因共表达腺病毒载体对MG-63人骨肉瘤细胞体外抑癌增效作用研究

目的用人肿瘤生长抑制因子(mING4)及人白介素24(IL-24)基因构建腺病毒双基因共表达载体(Ad-ING4-IL-24),并设Ad-ING4和Ad-IL-24单基因对照,研究双基因共表达载体对MG-63人骨肉瘤细胞体外抑癌增效作用.方法首先将已构建成功的Ad-ING4-IL-24,Ad-ING4及Ad-IL-24腺病毒感染QBI-293A细胞,经多轮扩增后获得高滴度的病毒子,测病毒效价后分别以100 MOI剂量感染MG-63细胞,用荧光显微镜、RT-PCR法检测ING-4及IL-24在MG-63细胞中的转录和表达;MTT法检测ING-4及IL-24基因表达对MG-63细胞的生长抑制效应;半定量RT-PCR法检测ING4及IL-24基因表达对MG-63细胞中的Bcl-2,Bax,Caspase3,p21,CD34等相关基因表达的影响.结果获得了高滴度的重组腺病毒Ad-ING4-IL-24,Ad-ING4,Ad-IL-24和Ad-GFP;治疗后MG-63细胞中ING-4及IL-24双基因表达对MG-63细胞增殖有明显抑制作用,ING-4及IL-24基因表达可通过上调细胞中Bax,Caspase3,p21和下调Bcl-2,CD34基因表达诱导细胞凋亡.在病毒子剂量为100 MOI时,Ad-ING4-IL-24对MG-63人骨肉瘤细胞抑制作用比对Ad-ING4和Ad-IL-24抑制效果更为显著.结论腺病毒介导的ING4或/和IL-24基因可明显抑制MG-63人骨肉瘤细胞的生长,且Ad-ING4-IL-24双基因比Ad-ING4和Ad-IL-24单基因的抑制效果更为显著,该现象可能是通过改变Bcl-2,Bax,Caspase3,p21,CD34基因表达水平来发挥抗肿瘤作用的.     

HMBA诱导人成骨肉瘤MG-63细胞分化过程中核基质蛋白表达变化

应用选择性抽提、双向聚丙烯酰胺凝胶电泳技术分析5mmol/LHMBA诱导处理前后人成骨肉瘤MG 63细胞核基质蛋白表达变化,鉴定与人成骨肉瘤细胞增殖分化相关的特异核基质蛋白.实验结果显示在HMBA诱导处理MG 63细胞分化过程中,核基质蛋白NMP 1、NMP 2、NMP 3、NMP 4、NMP 5、NMP 6和NMP 7等7个蛋白点表达发生显著变化.其中NMP 1仅在MG 63细胞中表达,NMP 6则为经HMBA诱导处理后新出现的蛋白质,NMP 2、NMP 7在HMBA诱导分化细胞中表达减弱,而NMP 3、NMP 4和NMP 5则在诱导分化后细胞中表达增强.首次表明在人成骨肉瘤MG 63细胞诱导分化过程中伴有核基质蛋白表达的差异,证实了与肿瘤细胞增殖分化相关的特异核基质蛋白的存在.这对于揭示核基质蛋白与细胞癌变和逆转的关系、阐明细胞增殖分化的基因表达调控原理,均具有重要意义.     

芒果多酚对宫颈癌Hela细胞的抑制作用及机制

用芒果多酚处理人宫颈癌细胞(Hela)后,采用MTT法检测细胞的存活率;Hoechst 33258荧光染色和流式细胞术检测细胞凋亡、细胞周期分布,同时用Western blot检测p53,p21,c-myc,cyclin D1蛋白质水平变化.结果显示：0.025,0.05, 0.1,0.2,0.4 mg/mL的芒果多酚溶液能明显抑制Hela细胞的增殖,并呈剂量和时间依赖性;荧光染色后,大多数细胞内可以看见浓密的荧光颗粒,并出现核收缩;流式细胞术分析发现,与未处理组比较,芒果多酚作用后的G1期Hela细胞数显著增多,G2期细胞数逐渐减少;凋亡率明显高于对照组;Western blot检测显示芒果多酚上调p53,p21蛋白水平,下调c-myc,cyclin D1蛋白水平. 以上结果表明芒果多酚能明显抑制人宫颈癌Hela细胞的增殖,诱导其凋亡.     

Effects of enhancing p21 waf1 on proliferation and expression of G1cyclins andCDKs in human breast cancer cells

The cyclin-dependent kinase inhibitor p21( waf1/cip1/sdil) is an important negative regulator in control of cell cycle. Its functions of inhibiting cancer cell growth and its effects on expression of G1 phase cyclins and related CDKs are a worthy topic for study. The plasmid expressing p2l with high level was transformed to human breast cancer cells, and the expression of p2l in cells was enhanced, then the cell growth rate, anchorage-independent growth and tu-morigenecity were tested, at the same time the expression levels of cyclinD1, CDK4, cyclinE and CDK2 were analyzed by Northern blot. The results showed that since the expression of p21 was enhanced in the cell, the rate of cell growth and anchorage-independent growth was inhibited, tumorigenecity was suppressed, the level of expression of cyclinE and CDK2 decreased while that of cyclinDl and CDK4 was not affected. It is suggested that the enhanced expression of p21 markedly inhibits the proliferation and lessens the tumorigenecity of breast cancer cells, and that p2l expression is not related to that of cyclinDl and CDK4, but affects the expression of cyclinE and CDK2 .     

Abrogation by c-myc of G1 phase arrest induced by RB protein but not by p53.

Inactivating mutations of the retinoblastoma gene (RB) are found in a wide variety of tumour cells. Replacement of wild-type RB can suppress the tumorigenicity of some of these cells, suggesting that the RB protein (Rb) may negatively regulate cell growth. As activation of c-myc expression promotes cell proliferation and blocks differentiation, it may positively regulate cell growth. The c-myc protein is localized in the nucleus and can physically associate with RB protein in vitro, hence c-myc may functionally antagonize RB function. Microinjection of Rb in G1 phase reversibly arrests cell-cycle progression. Here we co-inject RB protein with c-myc, EJ-ras, c-fos or c-jun protein. Co-injection of c-myc, but not EJ-ras, c-fos or c-jun, inhibits the ability of Rb to arrest the cell cycle. The c-myc does not inhibit the activity of another tumour supressor, p53 (ref. 12). Thus, c-myc and RB specifically antagonize one another in the cell.     

Effect of P15INK4b expression on the cell cycle and G1 phase related regulatory genes in human melanoma cells

Using the transfeetion teehnique. P15INK4b was introduced into P15INk4b gene deleted human melanoma A375 cells,and a cell model MLED6 overexpressing P15INK4b WAS CONSTRUCTED.Comparing with the control cells MLC2,MLEK6cells in G1phase increased by 11%,but those in Sphase decreased by 15%by FCM.By the method of thymidine(TdR)and N2O arresting,the proportions of synchronized Mphase cells of MLEK6 ana MLC23 were measured and found to be 89.1% and 76.8%respectively ,and the cells in G1phase were 74.3% for MLID6 AND 76. 4% forMLC2.The result of3 H-TdR incorporation indicated that the transition of G1/Sof MLEK6 cell was delayed 2h as compared with that of MLC2 cells,and incorporation rate also decreased.The observation on exprissions of some G1/ S-resates relatory rigusating genes showed that in MLIK6 cells the protein leves of P27KIPI increased with the decreasing expressions of cyclinD1,cyclinE and c-myc,especially cyclinD1 in late G1phade.The expression of cyclinE obviously decreased at G1/S transition ,and c-myc wad inhibited throughout all the process of G1 S phase.All the risults suggest that P15INK4b can delayG1/S transition of MLEK6 cells by inhibiting the cell cycle engine ,and by increasing the expression of Cdk ingibitor P27KIPI in different stages of G1 phase.     

KLF4沉默与阿霉素诱导的DNA损伤协同抑制肝癌HepG2细胞的生长

探究KLF4沉默与经不同作用浓度阿霉素处理诱导的DNA损伤对肝癌HepG2细胞增殖凋亡的影响及其作用机制.应用RNA干扰技术,采用siRNA转染HepG2细胞以沉默KLF4基因.采用MTT法检测KLF4沉默前后对HepG2细胞增殖的影响,使用流式细胞术检测KLF4沉默前后对HepG2细胞周期变化影响,应用Western blot法检测转染前后HepG2细胞中KLF4蛋白及细胞周期相关蛋白表达变化.Western blot检测到高浓度的阿霉素促进KLF4的表达,并且低浓度的阿霉素可使得细胞停滞在G2/M期,高浓度的阿霉素则使部分细胞凋亡(19.31%).将KLF4沉默后,发现细胞生长变缓,低浓度的阿霉素处理后,细胞随时间增加而出现更多的细胞凋亡;高浓度的阿霉素处理后,细胞数明显减少,更多的细胞发生凋亡(28.89%),且在KLF4沉默前后均发现低浓度阿霉素促进p53与p21表达,高浓度阿霉素抑制其表达.阿霉素诱导的DNA损伤可提高KLF4的表达,KLF4依赖于DNA损伤激活的p53促进p21的表达,进而引起G1/S期细胞周期阻滞.沉默KLF4与阿霉素诱导的DNA损伤可协同抑制肝癌细胞的增殖、促进凋亡,其在肝癌细胞 HepG2中扮演十分重要的角色.     

Correlation of chromosomal polysomy with overexpression of c-myc and c-erbB-2 in primary nasopharyngeal carcinoma: Tissue microarray study

Many genes may be involved in nasopharyngeal carcinoma (NPC) development and progression. Several known oncogenes, including c-myc and c-erb-B2, have been shown to have structural alteration and aberrant expression in NPC. Here, we constructed a tissue microarray to determine the status of c-myc and c-erbB-2 oncogenes at the DNA and protein levels using interphase fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) and to define the diagnostic, prognostic importance of the genetic changes. Results showed that amplification of c-myc and c-erbB-2 was not found in NPC. Polysomy 8 and 17 were observed in 43%-50% and 20%-27% of NPC tumors, respectively. Overexpressions of c-myc and c-erbB-2 oncoproteins were detected in 53.6% and 54.5% cases of NPC with polysomy 8 and 17, respectively. There was no significant correlation between c-myc and c-erbB-2 staining and the clinical stage. But overexpression of c-erbB-2 was associated with polysomy 17 in NPC. These findings suggest that chromosomal polysomy, not gene amplification, may be partially responsible for the upregulated expression of c-erbB-2 oncogene in NPC.