紫杉醇对TRAIL诱导胃癌SGC7901细胞凋亡的影响

目的探讨紫杉醇对TRAIL诱导的胃癌SGC7901细胞凋亡的影响。方法采用MTT法测定细胞活力、采用流式细胞仪检测细胞凋亡、Western blot检测蛋白表达。结果在胃癌SGC7901细胞中,100 ng/ml的TRAIL导致轻度的增殖抑制和细胞凋亡,TRAIL（100 ng/ml）联合紫杉醇（7.19μg/ml,24 h的IC50剂量）引起明显的细胞增殖抑制和诱导凋亡作用（P〈0.05）。TRAIL单药没有改变死亡受体5（DR5）的蛋白表达,而7.19μg/ml紫杉醇作用于胃癌SGC7901细胞后明显上调了DR5的蛋白表达。TRAIL和紫杉醇联合作用后,也有DR5蛋白的明显上调。结论紫杉醇通过上调DR5的蛋白表达增强了TRAIL诱导的胃癌SGC7901细胞凋亡。     

三氧化二砷协同肿瘤坏死因子相关细胞凋亡诱导配体抗胃癌细胞的作用及其机制

目的研究三氧化二砷（As2O3）联合肿瘤坏死因子相关细胞凋亡诱导配体（TRAIL）对胃癌细胞生长和凋亡的影响及其机制。方法人胃腺癌细胞株SGC7901以As2O3、rsTRAIL及两者联用处理；用MTT法检测细胞生长；用双染色流式细胞仪检测细胞凋亡；用间接免疫荧光染色结合流式细胞技术检测细胞表面TRAIL R1／DR4和TRAIL R2／DR5分子表达；RT—PCR方法检测TRAIL R1／DR4和TRAIL R2／DR5 mRNA表达。结果As2O3和rsTRAIL在单用或联用时均可以抑制胃癌细胞SGC7901生长，并在一定范围内呈时间、剂量依赖性；联合用药组抑制率显著高于单用As2O3或rsTRAIL组（P〈0．01），金正均方法分析提示两药联用有协同抑制细胞生长效应。As2O3和rsTRAIL在单用或联用时均可以诱导胃癌细胞SGC7901凋亡，联合用药组细胞凋亡率显著高于单用As2O3或rsTRAIL组（P〈0．01）。As2O3单独或联合rsTRAIL作用于SGC7901细胞24h后，死亡受体TRAIL R1／DR4和TRAIL R2／DR5在细胞表面的表达明显上调（P〈0．01），其mRNA表达水平亦相应增加（P〈0．01）。结论As2O3联合rsTRAIL可以协同抑制胃癌细胞SGC7901生长并显著增强两药诱导胃癌细胞凋亡的作用，其机制可能是As2O3增加细胞TRAIL R1／DR4和TRAIL R2／DR5 mRNA表达、上调细胞表面TRAIL死亡受体从而增加胃癌细胞SGC7901对rsTRAIL的敏感性。     

小干扰RNA沉默bcl-2基因抑制胃癌细胞生长的研究

目的利用RNA干扰技术,研究靶向bcl-2的小干扰RNA(siRNA)在体内外对胃癌细胞生长的影响,探讨其对胃癌治疗的可行性。方法化学合成bcl-2基因的siRNA,脂质体法将bcl-2 siRNA转染胃癌SGC 7901细胞,采用实时定量PCR和Western印迹观察bcl-2 siRNA转染前后胃癌细胞bcl-2基因的表达变化,四甲基偶氮唑盐(MTT)实验、流式细胞检测技术和端粒重复序列扩增法(TRAP)分别检测胃癌细胞增殖、凋亡和端粒酶活性变化。并将转染bcl-2 siRNA的胃癌SGC 7901细胞接种于裸鼠皮下,观察其在裸鼠体内成瘤及生长情况。结果数据经方差分析,bcl-2 siRNA转染胃癌SGC 7901细胞后,明显抑制bcl-2基因表达,并与其浓度和作用时间相关,以100 nmol/L bcl-2 siRNA对bcl-2基因表达抑制效果最佳。以该浓度的bcl-2 siRNA转染胃癌SGC 7901细胞后,bcl-2 mRNA和蛋白表达抑制分别为79．9%和85．3%；经bcl-2 siRNA作用的SGC 7901细胞生长明显缓于对照组(P＜0．05),细胞凋亡率高于对照组(P＜0．05),端粒酶活性明显低于对照组(P＜0．05)。转染100 nmol/L bcl-2 siRNA的胃癌SGC 7901细胞接种裸鼠皮下后,肿瘤出现时间晚,体积小．生长受到明显抑制(P＜0．05)。结论靶向bcl-2的siRNA可明显下调靶基因bcl-2的表达,在体内外抑制胃癌SGC 7901细胞的生长,为探索胃癌基因治疗提供新的策略。     

Periostin基因过表达对顺铂和5-氟尿嘧啶诱导人胃癌细胞凋亡的影响

背景：肿瘤细胞耐药性的产生是肿瘤化疗失败的主要原因之一。Periostin是一种基质特异性细胞黏附分子。既往研究发现,periostin除参与胃癌细胞的生长、侵袭、转移外,还能降低其对化疗药物的敏感性。目的：明确periostin基因过表达对顺铂和5-氟尿嘧啶（5．Fu）诱导人胃癌细胞凋亡的影响及其可能机制。方法：将表达人periostin全长序列的重组质粒稳定转染人胃癌细胞株SGC7901,同时设置空载体稳定转染组和未转染对照组,加入不同浓度顺铂或5-Fu处理24h,以流式细胞术检测细胞凋亡。以蛋白质印迹法检测经5μmol／L顺铂或10μmol／L5-Fu处理的SGC7901细胞的凋亡相关蛋白表达。结果：顺铂和5-Fu均能剂量依赖性地诱导SGC7901细胞凋亡,促进细胞色素c由线粒体释放至胞质,同时easpase-3激活,easpase-3底物PARP剪切增强。periostin稳定转染组SGC7901细胞对顺铂和5-Fu诱导的细胞凋亡具有显著抗性,细胞凋亡率显著低于经相同剂量化疗药物处理的空载体稳定转染组（P〈0．05）,胞质细胞色素C、cleavedcaspase-3、cleavedPARP蛋白表达亦明显下调。结论：过表达periostin基因可增强SGC7901细胞对顺铂和5-Fu诱导的细胞凋亡的抵抗能力,其抗凋亡活性与抑制线粒体依赖性凋亡途径有关。     

联合基因治疗体系pcDNA3.1(-)-shVEGF/yCDglyTK对胃癌细胞增殖的影响

目的:观察由磷酸钙纳米颗粒介导的联合基因治疗体系pcDNA3.1(-)-shVEGF/yCDglyTK对胃癌细胞增殖的影响.方法:以磷酸钙纳米颗粒为载体,介导pcDNA3.1(-)Null,pGenesil-1-hVEGF4-shRNA,pcDNA3.1(-)-CV-yCDglyTK,pcDNA3.1(-)-shVEGF/yCDglyTK转染胃癌细胞SGC7901,RT-PCR检测细胞中yCDglyTK及血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA的表达情况.Western blot分析yCDglyTK及VEGF蛋白的改变.MTT法检测融合基因对转染细胞的杀伤作用.流式细胞术检测转染细胞的凋亡情况.结果:SGC7901细胞转染pcDNA3.1(-)-shVEGF/yCDglyTK质粒后有yCDglyTKmRNA及相关蛋白表达,VEGF表达受抑制达30.3%.相对于转染pcDNA3.1(-)-CV-yCDglyTK质粒组,联合基因体系对胃癌细胞的抑制作用更强(P<0.05).pcDNA3.1(-)-shVEGF/yCDglyTK组细胞凋亡率达到67.9%±4.78%.结论:联合基因治疗体系pcDNA3.1(-)-shVEGF/yCDglyTK较单独的RNA干扰或自杀基因可更有效的杀伤胃癌细胞.     

HOXD10基因转染对人胃癌耐药SGC7901/VCR细胞增殖、凋亡及侵袭能力的影响

目的研究HOXD10基因转染人胃癌耐药细胞系SGC7901/VCR后表达情况及对细胞增殖、凋亡、侵袭能力的影响。方法将HOXD10基因表达质粒(pc DNA3.1-EGFP-HOXD10)转染至SGC7901/VCR中,利用Real-time PCR和Western blotting检测HOXD10基因转染后表达效果;MTT方法、平板单克隆实验、流式细胞术、Transwell方法分别检测HOXD10基因转染对细胞增殖、单克隆形成、细胞周期、凋亡、侵袭能力的影响;并用Western blotting检测HOXD10基因转染对肿瘤侵袭性相关因子MMP-2蛋白表达的影响。结果 HOXD10基因转染至人胃癌SGC7901/VCR细胞后其基因和蛋白表达水平均显著上升(P0.05),能够抑制SGC7901/VCR细胞增殖、单克隆形成、细胞周期,并提高顺铂作用下细胞凋亡率(P0.05),同时显著抑制细胞侵袭能力和MMP-2蛋白的表达(P0.05)。结论 HOXD10基因转染人胃癌耐药细胞系SGC7901/VCR后能够高表达,进而抑制细胞增殖、单克隆形成、细胞周期和侵袭能力并提高顺铂下细胞凋亡率,而其抑制细胞侵袭能力可能与MMP-2表达减少有关。     

可溶性人TRAIL基因表达载体的构建及其诱导肿瘤细胞凋亡的实验研究

目的 构建可溶性肿瘤坏死因子（TNF）相关凋亡诱导配体TRAIL基因表达载体并研究其瞬时转染对肿瘤细胞凋亡的影响。方法 采用重叠延伸PCR法克隆含有TNF-α信号肽的人TRAIL基因，并将其连入pUCm-1载体。测序正确后克隆入表达载体pcDNA3．1中。采用Gene-Companion^TM非脂质体型聚阳离子转染试剂体外瞬时转染人膀胱癌EJ细胞，RT—PCR法检测目的基因mRNA水平的表达，Western blot检测细胞培养上清中目的基因的表达，流式细胞术检测细胞凋亡，克隆形成实验检测细胞存活。结果 成功构建了可分泌表达的人可溶性TRAIL表达载体，体外转染TRAIL的EJ细胞凋亡率均显著高于未转染细胞及转染空载体细胞的凋亡率，并明显抑制细胞存活，细胞存活率为51.34％，明显低于末转染细胞及转染空载体的细胞存活率。结论 所构建的可溶性TRAIL表达载体转染肿瘤细胞可诱导细胞凋亡及抑制肿瘤细胞的生长存活。     

Survvin反义寡核苷酸诱导人胃癌细胞凋亡的作用

目的探讨生存素（Survivin）反义寡核苷酸（ASODN）诱导人胃癌细胞SGC7901凋亡的作用。方法设计合成特异性靶向Survivin的ASODN,将胃癌细胞株分为空白对照组（Sham组）、脂质体转染对照组（Lip组）、正义链转染对照组（Lip-SODN组）和ASODN转染组（Lip-ASODN组）。作用48h后,Westemblot法检测各组Survivin表达情况,流式细胞仪检测各组细胞凋亡率,免疫组化SP法检测细胞中PCNA表达情况。结果脂质体介导Survivin ASODN转染后的胃癌细胞Survivin蛋白表达明显下降;ASODN转染组细胞凋亡率明显高于各对照组（P均〈0．05）,各对照组间无统计学差异（P〉0．05）。ASODN转染后胃癌细胞中PCNA表达水平明显降低。结论 Survivin ASODN转染胃癌细胞能下调Survivin蛋白表达,诱导胃癌细胞凋亡,抑制细胞增殖,具有明显的抗癌作用。     

TRAIL体外诱导人肝星形细胞LX-2凋亡作用及调控机制

目的研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导肝星形细胞凋亡情况及调控机制。方法用RT-PCR检测LX-2中α-SMAmRNA和DR5mRNA的表达;MTT比色法、流式细胞术检测外源性TRAIL对LX-2细胞增殖和诱导细胞凋亡的影响;采用Westernblot检测Bax、Caspase3表达。结果培养的LX-2表达α-SMAmRNA和DR5mRNA逐渐增加,TRAIL可以抑制其细胞增殖,与剂量相关（P〈0.05）。与对照组比较,TRAIL诱导活化的LX-2细胞凋亡明显增加（P〈0.05）,Western blot分析显示TRAIL作用下,LX-2线粒体Bax、细胞质Caspase3表达上调。结论外源性TRAIL可以诱导活化的LX-2凋亡,其机制与DR5及线粒体Bax表达上调有关。     

胃肠富集Krüppel样因子基因转染对人胃癌细胞株SGC-7901及裸鼠移植瘤生长的影响

目的 探讨外源性胃肠富集Krüppel样因子(GKLF)基因转染对人胃癌细胞株SGC-7901在体内外的抗肿瘤效应.方法 荧光实时定量PCR和Western印迹法检测GKLF基因转染前后人胃癌细胞株SGC-7901中GKLF mRNA和蛋白的表达.应用四甲基偶氮唑盐(MTT)法、流式细胞技术、克隆形成实验和细胞侵袭实验分别检测GKLF基因转染后SGC-7901细胞增殖和侵袭力的变化.观察裸鼠移植瘤生长情况和应用免疫组织化学法检测移植瘤组织中微血管密度(MVD).结果 GKLF基因转染后,SGC7901-pcDNA3.1-GKLF组中GKLF mRNA(0.1216±0.0061)和蛋白(1.0547±0.0172)的表达明显高于SGC-7901组[分别为(0.0029±0.0012)和(0.6240±0.0177)]和SGC7901-pcDNA3.1组[分别为(0.0037±0.0013)和(0.5627±0.0510)],P＜0.05.与SGC-7901组和SGC7901-pcDNA 3.1组相比,从48 h开始,SGC7901-pcDNA 3.1-GKLF组细胞生长速度减慢、细胞出现G0/G1期部分阻滞、克隆形成率低、细胞侵袭力明显减弱(P值均＜0.05).SGC7901-pcDNA3.1-GKLF组皮下移植瘤生长速度减慢、瘤重轻、MVD低(P值均＜0.05).结论 GKLF基因转染可导致人胃癌细胞株SGC-7901的增殖活性和侵袭力降低,抑制裸鼠移植瘤生长和血管生成.

Abstract：

Objective To investigate the antitumour effects of transfected gut-enriched Krüppellike factor(GKLF) on human gastric carcinoma cell line SGC-7901 in vitro and in vivo. Methods The expression of GKLF mRNA and protein in human gastric carcinoma cell line SGC-7901 were detected before and after transfection by real-time fluorescence quantitative PCR and Western blot,respectively. Proliferation and invasion in SGC-7901 were measured respectively by MTT assay, flow cytometry, colony formation assay and cell invasion assay after transfected with GKLF. The growth of xenograft was observed, the microvessel density(MVD) of xenograft tissue was determined by immunohistochemistry. Results The GKLF mRNA and protein in SGC-7901 were overexpressed after transfected with GKLF(P＜0.05). The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups (P＜0.05). Transfected with GKLF caused part of the G0/G1 arrest, decreased clone formation rate and the invasion ability (P＜0.05). The growth speed of xenograft in SGC7901-pcDNA3.1-GKLF group was lower, the weight and MVD of xenograft tissue in SGC7901-pcDNA3. 1-GKLF group were less (P＜ 0. 05).Conclusion Transfected with GKLF maysuppress proliferation and invasion in human gastric carcinoma cell line SGC-7901, inhibit the growth and the angiogenesis of xenograft in nude mice.     

The therapeutic effects of recombinant adenovirus RA538 on human gastric carcinoma cells in vitro and in vivo

AIM:To evaluate the potential of RA-538 gene therapy for gastric carcinoma.METHODS:Human gastric carcinoma cell line SGC7901 treated with Ad-RA538 or Ad-LacZ were analysed by X-gal stain, MTT, DNA ladder, Tunel, flow cytometric analysis, PCR, and Western Blot in vitro. The tumorigenicity and experimental therapy in nude mice model were assessed in vivo.RESULTS:Ad-LacZ could efficiently transfer the LacZ gene into SGC7901 cells. X-gal-positive cells at MOI 25, 50, 100, and 200 were 90%, 100%, 100%, and 100% respectively. Ad-RA538 could strongly inhibit cell growth and induced apoptosis in SGC7901 cells.The proliferation of the Ad-RA538-infected SGC7901 cells was reduced by 76.3%.The mechanism of killing of gastric carcinoma cells by Ad-RA538 was found to be apoptosis by DNA ladder,Tunel and flow cytometric analysis.The tumorigenicity in nude mice using Ad-RA538 showed that all three mice failed to form tumor from 7 to 30 days compared with Ad-LacZ and parent SGC7901 cells. Experimental therapy on the nude mice model bearing subcutaneous tumor of SGC7901 cells showed that intratumor instillation of Ad-RA538 inhibited the growth of the tumors. Ad-RA538-treated tumors were inhibited by 60.66%, compared with that of the tumor injected with Ad-LacZ and mock.CONCLUSION: The expression of Ad RA538 can inhibit growth and induce apoptosis of gastric cancer cell in vitro and in vivo. Ad RA538 can be used potentially in gene therapy for gastric carcinoma.     

Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo

BACKGROUND AND AIMS: This study used a recombinant antisense c-myc adenovirus (Ad-ASc-myc) to evaluate how alterations of c-myc expression in the SGC7901 human gastric carcinoma cells could influence the proliferation, apoptosis and the growth of human gastric tumors in nude mice. METHODS: The human gastric carcinoma cell line, SGC7901, treated with Ad-ASc-myc or adenovirus recombinants carrying LacZ gene (Ad-LacZ) were analyzed by using X-gal stain, MTT, DNA ladder, TUNEL assay, flow cytometric analysis, polymerase chain reaction and western blot in vitro. The tumorigenicity and experimental therapy in nude mice models were assessed in vivo. RESULTS: The Ad-ASc-myc could strongly inhibit cell growth and induce apoptosis in SGC7901 cells. The proliferation of the Ad-ASc-myc-infected SGC7901 cells was reduced by 44.1%. The mechanism of killing gastric carcinoma cells by Ad-ASc-myc was found to be apoptosis, which was detected by the use of a DNA ladder, TUNEL and flow cytometric analysis. Infection of Ad-ASc-myc in nude mice showed that all three mice failed to form tumors from the 7 to 30 day period, compared with injection of Ad-LacZ and parent SGC7901 cells. Experimental therapy on the nude mice bearing subcutaneous tumors of SGC7901 cells showed that intratumor instillation of Ad-ASc-myc inhibited the growth of the tumors. Recombinant antisense c-myc adenovirus-treated tumors were inhibited by 68.9%, compared with tumors injected with Ad-LacZ and control (LacZ and phosphate-buffered saline). CONCLUSION: The expression of Ad-ASc-myc can inhibit growth and induce apoptosis of gastric cancer cells in vitro and in vivo and thus is a potential clinical utility in gene therapy for the treatment of gastric carcinoma.     

血管内皮生长因子小干扰核糖核酸联合双自杀基因抗胃癌作用的体外研究

目的 探讨联合应用靶向血管内皮生长因子(VEGF)小干扰RNA(siRNA)与双自杀基因yCDglyTK对人胃癌细胞的体外杀伤作用。方法 以磷酸钙纳米颗粒为载体介导空白质粒pcDNA3.1（-）null(空白质粒组)、靶向VEGF的干扰质粒pGenesil-shVEGF(干扰质粒组)、双自杀基因质粒pcDNA3.1（-）CV-yCDglyTK(双自杀基因组)及联合基因质粒pcDNA3.1（-）shVEGF-yCDglyTK（联合基因组）转染胃癌SGC7901细胞,未转染的胃癌细胞为空白对照组,经G418筛选稳定转染的胃癌细胞株,采用RT-PCR和免疫印迹法验证目的基因表达。给予前体药物5-氟胞嘧啶(5-FC)后,通过四甲基偶氮唑盐(MTT)生长曲线、旁观者效应实验、Hoechst 33258染色及流式细胞术,观察各组细胞的生物学特性变化、凋亡细胞形态及凋亡率。采用SPSS 13.0统计学软件进行数据处理分析,组间多重比较采用LSD检验。结果成功建立4种转染不同质粒的胃癌细胞株,联合基因组及双自杀基因组均可检测到双自杀基因yCDglyTK的表达。MTT生长曲线显示5-FC作用24 h后,与空白对照组和空白质粒组相比,干扰质粒组、双自杀基因组及联合基因组吸光度值明显降低(P＜0.01)。当稳定转染联合基因的SGC7901细胞占60％、80％和100％时,细胞相对存活率分别为13.09％±2.40％、9.74％±2.83％及5.68％±1.03％。荧光显微镜下见双自杀基因组及联合基因组大量细胞呈现凋亡形态改变。流式细胞检测结果示干扰质粒组、双自杀基因组以及联合基因组的凋亡率分别为16.40％±4.68％、57.63％±4.96％及69.07％±4.69％,与空白对照组相比,差异有统计学意义(P＜0.01)。结论采用靶向VEGF siRNA与双自杀基因联合治疗可有效杀伤胃癌SGC7901细胞,诱导凋亡是其杀伤瘤细胞的重要机制之一。     

核抗原Mina53在人胃癌细胞中的作用及其意义探讨

背景:核抗原Mina53基因为原癌基因Myc的下游直接靶基因之一,在一些消化系统恶性肿瘤中呈高表达,并与肿瘤增殖、侵袭、转移或患者生存期相关。目的:研究Mina53在人胃癌细胞中的作用及其对胃癌发生、发展的意义。方法:选择Mina53表达水平较高的人胃癌细胞株SGC7901和AGS,应用RNA干扰技术下调其Mina53表达,以转染无关序列siRNA的细胞作为对照组。采用CCK-8实验检测细胞增殖,流式细胞术检测细胞周期和细胞凋亡,细胞侵袭和迁移实验检测细胞侵袭、迁移能力。结果:与相应对照组相比,Mina53 siRNA转染组SGC7901、AGS细胞增殖受抑(96 h相对增殖率:60%和68%),并发生明显细胞周期G1期阻滞(SGC7901细胞G1/G2:2.76±0.12对1.86±0.06,P<0.05;AGS细胞G1/G2:1.78±0.13对1.34±0.05,P<0.05),细胞凋亡率分别增加9.8%±1.2%和10.6%±1.5%(P<0.05),穿透Transwell小室基质胶细胞数(SGC7901细胞:11.67±0.88对24.33±1.45,P<0.05;AGS细胞:8.00±1.15对20.33±1.73,P<0.05)和穿透Transwell小室微孔膜细胞数(SGC7901细胞:7.00±1.53对14.67±2.03,P<0.05;AGS细胞:8.00±1.16对15.33±1.45,P<0.05)均显著减少。结论:Mina53对人胃癌细胞的增殖、细胞周期、细胞凋亡以及侵袭、迁移能力具有调控作用,可能影响胃癌的生长、浸润和转移,有望作为胃癌基因治疗的靶点。     

Transduction of Fas gene or Bcl-2 antisense RNA sensitizes cultured drug resistant gastric cancer cells to chemotherapeutic drugs

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Effect of RNA Interference Inhibiting the Expression of the FUBP1 Gene on Biological Function of Gastric Cancer Cell Line SGC7901

Background: The research aimed to observe the effect of gene silencing on the proliferation, migration, cell cycle, apoptosis, and other biological functions of human gastric cancer cells with RNA interference inhibiting the expression of the far upstream element-binding protein 1 (FUBP1) in the gastric cancer cell line SGC7901.Methods: The shRNA lentivirus vector of the target gene FUBP1 was constructed to transfect the gastric cancer cell line SGC7901. The qRT-PCR and western blot assays were used to detect the expression levels of FUBP1 mRNA and protein in the gastric cancer cells. The CCK-8 assay was used to detect the proliferation of gastric cancer cells. The cell scratch assay and the transwell assays were used to detect the migration of gastric cancer cells. Flow cytometry was used to detect cell cycle distribution and apoptosis.Results: The shRNA lentiviral vector of FUBP1 was successfully transfected into the gastric cancer cell line SGC7901, and could effectively reduce the expression of mRNA and protein of FUBP1. The silencing of FUBP1 could inhibit the gastric cancer cell proliferation and affect the distribution of the cell cycle, resulting in S-phase arrest and cell growth inhibition. However, FUBP1 silencing has no significant effect on cell apoptosis and migration.Conclusions: The expression of FUBP1 can be inhibited specifically and effectively by RNA interference technology, which can significantly affect the biological function of the gastric cancer cell line SGC7901.     

Suppression of P-gp induced multiple drug resistance in a drug resistant gastric cancer cell line by overexpression of Fas

AIM:To observe the drug sensitizing effect and related mechanisms of fas gene transduction on human drug-resistant gastric cancer cell SGC7901/VCR (resistant to Vincristine).METHODS:The cell cycle alteration was observed by FACS. The sensitivity of gastric cancer cells to apoptosis was determined by in vitro apoptosis assay. The drug sensitization of cells to several anti-tumor drugs was observed by MTT assay. Immunochemical method was used to show expression of P-gp and Topo II in gastric cancer cells.RESULTS:Comparing to SGC7901 and pBK-SGC7901/VCR, fas-SGC7901/VCR showed decreasing G2 cells and increasing S cells, the G2 phase fraction of pBK-SGC7901/VCR was about 3.0 times that of fas -SGC7901/VCR but S phase fraction of fas -SGC7901/VCR was about 1.9 times that of pBK-SGC7901/VCR, indicating S phase arrest of fas-SGC7901/VCR. FACS also suggested apoptosis of fas-SGC7901/VCR.fas-SGC7901/VCR was more sensitive to apoptosis inducing agent VM-26 than pBK-SGC7901/VCR. MTT assay showed increased sensitization of fas-SGC7901/VCR to DDP, MMC and 5-FU, but same sensitization to VCR according to pBK-SGC7901/VCR. SGC7901, PBK-SGC7901/VCR and fas -SGC7901/VCR had positively stained Topo II equally. P-gp staining in pBK-SGC7901/VCR was stronger than in SGC7901, but there was little staining of P-gp in fas-SGC7901/VCR.CONCLUSION:fas gene transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree, possibly because of higher sensitization to apoptosis and decreased expression of P-gp.     

上调Periostin基因表达对人胃癌细胞增殖的影响

背景：有文献报道,在内源性periostin低表达的人肿瘤细胞株中,过表达periostin基因可抑制细胞的非贴壁依赖性生长,提示该基因具有肿瘤抑制功能。但亦有较多研究发现periostin在一些肿瘤细胞中呈高表达,并可促进其生长、侵袭和转移。目的：探讨上调periostin基因表达对人胃癌细胞增殖的影响。方法：构建、鉴定pcDNA3.1-periostin重组质粒并稳定转染人胃癌细胞株SGC7901和MGC-803,同时设置空载体稳定转染组和不予转染的对照组。蛋白质印迹法检测各组细胞periostin蛋白表达,MTT实验检测细胞活力。结果：pcDNA3.1-periostin重组质粒构建成功。periostin稳定转染组SGC7901和MGC-803细胞periostin蛋白相对表达量明显增高,分别约为相应空载体稳定转染组的2.5倍和4倍（P〈0.05）;MTT实验显示两组细胞在5 d培养过程中的细胞活力均与相应对照组相似,两组间差异亦无统计学意义。结论：稳定转染pcDNA3.1-periostin重组质粒能使人胃癌细胞过表达periostin基因,但periostin基因过表达对人胃癌细胞,至少是本研究检测的两株胃癌细胞的增殖能力无明显影响。     

RNAi‐mediated silencing of ezrin gene reverses malignant behavior of human gastric cancer cell line SGC‐7901

OBJECTIVE: To investigate the effects of ezrin targeting gene of RNA interference (RNAi) on human gastric cancer cell line SGC‐7901 in vitro. METHODS: The highly metastatic human gastric cancer cell line SGC‐7901 transfected with a small interfering (siRNA) lentivirus vector was selected for this research study. Expressions of ezrin mRNA and ezrin protein in the SGC‐7901 cells were detected using RT‐PCR and Western blot. Cell apoptosis was observed using flow cytometry. Transwell invasion and the cell adhesion test were used to verify the effect of RNAi on ezrin expression in the human gastric cancer cell line SGC‐7901 in vitro. RESULTS: Ezrin gene targeting via a RNAi‐mediated lentivirus vector had obvious inhibitory effects on ezrin expression in the human gastric cancer cell line SGC‐7901. The results of the RT‐PCR show the obvious inhibition of ezrin mRNA expression in Eai and Ebi groups (0.22 ± 0.01 vs 0.95 ± 0.04, P < 0.05; 0.31 ± 0.01 vs. 0.95 ± 0.04, P < 0.05). Western blot analysis revealed a 72.35 ± 3.74% reduction of the ezrin protein level after interference with the ezrin targeting gene. Moreover, the inhibition of ezrin expression clearly inhibited SGC‐7901 cell migration and invasion, and improved cell adhesion as well as increased sensitivity to camptothecin‐induced apoptosis. CONCLUSION: Ezrin gene targeting by RNAi can inhibit the metastatic growth and migration of SGC‐7901 human gastric cancer cells.