生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对抗肿瘤药物敏感性的研究

背景：生存素是凋亡抑制蛋白家族的重要成员，生存素基因有可能成为肿瘤反义基因治疗的理想靶基因。目的：研究生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对常用抗肿瘤药物敏感性的作用，探讨生存素反义核酸用于肿瘤基因治疗的可能性。方法：应用基因重组技术构建pEGFP-C1-生存素反义核酸重组质粒，以脂质体转染法转染SMMC．7721细胞，用逆转录聚合酶链反应(RT-PCR)检测生存素mRNA的表达，用流式细胞仪检测细胞凋亡。将生存素反义核酸分别与7种常用抗肿瘤药物共同作用于SMMC-7721细胞，用四唑蓝(MTT)比色法测定细胞杀伤率。结果：生存素反义核酸可抑制SMMC-7721细胞中生存素mRNA的表达，从而导致细胞凋亡增加，其作用呈剂量依赖性。生存素反义核酸可增加SMMC-7721细胞对7种常用抗肿瘤药物的敏感性，明显增强药物的杀伤作用。结论：生存素反义核酸能靶向抑制野生型生存素基因的表达，提高肝癌细胞对常用抗肿瘤药物的敏感性，有可能成为肿瘤基因治疗的新方法。     

下调XIAP表达增强化疗药物诱导胃癌细胞凋亡的作用

目的:观察下调X连锁凋亡抑制蛋白(XIAP)基因表达对胃癌细胞化疗敏感性的影响.方法:构建XIAP基因反义真核表达载体,稳定转染胃癌细胞株MKN-45,RT-PCR和Western blot法检测癌细胞XIAP基因表达.选用顺铂、丝裂霉素分别处理转染前后的胃癌细胞,采用MTT比色法、克隆形成抑制实验检测癌细胞体外生长活性:透射电镜、流式细胞术、TUNEL检测癌细胞凋亡及比率;Western blot和比色法检测细胞内caspase-3蛋白表达和活性水平.结果:RT-PCR和Western blot证实,稳定转染反义XIAP基因的胃癌细胞MKN-45的XIAP mRNA和蛋白表达水平分别降低84.75%(P<0.01)和89.75%(P<0.01),各浓度顺铂、丝裂霉素处理24 h后,转染反义XIAP基因的MKN-45细胞生长抑制率分别增加7.3%-25.3%(P<0.01),12.3%-16.3%(P<0.01).透射电镜下可见部分细胞发生典型的凋亡形态学改变,凋亡率分别为34.12%和32.5%,显著高于未转染对照组MKN-45细胞的凋亡率(14.2%,P<0.05).与MKN-45细胞比较,稳定转染反义XIAP基因的MKN-45细胞内caspase-3表达水平增高2.45倍(P<0.01),活性水平提高3.68倍(P<0.0 1).结论:通过反义RNA技术下调XIAP基因表达,能提高癌细胞中caspase-3的表达和活性,增强化疗药物对癌细胞的诱导凋亡作用.     

生存素基因突变体诱导胃癌细胞凋亡的实验研究

目的 生存素(survivin)基因在胃癌组织中高水平表达，而在正常胃黏膜中无表达。生存素表达的水平与胃癌的预后密切相关。通过基因重组构建生存素基因突变体(pcDNA3—survivin—mutant，Cys84Ala)质粒，并以脂质体转染的方法将质粒DNA转入胃癌细胞株，观察其对胃癌细胞的生物学特性及多药耐药性的影响。方法 应用RT—PCR、Western blot、免疫组化等方法检测胃癌细胞中生存素基因mRNA和蛋白的表达，流式细胞仪和吖啶橙染色检测胃癌细胞凋亡。结果 通过RT—PCR检测，在所有的胃癌细胞株中均有不同程度生存素基因表达，生存素基因突变体Cys84Ala通过抑制野生型生存素基因的功能诱导胃癌细胞凋亡，增加caspase—3活性和促进细胞色素C的释放，增加胃癌细胞对化疗药物的敏感性。结论 突变体Cys84A1a能诱导胃癌细胞凋亡和增加胃癌细胞对化疗药物的敏感性。因此，生存素基因突变体Cys84Ala有可能成为胃癌治疗的一个候选基因。     

胃癌相关基因GCRG213真核表达载体的构建及其对胃癌细胞MKN45的影响

目的 研究胃癌相关基因GCRG213正义、反义转染对胃癌细胞MKN45的影响.方法 采用分子克隆及基因转染技术将GCRG213基因转入哺乳动物细胞,并结合反义转染技术研究GCRG213基因对胃癌细胞恶性生物学行为的影响.结果结果 GCRG213正向和反向克隆正确插人真核表达载体pcDNA3.1(+);重组子pcDNA3.1-a、pcDNA3.1-b和空载体转染人胃癌细胞系MKN45细胞;正义转染其mRNA的表达上调,而反义转染下调;正义转染加快MKN45细胞生长增殖速度、降低细胞凋亡率,反义转染减慢MKN45细胞生长增殖速度,增加细胞凋亡率.结论 胃癌相关基因GCRG213可促进肿瘤细胞生长、分裂和转移,抑制肿瘤细胞的凋亡,可能是一个新发现的恶性肿瘤癌变的促进因素.     

反义生存素诱导胃癌细胞凋亡的实验研究

目的构建反义生存素真核表达载体(anti-pcDNA3-svv),探讨其对胃癌的凋亡诱导作用。方法采用反义生存素核酸瞬时电转染法转染胃癌细胞系Skov3,筛选出阳性细胞株。以Western blot检测生存素蛋白的表达,用流式细胞仪检测其对Skov3的凋亡诱导作用,并检测实验组细胞(Skov3-SVVanti)MDR-1 mRNA的表达。结果稳定表达anti-pcDNA3-svv的Skov3-SVVanti组生存素蛋白表达较Skov3-neo和Skov3组明显降低,Skov3-SVVanti组细胞流式细胞仪检测显示其凋亡率为19%。Skov3-SVVanti组MDR-1 mRNA表达较Skov3和Skov3-neo组明显减少(P<0.01)。结论反义生存素核酸可诱导胃癌细胞凋亡,并能降低MDR-1mRNA表达。     

人端粒酶反义寡核苷酸抑制三种不同分化程度胃癌细胞生长及作用机制研究

目的 研究特定序列人端粒酶反义寡核苷酸(AS-0DN)抑制三种分化程度不同的胃癌细胞(MKN-45、SGC-7901、MKN-28)生长的可能性，并阐述其抑制胃癌细胞生长的作用与胃癌细胞分化程度的相关性。方法 在指定的作用时问和浓度等条件下，以AS-0DN作用于不同分化程度的三种胃癌细胞，用改良端粒酶活性定量检测法测定AS-0DN片段作用前后胃癌细胞的端粒酶活性；用锥虫蓝染色法观察细胞活力；用倒置显微镜、电镜、流式细胞仪和原位末端标记(TUNEL)法观察细胞凋亡情况。结果以指定浓度的AS-ODN作用后，MKN-45和SGC-7901细胞出现明显的端粒酶活性和细胞生长抑制(P＜0．05)，但在同样浓度条件下，MKN-28胃癌细胞只出现端粒酶活性抑制。错义序列对照组则无明显变化。以10 μmol／L的AS-ODN连续作用三种胃癌细胞96h后，光镜、电镜和TUNEL法检测均发现MKN-45和SGC-7901细胞表现出特有的凋亡征象，流式细胞仪检测证实MKN-45和SGG-7901细胞的平均凋亡率在44．75％和33．56％，错义序列对照组则无明显变化(P＜0．05)。结论 AS-0DN能有效抑制胃癌细胞生长，其作用机制主要是抑制端粒酶活性和诱导细胞凋亡。AS-0DN对中分化胃癌细胞的生长抑制最显著，对低分化胃癌细胞的抑制作用略强于高分化胃癌细胞，提示端粒酶反义核酸的抑制作用不依赖于胃癌细胞的分化程度。     

ERβ重组质粒的构建及其在结肠癌细胞株Caco-2中的表达

目的: 基因重组技术构建重组质粒pEGFP-C1-ERbeta并检测其在结肠癌细胞株Caco-2中的表达. 方法: 应用RT-PCR从人结肠癌手术患者正常切缘组织中分离、扩增目的基因片段, 将所得cDNA定向克隆到真核表达载体pEGFP-C1中, 采用双酶切和测序分析鉴定插入基因的序列; 脂质体介导重组质粒pEGFP-C1-ERbeta瞬时转染Caco-2并上流式细胞仪分选, 获得比较单一的转染细胞; 分别采用RT-PCR、Western blot检测转染前后ERbeta基因不同分子水平表达. 结果: 酶切鉴定和测序分析表明重组表达质粒pEGFP-C1-ERbeta构建无误; RT-PCR和Western blot分析均表明, 与转染空质粒pEGFP-C1组和空白对照组细胞相比, 转染重组表达质粒pEGFP-C1-ERbeta组细胞ERbeta基因表达水平明显提高. 结论: 成功构建重组质粒pEGFP-C1-ERbeta并在Caco-2细胞株中表达, 为进一步研究ERbeta如何通过雌激素受体通路调控下游靶基因表达和参与结肠癌表遗传学发生机制奠定了基础.     

亚甲基四氢叶酸还原酶对胃癌细胞基因表达的影响

目的 分析亚甲基四氢叶酸还原酶（MTHFR）基因的真核表达质粒对人胃癌细胞生存力、DNA甲基化酶基因及肿瘤相关基因转录水平的影响。方法 分别构建含有人野生型（正义）MTHFR（W）和反义MTHFR（A）的真核表达质粒，将其转染入人胃癌MKN45细胞，噻唑蓝（MTT）法检测细胞生存活力，定量PCR检测DNA甲基化酶及胃癌相关基因的转录水平。结果 转染野生型MTHFR质粒的胃癌细胞生存活力增高（P〈0、01），而转染反义质粒者生存活力降低（P〈0．01）。野生型MTHFR质粒下调c-myc、p21WAF1a和hMLH1基因表达，反义MTHFR质粒的转染则无明显的基因调控作用。结论 重组MTHFR表达质粒可影响细胞生存活力及人胃癌细胞相关基因的表达，可望成为肿瘤基因防治的新靶点。     

ERβ重组质粒的构建及其在结肠癌细胞株Caco-2中的表达

目的:基因重组技术构建重组质粒pEGFP-C1ERβ并检测其在结肠癌细胞株Caco-2中的表达.方法:应用RT-PCR从人结肠癌手术患者正常切缘组织中分离、扩增目的基因片段,将所得cDNA定向克隆到真核表达载体pEGFP-C1中,采用双酶切和测序分析鉴定插入基因的序列;脂质体介导重组质粒pEGFP-C1-ERβ瞬时转染Caco-2并上流式细胞仪分选,获得比较单一的转染细胞:分别采用RT-PCR、Western blot检测转染前后ERβ基因不同分子水平表达.结果:酶切鉴定和测序分析表明重组表达质粒pEGFP-C1-ERβ构建无误:RT-PCR和Western blot分析均表明,与转染空质粒pEGFP-C1组和空白对照组细胞相比,转染重组表达质粒pEGFP-C1-ERβ组细胞ERβ基因表达水平明显提高.结论:成功构建重组质粒pEGFP-C1-ERβ并在Caco-2细胞株中表达,为进一步研究ERβ如何通过雌激素受体通路调控下游靶基因表达和参与结肠癌表遗传学发生机制奠定了基础.     

生存素反义寡核苷酸诱导结肠癌细胞株凋亡的实验研究

背景:生存素(survivin)是凋亡抑制蛋白家族的成员之一,在肿瘤组织和人类胚胎组织中有高水平表达,在分化成熟的组织中不表达或低水平表达。有研究显示,64.5%～85%的结肠癌组织中存在生存素基因表达,其表达与结肠癌患者的不良预后有关。目的:观察生存素反义寡核苷酸对结肠癌细胞株凋亡的影响。方法:通过基因工程技术构建生存素反义寡核苷酸质粒pcDNA3鄄sur鄄As,以脂质体转染的方法将质粒DNA转入结肠癌细胞株。应用逆转录聚合酶链反应(RT鄄PCR)和蛋白质印迹法(Westernblot)检测结肠癌细胞株中生存素mRNA和蛋白的表达,流式细胞仪和碘化丙啶(PI)染色检测细胞凋亡,比色测定检测caspase鄄3活性的变化。结果:SW1116、COLO205、HT鄄29和SW1417四株结肠癌细胞株中均有生存素mRNA和蛋白表达。瞬时转染生存素反义寡核苷酸后,4株结肠癌细胞株的细胞凋亡率均明显增加,并呈剂量依赖性(P<0.01);结肠癌细胞株SW1116在荧光显微镜下呈现典型的凋亡细胞形态,细胞中生存素蛋白表达水平明显降低,caspase鄄3活性显著增加(P<0.01)。结论:应用生存素反义寡核苷酸抑制生存素基因的表达能诱导结肠癌细胞株凋亡,以生存素为靶基因的治疗方案可能有助于结肠癌患者的治疗。     

Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

AIM: To study the effect of NF-κB, survivin, Bd-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells. METHODS: Gastric cancer cells of SGC-7901, MKN28, MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis indudng ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot. RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SC-C-7901cells respectively. Western blot revealed that the expressions of NF-EB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells. CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.     

Effect of NF-κB, survivin, Bcl-2 and Caspase3 on apoptosis of gastric cancer cells induced by tumor necrosis factor related apoptosis inducing ligand

AIM: To study the effect of NF-κB, survivin, Bcl-2 and Caspase3 on tumor necrosis factors related apoptosis inducing ligand (TRAIL) induced apoptosis of gastric cancer cells.METHODS: Gastric cancer cells of SGC-7901, MKN28,MKN45 and AGS lines were cultured in PRMI-1640 medium and the apoptosis rates of the cells of 4 lines were observed after treatment of tumor necrosis factors related apoptosis inducing ligand (TRAIL) with a flow cytometer. The expression of NF-κB, survivin, Bcl-2 and Caspase3 in gastric cancer cells of 4 lines was analyzed with Western blot.RESULTS: After the gastric cancer cells were exposed to TRAIL 300 ng/ml for 24 hours, the apoptosis rate was 36.05%, 20.27%, 16.50% and 11.80% in MKN28, MKN45,AGS and SGC-7901cells respectively. Western blot revealed that the expressions of NF-κB and survivin were lower in MKN28 cells than in MKN45, AGS and SGC-7901 cells. In contrast, the expression of Caspase3 was higher in MKN28 cells than in MKN45, AGS and SGC-7901 cells.CONCLUSION: There is a selectivity of TRAIL potency to induce apoptosis in gastric cancer cells of different cell lines.The anticancer potency of TRAIL is associated with the decreased expression of NF-κB and survivin and increased expression of Caspase3 of gastric cancer cells.     

Effect and mechanism of the Twist gene on invasion and metastasis of gastric carcinoma cells

AIM： To study the effect of the transfected Twist gene on invasion and metastasis of gastric carcinoma cells and the possible mechanisms involved. METHODS： Human gastric carcinoma MKN28 cells were stably transfected with Twist sense plasmid, and MKN45 cells were stably transfected with Twist antisense plasmid using the lipofectamine transfection technique. RT-PCR, Western blotting, EMSA, gelatin zymography assay, and in vitro invasion and migration assays were performed. Nude mice metastasis models were established by the abdominal cavity transfer method. RESULTS： Cell models （TwistS-MKN28） that steadily expressed high Twist protein were obtained. Compared with MKN28 and pcDNA3-MKN28 cells, adherence, migration and invasion ability of TwistS -MKN28 cells were clearly raised. The number of cancer nodules was increased significantly in the abdominal cavity and liver of nude mice inoculated with TwistS-MKN28 cells. Overexpression of Twist in MKN28 cells increased Tcf-4/ Lef DNA binding activity, and promoted expression of Tcf-4’s downstream target genes cyclin D1 and MMP-2. However, suppression of Twist （TwistAS-MKN45） inhibited MKN45 cell invasion and the expression of cyclin D1 was reduced. The activity of MMP-2 was also decreased. CONCLUSION： These results indicate that Twist promotes gastric cancer cell migration, invasion and metastasis, and Twist may play an important role in Wnt/ Tcf-4 signaling.     

Survivin antisense oligodeoxynucleotide inhibits growth of gastric cancer cells

AIM: To investigate the effect of transfected survivin antisense oligonucleotide (ASODN) on proliferation and apoptosis of gastric cancer cells. METHODS: The authors designed ASODNs targeting different regions of survivin mRNA, including surviving ASODN1, ASODN2 and ASODN3. ASODNs were transfected into gastric cancer cell line SGC 7901, cell growth was detected by MTT assay. Cells exposed to the potent oligonucleotide were also examined for apoptosis induction by FCM and fluorescence microscopy. Semiquantitive RT-PCR and Western blot examinations were carried for expression of survivin mRNA and protein. RESULTS: ASODN3 caused a statistically significant reduction of cell viability to 60.6% (+/-2.9%) (P<0.01), while ASODN1 and ASODN2 had no such changes (P>0.05). The cell growth was also significantly inhibited by ASODN3, compared with reversal and scrambled sequence. A significant loss of survivin mRNA was presented in ASODN3 treated cells and this was not seen in treatment with sense ODN or scramble ODN. Protein level was significantly decreased 48 h after survivin ASODN trasfected by approximately 2-fold decrease compared with untreated controls. However, ASODN3 did not induce significant apoptosis response until 48 h after transfection (P>0.05). CONCLUSION: ASODN3, which targets translation initiation part, can be identified as a most potent antisense compound. Srvivin ASODN3 may provide a novel approach to therapy of gastric cancer.     

胃癌相关基因GCRG213正反义真核表达载体的构建及鉴定

目的:构建胃癌相关基因GCRG213正、反义真核表达载体．方法:从pGEM-T质粒上扩增出的胃癌相关基因GCRG213的DNA片段,两端分别引入限制性内切酶KpnI,BamHI和EcoRI, BamHI识别位点．按正向、反向克隆入真核表达载体pcDNA3．1( )．测序正确的重组子pcDNA3．1-a(含GCRG213正向克隆), pcDNA3．1-b(含GCRG213反向克隆)和空载体经脂质体转染人胃癌细胞系MKN45细胞, G418筛选获得稳定转染的细胞株,采用半定量RT-PCR及Wlestern blot比较转染不同质粒的 MKN45细胞中GCRG213在mRNA和蛋白质水平上的表达差异．结果:经测序证实,GCRG213正向克隆和反向克隆正确插入真核表达载体 pcDNA3．1( ),组成重组子pcDNA3．1-a(含正向克隆),pcDNA3．1-b(含反向克隆)．重组子 pcDNA3．1-a,pcDNA3．1-b和空载体经脂质体转染人胃癌细胞系MKN45细胞,G418筛选获得稳定转染的细胞株．与对应的空载体比较,RT-PCR结果显示转染pcDNA3．1-a的 MKN45细胞中其mRNA的表达上调35．4％,而转染pcDNA3．1-b的MKN45细胞中其mRNA 的表达下调32．1％;Western blot结果显示转染 pcDNA3．1-a的MKN45细胞中其蛋白的表达上调49．4％,而转染pcDNA3．1-b的MKN45细胞中其蛋白的表达下调50．3％．结论:成功构建胃癌相关基因GCRG213正、反义真核表达载体．     

YM155对人胃癌细胞株MKN28的作用及其机制研究

背景:研究发现生存素(survivin)在胃癌组织中高表达,YM155是survivin的特异性抑制剂。目的:探讨YM155对人胃癌细胞株MKN28的作用及其机制。方法:以不同浓度YM155作用于人胃癌细胞株MKN28。采用甲基噻唑基四唑(MTT)法检测细胞增殖抑制率;以原位末端标记(TUNEL法)检测细胞凋亡率;以逆转录聚合酶链反应(RTPCR)、蛋白质印迹法分别检测survivin mRNA和survivin、多聚ADP核糖聚合酶(PARP)、caspase-3蛋白表达。结果:YM155作用后,MKN28细胞增殖抑制,凋亡增加。随着YM155浓度升高,survivin mRNA和蛋白表达水平明显降低,并伴随PARP、caspase-3蛋白裂解。结论:YM155可抑制人胃癌细胞株MKN28增殖,并诱导其凋亡,此机制可能与抑制survivin表达,继而激活caspase凋亡信号通路有关。     

生存素基因突变体诱导胃癌细胞有丝分裂障碍的实验研究

目的 观察生存素基因突变体质粒(Cys84Ala)对胃癌细胞有丝分裂过程中细胞动力学行为的影响。方法 应用基因重组技术构建Cys 84 Ala，以脂质体转染的方法将质粒DNA转入胃癌细胞株，应用免疫组化法检测胃癌组织中生存素基因表达，流式细胞仪检测胃癌细胞凋亡及细胞周期，Western blot检测胃癌细胞中多聚ADP核糖聚合酶(PARP)和细胞色素C蛋白的表达，免疫荧光染色检测有丝分裂危象情况。结果 Cys84Ala可通过抑制野生型生存素基因的功能诱导胃癌细胞凋亡，增加caspase-3活性，促进PARP裂解和细胞色素C的释放，亦可导致胃癌细胞发生有丝分裂障碍而死亡。结论 cys84Ala能通过不同的途径诱导胃癌细胞凋亡和有丝分裂危象，并有可能成为胃癌治疗的一个候选基因。