肿瘤易感基因101在胃癌耐药细胞中的表达及作用

目的 探讨肿瘤易感基因(TSG)101在多药耐药胃癌细胞中的表达和作用。方法 应用半定量RT-PCR和Western blot方法观察TSG101在胃癌细胞SGC7901及其长春新碱(VCR)耐药亚系SGC7901／VCR中的表达。将TSG101真核表达载体转染SGC7901细胞，通过Western blot方法鉴定转染细胞TSG101蛋白的表达。用流式细胞仪检测细胞周期的变化和细胞内阿霉素(ADR)的平均荧光强度。用MTT法检测细胞生长曲线和对VCR、ADR的药敏性。结果 胃癌耐药细胞SGC7901／VCR与亲本细胞SGC7901相比，TSG101的表达明显增高。SGC7901细胞转染TSG101真核表达载体后其表达较对照细胞明显增高，G1期细胞比例减少而S期比例增加，细胞增殖加快，且对VCR、ADR的敏感性减低，细胞内阿霉素蓄积和潴留减少。结论 TSG101真核表达载体转染SGC7901后其耐药性明显增强，提示TSG101在胃癌多药耐药中发挥了一定的作用。     

Ss-A/Ro核蛋白60 ku亚单位反义核酸逆转胃癌细胞多药耐药

目的:对Ro 60反义核酸在逆转胃癌细胞多药耐药中作用进行研究．方法:克隆Ro 60编码基因,构建Ro 60编码基因的反义真核表达载体,将其转导入SGC7901细胞,应用半定量RT-PCR技术,对基因转染细胞进行鉴定,通过MTT法进行体外药物敏感性分析,借助流式细胞仪检测细胞内蓄积的阿霉素．结果:成功构建了Ro 60反义真核表达载体,应用脂质体介导法将其转导入SGC7901-VCR, Ro 60反义真核表达载体转染SGC7901-VCR细胞后,Ro 60的表达量明显下降,体外药物敏感性实验提示其对长春新碱、丝裂霉素、顺铂、阿霉素的敏感性增加,转染反义表达载体的SGC7901-VCR细胞与未转染和转染空载体的细胞相比,IC50值(mg/L)有显著的下降(7．66±0．45 vs 19．56±0．38,17．48±0．85;0．84±0．03 vs 1．62±0．06．1．80±0．03;0．51±0．03 vs 0．87±0．03．0．88±0．03;0．22±0．01 vs 0．52±0．02,0．43±0．03,均P<0．01),细胞内阿霉素蓄积有显著的增加(51．94±1．26 mg／L vs 36．27±0．98,37．01±0．91 mg／L,P<0．01)．结论:Ro 60反义真核表达载体转染SGC7901后能够抑制胃癌细胞的多药耐药表型．     

人核糖体蛋白S13与胃癌细胞多药耐药性的实验研究

目的 探讨人核糖体蛋白S13(RPS13)在胃癌多药耐药(MDR)机制中的作用。方法 采用RT-PCR法扩增RPS13 cDNA片段编码区序列全长，DNA重组技术构建正反义真核表达载体，经脂质体介导转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，斑点杂交检测转染细胞mRNA水平的变化；MTT法测定细胞对化疗药物的敏感性，流式细胞仪检测细胞周期。结果 RT-PCR法成功扩增出RPS13 cDNA片段编码区序列全长，并构建正反义真核表达载体；斑点杂交试验证实：正义转染细胞RPS13 mRNA水平上调，反义转染细胞其mRNA水平下调。RPS13正义核酸转染SGC7901细胞后，细胞对阿霉素、5-氟尿嘧啶和长春新碱的敏感性降低；转染反义核酸后，耐药细胞对丝裂霉素和长春新碱的敏感性增加。细胞周期测定表明高表达RPS13后，G1期、S期和G2期细胞的比例分别为47．0％、33．2％和19．8％；低表达RPS13后，G1期、S期和G2期细胞的比例分别为62．9％、1、0％和36．1％。结论 RPS13参与胃癌耐药细胞SGC7901／VCR的多药耐药。     

人宫颈癌基因反义核酸抑制肝癌细胞生长作用的实验研究

目的探讨针对人宫颈癌基因(HCCR)反义核苷酸影响肝癌细胞增殖、凋亡的作用。方法构建pcD-NA3.1-HCCR反义真核表达质粒,转染HepG2细胞,实时定量PCR检测HCCR mRNA表达水平,Western blot检测其蛋白表达水平,流式细胞仪观察HepG2细胞凋亡和细胞周期的变化,MTT法检测细胞的增殖活性。结果HCCR反义核酸可有效抑制HepG2细胞HCCR的表达,与转染前相比,转染后24小时HCCR mRNA水平下降到16%,其蛋白表达水平在24小时也明显降低。对细胞的增殖与凋亡活性检测显示:转染HCCR反义质粒后,HepG2细胞增殖受到抑制,转染后24小时抑制率为47.62%(P<0.05),细胞凋亡率为14.34%±0.91%,较对照组明显增多(t=21.799,P<0.05),细胞分裂多停止在G0G1期。结论HCCR反义核酸可以抑制HepG2细胞中HCCR的表达,抑制细胞增殖,促进细胞凋亡,使细胞停滞于G1期。HCCR反义核酸用于肝癌细胞的基因治疗具有一定的潜在意义。     

Bcl-2反义核酸对胃癌细胞SGC-7901增殖的影响

目的观察Bcl-2反义核酸对胃癌细胞SGC-7901增殖及细胞周期的影响.方法利用转染Bd-2反义核酸的胃癌细胞antibcl-27901与SGC-7901细胞比较,MTT方法及流式细胞仪观察转导细胞的增殖能力和细胞周期.结果转染Bcl-2反义核酸使胃癌细胞增殖受抑,细胞周期未受影响.结论Bd-2反义核酸对胃癌细胞具有一定的抑瘤效应.     

反义Survivin核酸诱导凋亡及逆转胃癌耐药机制的实验研究

目的:探讨反义Survivin核酸(anti-pcDNA3-svv)对胃癌细胞系SGC7901的凋亡诱导、对泰索帝的化疗增敏作用及对化疗耐药的逆转作用.方法:采用脂质体转染法转染胃癌细胞系SGC7901,并筛选阳性克隆,以Westernblot检测Survivin蛋白的表达,RT-PCR检测MDR-1mRNA的表达,用透射电镜及TUNEL法观察并检测转染后SGC7901的凋亡变化,采用MTT法检测转染细胞对泰索帝的敏感性.结果:转染anti-pcDNA3-svv的SGC7901细胞系(SGC7901-SVVanti)其Survivin蛋白比未转染者明显降低,仅为其29.9%(P<0.01);透射电镜下可见转染组细胞呈凋亡晚期变化,TUNEL显示转染组与未转染组AI分别为0.241和0.083(P<0.01);转染组MDR-1mRNA较未转染组明显降低,转染组与未转染组MDR指数分别为:0.196±0.013和3.126±0.019(P<0.01);转染组对泰索帝的IC50值为16.7±1.98μg/L,而未转染组为55.7±1.89μg/L,差异具有显著的统计学意义(P<0.01).结论:反义Survivin核酸可诱导SGC7901细胞凋亡,增强泰索帝化疗敏感性,逆转耐药.     

hPOT1基因正反义真核表达载体的构建及鉴定

目的 构建人端粒保护蛋白（human protection of telomeres，hPOT1）基因的正、反义真核表达载体，以进一步研究hPOT1蛋白的功能及作用机制。方法用限制性内切酶将hPOT1 cDNA从pLPCNMyc POT1质粒上切下来，经适当改造后以正、反方向插入到质粒pcDNA3．1（-）中，构建hPOT1基因的正、反义真核表达载体；用PCR、酶切及DNA测序的方法鉴定hPOTI基因正、反义表达载体序列和方向的正确性。结果PCR、酶谱分析及DNA测序结果表明hPOT1正，反义真核表达载体序列和方向正确。结论成功地构建了hPOT1正、反义真核表达载体，为进一步研究hPOT1在端粒保护，细胞衰老和凋亡中的作用奠定了基础。     

PSME1基因对人胃腺癌细胞株SGC-7901生物学特性的影响

背景:PSME1基因编码蛋白PA28α是26S蛋白酶体的重要组成部分,研究显示其在多种人类恶性肿瘤中表达下调,可能影响肿瘤细胞的生物学特性。目的:探讨PSME1基因对胃癌细胞恶性表型相关生物学特性的影响。方法:将PSME1基因克隆入pcDNA3.1载体,构建PC-PSME1表达载体,以脂质体法转染人胃腺癌细胞株SGC-7901,筛选并鉴定稳定转染细胞株SGC-PSME1。以生长曲线法、克隆形成实验、流式细胞术和细胞迁徙/侵袭实验分析SGC-PSME1细胞株的生长、增殖、细胞周期、细胞凋亡和侵袭转移情况。结果:与转染空载体的SGC-PC细胞和空白对照组SGC-7901细胞相比,SGC-PSME1细胞的生长速度和克隆形成率降低,G0/G1期细胞比例增高,S期细胞比例降低,细胞凋亡率增高,差异均有统计学意义(P0.05);SGC-PSME1细胞的迁徙率与两对照组相比无明显差异。结论:PSME1基因对抑制胃癌细胞的恶性表型有一定意义。其可抑制胃癌细胞的生长、增殖,同时影响细胞周期,对细胞的侵袭转移能力可能无明显影响。     

shRNA沉寂HK-Ⅱ表达对胃癌SGC7901细胞增殖、凋亡的影响

目的:观察shRNA沉寂HK-Ⅱ基因的表达对胃癌细胞株SGC7901细胞增殖、凋亡的影响, 初步评价HK-Ⅱ基因治疗胃癌的应用前景.方法:取胃癌细胞株SGC7901及胃上皮细胞株GES-1细胞为研究对象, 将每株细胞分别分成5组(A: 干扰组; B: 阳性对照组; C: 阴性对照组; D: 脂质体组; E: 空白对照组). 用shRNA沉寂胃癌细胞株SGC7901及胃上皮细胞株GES-1细胞中HK-Ⅱ的表达. 用逆转录-聚合酶链反应(RT-PCR)检测转染shRNA后每株细胞中各组HK-Ⅱ mRNA的表达变化; 分别用MTT及流式细胞仪检测转染后细胞增殖、凋亡的变化.结果:HK-Ⅱ mRNA在胃癌细胞株SGC7901细胞中的表达明显高于胃上皮细胞株GES-1细胞(t = 12.119, P <0.01), HK-Ⅱ shRNA可以明显抑制两株细胞中HK-Ⅱ的表达(P <0.01).沉寂HK-Ⅱ的表达可抑制SGC7901细胞的增殖(F = 159.811, P <0.01), 并促进其凋亡(χ2= 21.324, P <0.01); 但对胃上皮细胞增殖(F =0.704, P = 0.592)及凋亡(χ2 = 1.007, P = 0.909)无明显影响.结论:沉寂HK-Ⅱ表达可以抑制胃癌细胞株SGC7901的增殖, 促进其凋亡; 但对胃上皮细胞株GES-1无明显影响. HK-Ⅱ可能成为胃癌治疗的一个靶点.     

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.     

环氧合酶-2反义核酸抑制胃癌细胞的恶性表型

目的　通过环氧合酶 2 (COX 2 )反义核酸基因转染 ,逆转COX 2高表达的人胃癌细胞系中其表达水平 ,观察细胞生物学行为的变化情况 ,以初步探讨COX 2表达在胃癌发生中的某些具体机制。方法　使用脂质体介导的方法 ,用反义重组质粒及空载体分别转染COX 2异常高表达的人胃癌细胞系SGC 790 1(转染细胞分别命名为 790 1 AS及 790 1 P细胞 )。通过免疫细胞化学及RNA斑点杂交试验检测反义核酸转染的细胞中COX 2的蛋白及mRNA表达水平。四唑盐 (MTT)比色试验检测转染细胞的体外增殖速度。应用裸鼠成瘤试验比较转染前后细胞体内成瘤能力的差别。结果　免疫细胞化学及RNA斑点杂交试验证实 :在反义核酸转染的 790 1 AS细胞中COX 2的蛋白及mRNA水平均显著下调。MTT比色试验显示 790 1 AS细胞的增殖速度低于亲本SGC 790 1细胞。裸鼠成瘤试验表明 ,反义核酸转染细胞成瘤潜伏期延长 ,成瘤体积减小。 3组细胞接种裸鼠 30d后 ,瘤体的平均重量( x±s)分别为 (82 6 6 7± 77 6 7)mg(SGC 790 1细胞 ) ,(776 6 7± 30 0 0 6 )mg(790 1 P细胞 )和 (486 6 7±15 2 8)mg (790 1 AS细胞 )。转染反义核酸的细胞成瘤性显著低于未转染细胞及转染载体对照细胞(P <0 0 1)。结论　胃癌细胞中过表达的COX 2与细胞的恶性表型相关。用     

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.     

Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

AIM： To study the effect of type 1 Na^＋/H^＋ exchanger （NHE1 ） antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901. METHODS： Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method. Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH （pHi）, cell cycle, clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined. RESULTS： Antisense eukaryotic expressing vectors were successfully constructed and transfected into SGC-7901. The transfectant obtained named 7901 -antisense （7901-AS） stablely produced antisense NHE1. There was a significant difference between the pHi of 7901-AS cells （6.77 ± 0.05） and that of 7901-zeo cells and SGC-7901 cells （7.24 ± 0.03 and 7.26 ± 0.03, P 〈 0.01）. Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/G0 phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were clearly inhibited. CONCLUSION： NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901 . These results suggest a potential role for human tumor gene therapy.     

应用基因转染方法调节HSP70在人胃癌细胞株SGC7901中的表达

目的　为探讨 ＨＳＰ７０ 在人胃癌中过度表达的意义, 调节ＨＳＰ７０ 在人胃癌细胞系ＳＧＣ７９０１ 中的表达．方法　应用脂质体介导的基因转染方法,将表达针对ＨＳＰ７０的反义ＲＮＡ 表达载体及ＨＳＰ７０ 表达载体转入ＳＧＣ７９０１ 细胞,通过ｈｙｇｒｏ ｍ ｙｃｉｎｅ 抗性筛选, 挑选阳性克隆;对阳性克隆从ＲＮＡ 水平（ 点杂交及ＲＮａｓｅ 保护试验） 及蛋白质水平（ Ｗｅｓｔｅｒｎｂｌｏｔ 及免疫组化） 进行研究．结果　ＨＳＰ７０ 在转染细胞中的表达得到了调节． 为进一步研究ＨＳＰ７０ 对胃癌形成与发展的作用创造了条件．结论　经过ＨＳＰ 反义或正义ＲＮＡ 转染的ＳＧＣ７９０１ 细胞具有不同的ＨＳＰ７０ 表达水平可用于胃癌发生机制的研究．     

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.     

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

ＩＮＴＲＯＤＵＣＴＩＯＮＣｈｅｍｏｔｈｅｒａｐｙｉｓｏｎｅｏｆｔｈｅｍａｊｏｒｍｅｔｈｏｄｓｉｎｔｕｍｏｒｔｒｅａｔｍｅｎｔ,ｂｕｔｉｔｏｆｔｅｎｄｏｅｓｎｏｔｗｏｒｋｄｕｅｔｏｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｃｅ（ＭＤＲ）．Ｒｅｃｅｎｔｓｔｕｄｉ...     

Antisense angiopoietin-1 inhibits tumorigenesis and angiogenesis of gastric cancer

AIM: To investigate the effect of angiopoietin-1 (Ang-1) on biological behaviors in vitro and tumorigenesis and angiogenesis in vitro of human gastric cancer cells. METHODS: Human full-length Ang-1 gene was cloned from human placental tissues by RT-PCR method. Recombinant human Ang-1 antisense eukaryotic expression vector was constructed by directional cloning, and transfected by lipofectin method into human gastric cancer line SGC7901 with high Ang-1 expression level. Inhibition efficiency was confirmed by semi- quantitive PCR and Western blot method. Cell growth curve and cell cycle were observed with MTT assays and flow cytometry, respectively. Nude mice tumorigenicity test was employed to compare in vitro tumorigenesis of cells with Ang-1 suppression. Microvessel density (MVD) of implanted tumor tissues was analyzed by immunohistochemistry for factorⅧstaining. RESULTS: Full-length Ang-1 gene was successfully cloned and stable transfectants were established, namely 7Angl- for antisense, and 7901P for empty vector transfected. 7Angl- cells showed down-regulated Ang-1 expression, while its in vitro proliferation and cell cycle distribution were not significantly changed. In contrast, xenograft of 7Angl- cells in nude mice had lower volume and weight than those of 7901P after 30 days' implantation (p<0.01, 293.00±95.54 mg vs. 624.00±77.78 mg) accompanied with less vessel formation with MVD 6.00±1.73 compared to 7901P group 8.44±1.33(p<0.01). CONCLUSION: Ang-1 may play an important role in tumorigenesis and angiogenesis of gastric cancer, and targeting its expression may be beneficial for the therapy of gastric cancer.     

survivin基因shRNA真核表达载体的构建及其对人胃癌细胞株中survivin表达的影响

背景:胃癌是我国最常见的恶性肿瘤之一,survivin是凋亡抑制蛋白家族的新成员,在胃癌组织中高表达。目的:构建survivin基因短发夹RNA(shRNA)真核表达载体并观察其对人胃癌细胞株BGC823和SGC7901中survivin表达的影响。方法:根据GenBank中survivin基因序列设计并合成能转录shRNA的双链DNA序列,插入含有绿色荧光蛋白(GFP)基因和U6启动子的真核表达载体pRNAT-U6.3中,构建重组载体pRNA-shSUR。重组载体经鉴定后转染胃癌细胞株BGC823和SGC7901,以转染pRNA-shControl作为阴性对照。荧光显微镜下观察转染情况,蛋白质印迹法检测survivin蛋白表达,Annexin V-FITC/PI双染法检测胃癌细胞凋亡情况。结果:成功构建了针对survivin基因的shRNA表达载体。转染胃癌BGC823和SGC7901细胞48 h后,与阴性对照组相比,pRNA-shSUR组GFP表达增强,survivin蛋白表达受到明显抑制(P<0.05),胃癌细胞早期凋亡率明显增加。结论:成功构建靶向survivin基因的特异性shRNA真核表达载体,转染胃癌细胞后可抑制survivin蛋白表达并促进细胞凋亡,为进一步研究survivin基因与胃癌生物学行为以及化疗耐药等的相关性奠定了基础。