反义c-myc涂层支架对兔颈动脉细胞凋亡的影响

目的 :探讨铂 铱合金明胶蛋白涂层支架局部导入c myc反义寡核苷酸 (ASODN)对兔颈动脉细胞凋亡的影响 ,寻求防治支架内再狭窄的途径。方法 :将携带c mycASODN的国产铂 铱合金明胶蛋白涂层支架置入兔颈动脉 (给药组 ,n =16 ) ,在术后 7、14、30、90d处死动物行苏木精 伊红和Weigert染色 ,图像分析测量新生内膜厚度和面积 ,c myc蛋白免疫组化染色 ,采用末端脱氧核苷酸酶介导的dUTP缺口末端标记法检测细胞凋亡 ,并与对照组 (n =16 )进行对比分析。结果 :两组支架术后 7、14d均未观察到平滑肌细胞凋亡 ,术后 30d在新生内膜中观察到明显的细胞凋亡 ,90d时显著高于 30d ;同时给药组平滑肌细胞的凋亡显著高于对照组。结论 :c mycASODN可诱导支架置入后平滑肌细胞凋亡 ,可用于防治再狭窄     

腺病毒介导反义c-myc RNA对人胃癌细胞系作用的分子机制研究

序列特异的ＤＮＡ结合蛋白ｃｍｙｃ在细胞的增殖与分化过程中起着十分重要的调节作用,并能诱导细胞凋亡。反义ｃｍｙｃＲＮＡ,采用ｃｍｙｃ基因含有起始密码子的第二外显子及其两侧部分内含子序列,共1.53ｋｂ片段构建而成,具有抑制ｃｍｙｃ表达的作用[1]。本研究以腺病毒介导,胃癌细胞系作为靶细胞,研究反义ｃｍｙｃ的抑癌作用及其机制。材料与方法一、细胞系ＳＧＣ7901细胞为人胃腺癌细胞系。293细胞为腺病毒Ｅ1基因转化的人胚肾细胞系。二、重组体腺病毒的制备反义ｃｍｙｃ及ＬａｃＺ重组体腺病毒(ＡｄｅｎｏｖｉｒｕｓＡｎｔｉｓｅｎｓｅ…     

反义寡核苷酸转染对SGC-7901胃癌细胞端粒酶活性及细胞凋亡的影响

目的：探讨端粒酶反义寡核脱氧核苷酸（PS－ASODN）对SGC－7901胃癌细胞端粒酶活性及细胞凋亡的影响。方法：脂质体介导的PS－ASODN作用于SGC－7901细胞后，台盼蓝拒染法计算细胞生长抑制剂，采用半定量TRAP－银染法检测端粒酶活性，用流式细胞仪观察细胞凋亡率及细胞周期变化，采用光镜及电镜观察细胞凋亡形态。结果：终浓度为3、2及1μM的PS－ASODN对SGC－7901细胞均有抑制作用，抑制率与对照组比较，差异有显著性（P＜0．01）；PS－ASODN转染后48h，端粒酶活性明显降低（P＜0．05）。以上抑制作用呈剂量依赖性及序列特异性。流式细胞仪检测到凋亡峰，细胞受阻于G0/G1期；光镜及电镜显示典型的细胞凋亡形态改变。对照寡核苷元上述作用。结论：以端粒酶RNA模板区为靶点的反义寡核苷不但明显抑制胃癌细胞的增殖，降低端粒酶活性，而且具有促凋亡作用，对胃癌具有重要治疗价值。     

腺病毒介导RA538及反义c-myc在不同细胞系中作用及其机制

目的比较重组RA538,反义c-myc及LacZ腺病毒(adenovirus,AV)对不同靶细胞的转染效率、生物学特性并探讨其作用的分子机制.方法以人胃癌细胞(SGC7901)、食管癌细胞(EC109)及人胚肺二倍体细胞(2BS)系为靶细胞,采用LacZ基因转染X-gal染色、形态学观察、MTT,RT-PCR等方法,研究重组RA538,反义c-myc及LacZ AV对上述细胞的转染效率,生物学作用及其分子机制.结果 AV-LacZ进行重组腺病毒转导效率检测显示其对SGC7901,2BS细胞具有很高的转导效率,对EC109细胞转导效率较低.AV-RA538及AV-ASc-myc对SGC7901细胞能产生明显的生长抑制效应并诱导凋亡,其生长抑制率分别为76.3%和44.1%.AV-RA538及AV-ASc-myc对SGC7901细胞内源性c-myc,bcl-2基因的表达具有抑制作用.AV-RA538及AV-ASc-myc对EC109细胞及2BS细胞无明显的生长抑制及凋亡诱导作用,AV-RA538对EC109及2BS细胞中内源性c-myc,bcl-2基因的表达无调节作用.结论 AV载体转导效率很高,能实现目的基因在转导细胞中的高水平表达,但对不同靶细胞的转染效率存在差别.AV-RA538,AV-ASc-myc对SGC7901的生长抑制及凋亡诱导作用可能是通过AV的高效转导及抑制c-myc,bcl-2的表达而实现的.AV-RA538,AV-ASc-myc对食管癌、2BS细胞系无类似作用可能与其对上述细胞的转导的作用及内源性基因表达的作用有关.     

反义c-myc寡核苷酸对转染FHIT基因胃癌细胞MKN28的影响

目的: 探讨脂质体介导的c-myc反义寡核苷酸对导入脆性组氨酸三联体(FHIT)基因的胃癌细胞增殖及凋亡的影响.方法: 通过脂质体将重组FHIT基因PRC/CMV质粒和空载体转染到人类胃癌细胞系MKN28,并分别转染c-myc反义寡核苷酸,RT-PCR和Westen b1ot法检测FHIT基因的转染,Westem b1ot法检测细胞c-mHyc的表达,MTT法分析细胞增殖,AO/EB染色法和流式细胞分析技术检测细胞凋亡.结果: 转染FHIT基因后,MKN28细胞检测到FHIT基因片段和FHIT蛋白,而未转染的细胞及转染空载体的细胞未检测到FHIT基因片段及FHIT蛋白.转染c-myc反义寡核苷酸后.对MKN28细胞c.myc的表达有明显的抑制作用,并呈明显的时间依赖性;c-myc asODN对FHIT MKN28细胞抑制率(F=177.480,P<0.05),凋亡率(F=41.500,P<0.05)和凋亡比例明显高于FHIT MKN28细胞.结论: 癌基因c-myc的表达抑制联合FHIT基因的表达可以发挥较强的抗肿瘤细胞作用,为多基因治疗肿瘤提供了理论基础.     

c-myc反义寡核苷酸对结肠癌HT-29细胞增殖、凋亡及化疗敏感性的影响

目的:研究体外c-myc反义寡核苷酸(ASODN)对人结肠癌HT-29细胞增殖、凋亡及化疗敏感性的影响.方法:利用脂质体LipofectamineTM2000介导将c-myc ASODN转染入大肠癌HT-29细胞中, 逆转录多聚酶链反应(RT-PCR)、Westernblot方法检测c-myc基因mRNA及蛋白的表达,MTT、流式细胞仪(FCM)检测c-myc ASODN对人结肠癌HT-29细胞增殖抑制及其对奥沙利铂敏感性影响.结果:转染c-myc ASODN后, HT-29细胞内c-myc mRNA水平显著降低(0.464±0.029 vs0.974±0.027, 0.945±0.012, 均P<0.01). 在HT-29细胞中存在分子质量62 kDa的特异性条带, 与c-myc分子质量相符, ASODN组在PVDF膜上的特异性条带明显弱于对照组; MTT结果显示转染c-myc ASODN 48 h后的HT-29细胞的增殖速度较对照组细胞明显减慢. FCM显示c-myc ASODN转染后72 h后, 奥沙利铂+ c-myc ASODN组细胞凋亡率显著高于对照组( P<0.05).结论:体外c-myc ASODN可抑制c-myc mRNA及蛋白的表达, 阻断c-myc可抑制HT-29细胞增殖并增强其对奥沙利铂的敏感性, 可能为大肠癌的基因治疗提供新的靶点.     

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。     

生存素反义寡脱氧核苷酸对人胃癌细胞株凋亡的影响

背景：生存素（survivin）是凋亡抑制蛋白（IAP）基因家族成员之一，在多数肿瘤组织中高表达。目的：观察生存素反义寡脱氧核苷酸（ASODN）对人胃癌细胞株凋亡的影响。方法：将体外培养的人胃癌细胞株SGC-7901分为不同浓度生存素ASODN组、无关寡脱氧核苷酸（N-ODN）组和对照组。以四甲基偶氮唑蓝（MTT）试验检测生存素ASODN对SGC-7901细胞生长的影响；通过形态学观察、DNA电泳和流式细胞仪分析反映细胞凋亡情况；以端粒重复序列扩增-酶联免疫吸附测定（TRAP-ELISA）方法检测端粒酶活性。结果：生存素ASODN能抑制SGC-7901细胞生长，诱导细胞凋亡，抑制端粒酶活性。凋亡细胞形态学上表现为细胞膜起泡、染色质固缩、核碎裂、凋亡小体形成；DNA电泳呈现凋亡特征性阶梯状条带；流式细胞仪分析显示G1期前出现亚二倍体凋亡峰。结论：生存素ASODN能诱导人胃癌细胞株SGC-7901凋亡，抑制细胞生长及其端粒酶活性。     

脂质体介导的反义寡核苷酸抑制人胃癌细胞端粒酶活性

目的：探讨特异性反义寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅ ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,ＡＳＯＤＮ）对人胃癌细胞端粒酶活性的影响。方法应用脂质体包裹与端粒酶ＲＮＡ模板区互补的ＡＳＯＤＮ,处理人胃癌细胞系ＦＧＣ８５。用端粒重复扩增分析（ｔｅｌｏｍｅｒｅ ｒｅｐｅａｔ ａｍｐｌｉｆｉｃａｔｉｏｎ ｐｒｏｔｏｃｏｌ,ＴＲＡＰ）－ＰＣＲ－ＥＬＩＳＡ检测法观察端粒酶活性的变化,结果经脂质体包裹ＡＳＯ     

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.     

XAF1基因重组腺病毒载体的构建及其在人肝癌细胞体内外的表达

目的利用携带35型腺病毒纤毛的嵌合型5型腺病毒载体系统Ad5/F35构建XAF1基因重组腺病毒,体内外感染人肝癌细胞SMMC7721并使XAF1基因有效表达。方法将真核表达质粒pcDNA3.1-XAF1和穿梭质粒pDC316用BamHⅠ和EcoRⅠ双酶切、筛选、测序获得重组穿梭质粒pDC316-XAF1。将测序正确的pDC316-XAF1和骨架质粒pBHG-fiber5/F35用Lipofectamine2000共转染HEK293细胞,进行细胞内同源重组,得到重组腺病毒Ad5/F35-XAF1。予终点稀释法测定重组腺病毒的感染滴度。用同样的方法得到携带增强型绿色荧光蛋白(EGFP)的报告病毒Ad5/F35-EGFP。建立人肝癌细胞株SMMC7721裸鼠移植瘤模型,将Ad5/F35-XAF1和Ad5/F35-EGFP重组腺病毒分别感染人肝癌细胞株SMMC7721和瘤内注射;荧光显微镜观察EGFP在细胞和移植瘤冰冻切片中的表达;RT-PCR和Westrenblot法检测XAF1的mRNA和蛋白在细胞和移植瘤组织的表达。结果重组腺病毒Ad5/F35-EGFP感染肝癌细胞和瘤内注射后,予荧光显微镜均可见细胞和冰冻切片中呈现绿色荧光;Ad5/F35-XAF1感染肝癌细胞和瘤内注射后,XAF1mRNA和蛋白表达均显著高于对照组和报告病毒组。结论成功构建重组腺病毒Ad5/F35-XAF1和Ad5/F35-EGFP。该腺病毒载体可携带目的基因在人肝癌细胞株SMMC7721体内和体外进行有效表达。     

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.     

Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo

BACKGROUND/AIMS: Gastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer. METHODS: The responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo. RESULTS: p53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours. CONCLUSIONS: Adenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers.     

大蒜素对人肺癌H460细胞增殖及凋亡的影响

目的观察大蒜素对人肺癌H460细胞增殖及凋亡的影响。方法将体外培养的人肺癌H460细胞与不同浓度的大蒜素共育,采用MTT法测算H460细胞增殖率,采用流式细胞仪检测H460细胞凋亡率。结果随着大蒜素浓度增大、作用时间延长,H460细胞的增殖率逐渐降低,呈剂量、时间依赖性（P均〈0.05）;随着大蒜素浓度增大,H460细胞凋亡率逐渐升高,呈一定剂量依赖性（P〈0.05）。结论大蒜素能够抑制人肺癌H460细胞的增殖,并诱导其凋亡。     

CD95基因抑制胃癌细胞生长的体外抑瘤效应

目的　将CD95基因导入胃癌细胞 ,建立CD95基因表达株 ,并比较转导前后mRNA与蛋白的表达水平。观察CD95蛋白对体外培养胃癌细胞的抑制作用。方法　采用分子克隆技术将CD95基因插入真核表达载体pBK CMV的多克隆克隆位点之间 ,以脂质体介导法将目的基因导入受体细胞SGC790 1,用G418筛选克隆细胞 ;以Northernblot,Westernblot检测CD95基因的表达。MTT法检测转导株对化疗药物的敏感性 ;直接记数法描述转导株的细胞生长曲线 ;软琼脂集落形成实验观察基因转导前后细胞的克隆形成力。结果　成功地构建了真核表达载体pBK CD95cDNA。转导细胞后 ,从 1× 10 5细胞中筛选出 10 0个抗性克隆以上 ,转导率大于 0 1% ,随机挑选 2个克隆扩增培养 ,获得了 1株稳定的抗性细胞 ,从而有效地建立了CD95基因表达株 (SGC790 1CD95cells)。杂交结果表明 ,转导株在mRNA及蛋白水平的表达均明显高于非转导株。转导细胞的细胞倍增时间、对数生长期等均体现了比非转导株更为缓慢和处于抑制状态 ,集落形成能力低下 ,而对VCR、5 FU等化疗药物的敏感性明显增强。结论　CD95基因在胃癌细胞中处于低表达状态 ;通过真核表达载体的介导 ,CD95基因导入胃癌细胞后 ,能有效地表达CD95mRNA及其蛋白。CD95基因转导株的表达蛋白能有效地抑制体外培     

Poor outcome of adenovirus infections in adult hematopoietic stem cell transplant patients with sustained adenovirus viremia

H. Omar, Z. Yun, I. Lewensohn‐Fuchs, L. Pérez‐ Bercoff, C. Örvell, L. Engström, G.‐K. Vuong, P. Ljungman. Poor outcome of adenovirus infections in adult hematopoietic stem cell transplant patients with sustained adenovirus viremia.
Transpl Infect Dis 2010: 12: 465–469. All rights reserved Abstract: The outcome of adenovirus (ADV) infections in adult hematopoietic stem cell transplant (HSCT) patients remains poorly characterized. We studied 14 adults and 3 children, who had undergone HSCT and had developed ADV viremia. Peak ADV DNA levels were significantly higher in patients with ADV diseases than in those without (P=0.03). All children survived the ADV infections. Among the 14 adult HSCT patients, 11 were treated with cidofovir, 2 with ribavirin, and 1 did not receive antiviral treatment. Six of the 13 (46%) treated patients developed ADV diseases and 3 of them (23%) died of ADV infections. Sustained viremia (≥3 positive polymerase chain reaction assays during follow‐up) was detected in all patients who finally died of ADV infections. However, 2 adults having had transient ADV viremia either survived or died of diseases other than ADV infections. Our study indicates that the outcome of adult HSCT patients with sustained ADV viremia may be poor, even for those who have received anti‐ADV treatment.