菟丝子醇提物对胃癌SGC7901细胞凋亡的影响

目的 探讨菟丝子(CCL)醇提物诱导人胃癌SGC7901细胞凋亡的作用.方法 以CCL(10、50、100 mg/L)作用于人胃癌SGC7901细胞,同时设DMSO和培养液作为对照组.采用HE染色法观察细胞形态变化,琼脂糖凝胶电泳法分析SGC7901细胞的DNA片段化.结果 HE染色镜下可见较为典型的凋亡形态学变化:细胞变小,核皱缩,荧光强度增强,核仁消失,新月体样改变,凋亡小体形成等;琼脂糖电泳出现明显的梯状条带,并存在剂量依赖性,而对照组未出现梯状条带.结论 CCL能够诱导人胃癌SGC7901细胞凋亡.     

Twist基因沉默促进人胃癌SGC7901细胞凋亡的机制

目的探讨Twist基因沉默促进人胃癌SGC7901细胞凋亡的机制。方法以稳定转染pGenesil-Twist质粒的人胃癌SGC7901细胞作为研究对象,实验分为对照组、空载体组、Twist基因沉默组。采用AnnexinV-FITC/PI染色观察细胞凋亡情况;通过检测细胞Caspase3、9活性、p53、Bcl-2、Bax蛋白表达情况和Ca2+含量探讨细胞的凋亡机制。结果激光共聚焦显微镜检测结果显示Twist基因沉默组中有较多的细胞呈现绿色荧光,未见明显的呈现红色荧光的细胞。流式细胞仪检测显示,Twist基因沉默组AnnexinV-FITC阳性细胞的比例明显高于SGC7901组和空载体组(P<0.01);Twist基因沉默组细胞中p53蛋白表达量显著高于对照组和空载体组(P<0.01),Bcl-2蛋白表达量显著低于对照组和空载体组(P<0.01),而Twist基因沉默组中Bax蛋白表达比例显著高于对照组和空载体组(P<0.01);Twist基因沉默组Caspase3、9酶活性明显高于对照组和空载体组(P<0.01,P<0.05);Twist基因沉默组SGC7901细胞中Ca2+含量显著高于对照组和空载体组(P<0.05)。结论 Twsit基因通过线粒体介导的细胞凋亡途径调节细胞凋亡,Twist基因有望作为靶基因发挥抗肿瘤作用。     

RNA干扰沉默Twist基因对人胃癌细胞系SGC7901细胞增殖能力的影响

目的 探讨Twist基因沉默后对人胃癌SGC7901细胞增殖能力的影响.方法 利用脂质体转染法将携带有Twist干扰片段的pGenesil/Twist载体导入人胃癌SGC7901细胞,G418筛选阳性克隆,经RT-PCR,Western印迹法检测干扰效果.通过绘制生长曲线、流式细胞术检测细胞周期等反映Twist对SGC7901细胞增殖能力的影响.结果 生长曲线结果显示Twist基因沉默组细胞生长速度较两对照组缓慢.细胞周期结果显示Twist基因沉默组S期细胞数明显较对照组减少,增殖能力下降.结论 Twist基因沉默后可抑制SGC7901细胞的分裂增殖,可能成为临床胃癌基因治疗的新靶点.     

钒化钠抑制人胃癌SGC7901细胞增殖的作用机理

目的通过观察钒化钠对人胃癌SGC7901细胞周期蛋白cyclinD1表达的影响,探讨其对人胃癌SGC7901细胞增殖抑制的作用机理。方法应用MTT法测定钒化钠对人胃癌SGC7901细胞的生长抑制作用;用Western-blot法检测钒化钠对人胃癌SGC7901细胞周期蛋白cyclinD1表达水平的变化。结果一定浓度的钒化钠使人胃癌SGC7901细胞增殖周期密切相关的cyclinD1蛋白表达降低。结论一定浓度的钒化钠能明显抑制人胃癌SGC7901细胞的生长,钒化钠的抗肿瘤机制可能与cyclinD1蛋白表达降低有关。     

Adv-shRNA抑制NRP2表达对胃癌SGC7901细胞增殖的影响

目的通过特异性抑制胃癌细胞株SGC7901中神经纤毛蛋白2(NRP2)基因的表达,检测RNA干扰片段对SGC7901细胞增殖能力的影响。方法将NRP2的特异性RNA干扰腺病毒载体转染至胃癌细胞SCG7901 72 h后荧光显微镜观察转染情况;采用RT-PCR法检测转染后NRP2基因mRNA表达情况;MTT法检测胃癌细胞增殖情况;Western印迹检测转染后NRP2蛋白表达情况。结果成功将NRP2-shRNA重组腺病毒载体转染至胃癌细胞SGC7901。各重组腺病毒转染组NRP2 mRNA水平均低于阴性对照组和空白组,其中以NRP2-shRNA-3抑制效应最强。阴性对照组与空白组细胞增殖迅速,两组间无显著差异(P>0.05),实验组经腺病毒载体转染后细胞增殖程度显著减少,与前两组相比有显著差异(P<0.05)。阴性对照组与空白组NRP2蛋白表达量明显高于实验组(P<0.01),而阴性对照组与空白组之间无显著差异(P>0.05)。结论重组腺病毒载体NRP2-shRNA可有效抑制胃癌细胞SCG7901中NRP2基因的mRNA和蛋白的表达,并在一定程度上抑制癌细胞增殖,可作为动物实验的理论基础和前期条件。     

胰岛素样生长因子Ⅱ与胃癌细胞SGC7901 c-fos和c-jun表达的关系

目的:探讨胰岛素样生长因子Ⅱ(insulin-like growth factor-Ⅱ,IGF-Ⅱ)与胃癌细胞SGC7901 c-fos和c-jun表达的关系.方法:体外细胞培养,分别用MTT法和免疫组织化学以及逆转录聚合酶链反应(RT-PCR)的方法检测不同浓度IGF-Ⅱ(0,10,50,100 mg/L)作用胃癌细胞后细胞的增殖率和c-fos和c-jun蛋白及mRNA的表达情况.结果:IGF-Ⅱ作用于细胞SGC7901的增殖效应呈浓度和时间依赖性:随着药物浓度的升高,c-fos和c-jun蛋白,mRNA表达均增加.在IGF-Ⅱ10,50,100 mg/L时,c-fos和c-jun蛋白分别为41.32±1.28 mg/L,50.43±0.57 mg/L,64.22±1.76 mg/L;52.00±0.67 mg/L,63.20±0.95 mg/L,76.31±1.16 mg/L;c-fos,c-jun mRNA与GAPDH mRNA比值分别为0.316±0.021,0.392±0.0357,0.478±0.028;0.379±0.006,0.412±0.022.0.494±0.048,均与相应对照组(30.00±1.01 mg/L,41.14±2.02 mg/L,0.220±0.037,0.290±0.064)相比有统计学意义(P<0.05,P<0.01).结论:胰岛素样生长因子Ⅱ可能通过旁分泌上调c-fos和c-jun的表达而诱导胃癌细胞增殖.     

RNA干扰沉默CXCR4基因对胃癌细胞SGC7901增殖和侵袭力的影响

目的探讨特异性抑制胃癌细胞SGC7901中趋化因子受体4(CXCR4)的表达及RNA干扰对胃癌细胞SGC7901细胞增殖及侵袭力的影响。方法将腺病毒载体CXCR4-shRNA转染至胃癌细胞SGC7901,RT-PCR检测转染后CXCR4 mRNA的表达量;MTT法检测癌细胞增殖情况;Transwell小室侵袭实验对胃癌细胞侵袭力进行检测。结果 (1)成功将CXCR4-shRNA重组腺病毒载体转染至胃癌细胞SGC7901;(2)空白组与对照组细胞增殖迅速,两组之间无显著性差异(P>0.05),实验组经腺病毒载体转染后细胞增殖程度显著减少,与前两组相比有显著性差异(P<0.05);(3)shRNA-CXCR4腺病毒载体和空白组及对照组相比能显著抑制SGC7901细胞的侵袭力(P<0.05),抑制率为57.01%。空白组与对照组相比无显著性差异(P>0.05)。结论重组腺病毒载体CXCR4-shRNA能有效抑制胃癌细胞SGC7901的侵袭,并在一定程度上抑制癌细胞增殖。     

ER-α36对胃癌SGC7901细胞在裸鼠体内生长的影响

目的:观察ER-α36分子对人胃癌细胞SGC7901在裸鼠体内生长的影响.方法:利用慢病毒转染技术构建稳定高表达和低表达ERα-36分子的重组人胃癌SGC7901细胞株.实验分为空白对照组(SGC7901-Control)、ER-α36低表达组(SGC7901-Low36)和ER-α36高表达组(SGC7901-High36),每组各3只雄性裸鼠.将上述细胞(5×106个/mL)接种裸鼠皮下,建立裸鼠荷瘤模型,连续观测30d.采用瘤结节体积测量和称质量、瘤组织HE染色及免疫组织化学法检测Ki67指数和E-cadherin蛋白表达等方法,鉴定各组细胞生长的情况.结果:全组实验动物均出现移植瘤.第16天开始,SGC7901-High36组裸鼠肿瘤的体积>SGC7901-Control组裸鼠肿瘤的体积>SGC7901-Low36组裸鼠肿瘤的体积,两两之间有显著性差异(P<0.05).第30天,SGC7901-High36组肿瘤的质量(2.58g±0.014g),明显大于SGC7901-Control组(1.32g±0.0245g)和SGC7901-Low36组(0.471g±0.021g),两两之间有显著性差异(P...     

苦参碱对胃癌SGC7901/DDP细胞耐药相关miRNA表达的影响和靶点分析

目的探讨苦参碱对化疗药物的增敏作用和机制。方法以非毒性剂量的苦参碱联合长春新碱、阿霉素、5-氟尿嘧啶干预胃癌SGC7901/DDP细胞,观察苦参碱对胃癌SGC7901/DDP细胞耐药性的增敏作用;采用实时荧光定量PCR观察苦参碱干预后耐药相关miRNA的表达,并应用生物信息学方法预测和分析相关miRNA的靶基因和信号通路,应用GO分析方法进行了靶基因的功能注释。结果苦参碱可以增强胃癌SGC-7901/DDP细胞对化疗药物的敏感性,逆转耐药;苦参碱可以剂量依赖性上调耐药相关mir-7、mir-125b、mir-200a、mir-200b、mir-200c、mir-146a的表达,生物信息学方法预测和分析了其靶基因和相关信号通路,并筛选出耐药相关miRNA靶向调控的耐药基因和DNA修复基因,为苦参碱克服胃癌化疗多药耐药提供了靶点。结论苦参碱可以增强胃癌SGC-7901/DDP细胞对化疗药物的敏感性;其作用可能与苦参碱可以上调耐药相关mir-7、mir-125b、mir-200a、mir-200b、mir-200c、mir-146a的表达,并通过miRNA靶向调控的耐药基因和DNA修复基因有关。     

Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901 cells

AIM:To study the role of Twist gene in gastric cancer by gene silencing,including the potential of induction of apoptosis,cell cycle arrest,and proliferation inhibition in human malignant gastric SGC7901 cells.METHODS:The expression level of Twist in gastric cancer samples was measured by immunohistochemistry.The effects of Twist gene silencing were detected at both m RNA and protein levels by RT-PCR and Western blot.We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry.We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit.Cell cycle distribution was analyzed by flow cytometry.Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay.RESULTS:Twist protein was highly expressed in gastric cancer samples.Twist gene silencing significantly induced apoptosis,cell cycle arrest at G0/G1 phase,proliferation inhibition,and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells.Meanwhile,both caspase-3 and caspase-9 were activated.CONCLUSION:The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.     

Antisense expression of PKCα improved sensitivity of SGC7901/VCR cells to doxorubicin

AIM: To explore whether antisense blocking of protein kinase C alpha (PKCα) would reverse multi-drug resistance (MDR) in the vincristine (VCR)-resistant human gastric cancer cell line SGC7901/VCR.METHODS: SGC7901/VCR cells expressing antisense PKCα, SGC7901/VCR/aPKC, were established by transfection with a recombinant plasmid reversely inserted with PKCα cDNA. Empty vector (PCI-neo)-transfected cell clones, SGC7901/VCR/neo, served as the control. Western blot method was used to detect PKCα content in SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells, using PKCα-specific antibody. The sensitivity of SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin (DOX) in vitro was determined by MTT assay. The uptake of DOX in these cells was detected with fluorescence spectrophotometer.RESULTS: Western blot analysis showed that the PKCα protein level was about 8.7-fold higher in SGC7901/VCR cells than that in SGC7901 cells, whereas the protein expression of PKCα was reduced by 78% in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells. SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity, accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells.CONCLUSION: PKCα positively regulates MDR in SGC7901 cells, and inhibition of PKCα can partially attenuate MDR in human gastric cancer cells.     

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM： To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action.
METHODS： The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MI-I-） assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential （A~Pm） were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases （PI3K）.
RESULTS： Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 pmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt.
CONCLUSION： Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.     

Activator protein-1 involved in growth inhibition by RASSF1A gene in the human gastric carcinoma cell line SGC7901

AIM：To investigate the role of Ras association domain family protein 1 isoform A （RASSF1A） in gastric tumorigenesis. METHODS：Through over-expression of RASSF1A gene in the SGC7901 cell line which was induced by a lipofectamine-mediated gene transfer approach. Activator protein-1 （AP-1） DNA binding activity was measured by electrophoretic mobility shift assay （EMSA）. RESULTS：Compared with the control clones, cells over- expressing RASSF1A exhibited significant inhibition of cell growth with G1 cell cycle arrest in vitro and in vivo. The over-expression of RASSF1A significantly inhibited AP-1 activity in SGC7901 cells （0.981±0.011 vs 0.354±0.053, P〈0.001）. In addition, both Western blot analysis and immunocytochemistry demonstrated that RASSF1A down-regulated the expression of c-Fos （0.975± 0.02 vs 0.095±0.024, P〈0.001） but not c-Jun.
CONCLUSION： Over-expression of RASSF1A inhibits the growth of SGC7901 cells by negatively regulating the AP-1 activity, the latter in turn negatively signals cell proliferation.     

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines

AIM: To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines. METHODS: The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes' proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals. RESULTS: The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes' proliferations were remarkably increased than their parental dendritic cells. CONCLUSION: The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their anti-tumor biotherapies.     

幽门螺杆菌依赖丝裂原活化蛋白激酶信号通路诱导SGC7901细胞表达分泌白细胞介素-8

背景:已知前炎症细胞因子白细胞介素(IL)鄄8在慢性活动性胃炎、消化性溃疡等幽门螺杆菌(H.pylori)感染相关疾病的发生、发展中起重要作用,但H.pylori诱导胃上皮细胞表达、分泌IL鄄8的分子机制尚不明确。目的:通过体外实验了解H.pylori对人胃癌细胞株SGC7901表达、分泌IL鄄8的影响,探讨p38丝裂原活化蛋白激酶(MAPK)信号通路在H.pylori诱导SGC7901细胞IL鄄8mRNA表达和IL鄄8蛋白分泌中的作用。方法:SGC7901细胞经p38MAPK特异性抑制剂SB203580预作用2h,再加入H.pylori标准菌株CCUG17874共培养。采用逆转录聚合酶链反应(RT鄄PCR)检测IL鄄8mRNA的表达,采用酶联免疫吸附测定(ELISA)检测IL鄄8蛋白的分泌,观察SB203580对H.pylori诱导SGC7901细胞表达、分泌IL鄄8的影响。结果:H.pylori能诱导SGC7901细胞表达、分泌IL鄄8。SB203580能以剂量依赖的方式抑制H.pylori诱导的SGC7901细胞IL鄄8蛋白分泌,经终浓度为0.3、1、3和10μmol/L的SB203580预作用2h,SGC7901细胞的IL鄄8蛋白分泌与未经SB203580预作用组相比分别减少了26%、51%、64%和73%(P<0.05);SB203580也能使H.pylori诱导的IL鄄8mRNA表达显著减弱。结论:H.pylori能诱导胃上皮细胞表达、分泌IL鄄8,该作用在一定程度上依赖于MAPK信号通路。     

幽门螺杆菌致胃上皮细胞株GES-1和胃癌细胞株SGC-7901的氧化性损伤

目的: 探讨H pylori对人正常胃黏膜上皮细胞永生细胞株GES-1和人淋巴结转移胃腺癌细胞株SGC-7901的氧化性DNA损伤作用.方法: 采用细菌-细胞共培养的方法, 比较H pylori作用前后GES-1和SGC-7901细胞株的形态学变化;采用激光扫描共聚焦显微镜方法, 比较H pylori作用前后GES-1和SGC-7901细胞株8-羟基脱氧鸟苷(8-OhdG)的表达.结果: H pylori对GES-1和SGC-7901细胞株均具有损伤作用;8-OHdG表达升高, 加菌组与对照组相比差别具有统计学意义(64.9396±17.8142 vs 32.3010±7.3620;102.8344±30.2632 vs 77.1336±32.3223, 均P = 0.000);而且8-OHdG表达的变化程度GES-1细胞显著高于SGC-7901细胞.结论: H pylori能够诱导GES-1和SGC-7901细胞DNA氧化性损伤显著增加;在H pylori氧化损伤的相关研究中, 更适宜选择对损伤作用敏感的GES-1细胞株作为研究对象.     

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.