PCNA反义cDNA基因转导抑制人膀胱癌细胞增殖的研究

目的 探讨增殖细胞核抗原（PCNA）基因反义cDNA对人膀胱癌细胞体外增殖活性的抑制作用。方法 构建PCNA反义cDNA真核表达载体并转染膀胱癌DJ细胞,通过细胞生长曲线、MTT比色分析、H^3-TdR掺入法检测癌细胞增殖活性,流式细胞仪（FCM）分析细胞周期时相变化,SABC免疫组化观察癌细胞PCNA蛋白表达水平。结果 PCNA反义cDNA导入后,膀胱癌DJ细胞生长速率显著减慢（P＜0．01）,增殖活性抑制率59．02％（P＜0．05）,DNA合成速率降低52．31％（P＜0．01）,S期细胞减少,细胞周期阻滞于G0／G1期,PCNA蛋白表达显著减弱（P＜0．05）,差别均有显著性意义。结论 PCNA反义cDNA基因转导能有效抑制膀胱癌细胞的PCNA蛋白表达及体外增殖活性,为肿瘤基因治疗提供了有效途径。     

PCNA反义寡脱氧核苷酸对BIU-87细胞体外增殖活性的影响

目的为探讨基因治疗膀脱癌的新途径,使用增殖细胞核抗原（PCNA）反义寡聚核苷酸（ASODN）作用于膀优癌细胞系BIU-87。方法应用细胞生长曲线和MTT比色法检测细胞增殖活性,并采用免疫组织化学方法（SABC法）检测PCNA蛋白的表达。结果ASOND（30urn）处理组细胞与对照组比较生长曲线差异有显著性,增殖显著受抑（P＜0．05）,PCNA蛋白表达完全抑制。而SODN组则无此作用（P＞0．05）。结论PCNA反义寡聚核苷酸可以显著抑制BIU-87细胞体外增殖活性,其抑制作用可能通过阻断PCNA蛋白表达实现。     

反义增殖细胞核抗原联合p16基因转染抑制膀胱癌细胞生长的研究

目的 探讨膀胱癌联合基因治疗的新策略。方法 反义增殖细胞核抗原(PCNA)联合p16转染膀胱癌细胞，观测共转染1—7d后癌细胞增殖活性、DNA合成速率、细胞周期时相、克隆形成能力、细胞凋亡和PCNA、p16基因表达情况。结果 联合转染后癌细胞PCNA表达减弱，p16表达显著增强，增殖活性抑制15．45％—68．47％(P＜0．01)，DNA合成速率减慢65．77％(P＜0．01)，细胞周期阻滞于G0／G1期，克隆形成率抑制64．49％(P＜0．01)，凋亡率为22．00％(P＜0．01)。结论 反义PCNA与p16共转染具有抑制增殖、诱导凋亡的双重作用，有望成为膀胱癌联合基因治疗的新途径。     

bcl-2基因表达在人膀胱癌多药耐药现象中的意义

目的探讨ｂｃｌ２基因在人膀胱癌多药耐药中的作用。方法应用原位ＤＮＡ末端转移酶标记法（ＴＵＮＥＬ）、免疫细胞化学及逆转录多聚酶链式反应（ＲＴＰＣＲ）技术,分别对阿霉素诱导的人膀胱癌耐药细胞株（ＢＩＵ８７／ＡＤＭ）及敏感细胞株（ＢＩＵ８７）的细胞凋亡及其ｂｃｌ２基因表达进行研究。结果（１）在相同条件下,与敏感细胞株相比,耐药细胞株中细胞凋亡明显受到抑制;（２）耐药细胞株中ｂｃｌ２基因表达明显强于敏感细胞株。结论凋亡抑制基因ｂｃｌ２在人膀胱癌多药耐药细胞中的高表达,是导致其细胞凋亡受抑制的重要原因,对于人膀胱癌多药耐药的产生具有重要意义。     

反义人端粒酶逆转录酶基因转染对人胃癌细胞系的影响

目的探讨反义人端粒酶(hTERT)基因治疗的可行性。方法构建hTERT基因的反义表达载体,经脂质体介导转染人未分化胃癌细胞系HGC-27,通过Southernblot检测外源反义基因的整合;RT-PCR及DNA测序法检测反义基因的转录;RT-PCR半定量方法检测被封闭目的基因mRNA的转录水平;TRAP及PCRELISA方法检测细胞的端粒酶活性;流式细胞仪检测细胞周期变化。结果外源反义hTERT基因已整合入细胞并获稳定转录,且能显著封闭目的基因转录的mRNA,并显著抑制HGC-27细胞的端粒酶活性,抑制HGC-27细胞的增殖并促进其凋亡。结论端粒酶反义hTERT基因可有效地应用于胃癌的基因治疗。     

PCNA反义寡核苷酸抑制人肝癌细胞体外增殖的研究

目的 观察增殖细胞核抗原（PCNA）反义寡核苷酸对人肝癌细胞株体外增殖的影响及生物学特性的改变，方法 MTT法，集落形成试验观察PCNA反义寡核苷酸对人肝癌细胞的生长抑制，免疫组化技术检测PCNA的蛋白表达，流式细胞仪分析细胞周期变化。结果 PCNA反义核酸作用后的肝癌细胞生长明显受到抑制，抑制效应于作用后48h最为明显，并于72h后逐渐减弱，集落形成率明显下降，PCNA蛋白表达明显下降，细胞周期中S期细胞数明显减少。结论 PCNA反义寡核苷酸能显著抑制人肝癌细胞的生长。     

反义增殖细胞核抗原cDNA对人膀胱癌裸鼠异体移植瘤生长的抑制作用

目的　探讨增殖细胞核抗原 (PCNA)反义cDNA对人膀胱癌裸鼠移植瘤模型生长的影响。　方法　脂质体介导反义PCNA真核表达载体转染人膀胱癌EJ细胞后 ,接种到裸鼠皮下组织 ,观察体内致瘤及生长情况 ;免疫组化检测瘤体PCNA蛋白表达 ,RT PCR分析瘤体PCNA、wt p5 3、c myc基因mRNA水平 ;并进行瘤体病理学评估。 　结果　反义PCNAcDNA导入后 ,EJ细胞体内致瘤活性降低 ,同对照组比较瘤体体积缩小 5 4 .2 3% (P <0 .0 5 ) ,重量减轻 4 2 .5 4 % (P <0 .0 1) ,瘤组织PCNA蛋白及mRNA水平分别减少 6 1.6 2 % (P <0 .0 1)和 72 .13% (P <0 .0 1) ,c myc基因表达下调4 7.34% (P <0 .0 1) ,wt p5 3表达活性无显著性改变 (P >0 .0 5 ) ,瘤体病理学特征改善。 　结论　转导反义PCNAcDNA能有效逆转肿瘤的恶性表型 ,是膀胱癌基因治疗的合理策略之一     

反义人类端粒酶 RNA逆转录病毒载体构建及其对结直肠癌细胞的抑制作用

目的 研究反义人类端粒酶RNA逆转录病毒载体对结直肠癌HT29细胞端粒酶活性及细胞生长的抑制作用。探讨以端粒酶为酸点的结直肠癌基因治疗的可能性。方法 将端粒酶hTR的cDNA反向插入逆转录病毒载体pLXRN中，转染包装细胞PT67后获得反义重组病毒，感染结直肠癌HT29细胞，采用RT-PCR检测hTR表达。端粒酶重复扩增法（telomerase repeat amplification protocol,TRAP)检测端粒酶活性，绘制生长曲线了解细胞生长状态，倒置显微镜观察和DNA片段电泳检测细胞凋亡。结果 反义hTR作用后的结直肠癌细胞hTR表达下降，端粒酶活性和细胞生长受到明显抑制，细胞出现凋亡。结论 反义hTR对结直肠癌HT29细胞的生长和端粒酶活性具有明显的抑制人舰艇可能以hTR为靶点对结直肠癌进行基因治疗。     

去甲斑蝥素对人原发性胆囊癌GBC-SD细胞系增殖及相关基因的干预效应

目的　研究去甲斑蝥素 (NCTD)对人原发性胆囊癌GBC SD细胞系增殖及相关基因的干预效应。方法　实验分NCTD组 (7个浓度梯度组 ,每组 6孔 )和对照组 (n =6) ,分别应用四唑盐比色法和链霉素亲合生物素复合物法测定NCTD对体外培养GBC SD细胞的体外杀伤效应和对增殖细胞核抗原 (PCNA)、Ki 67表达的影响。结果　NCTD分别在浓度 10mg/L、时间 6h时 ,即对GBC SD细胞增殖有抑制作用 ,且随浓度升高、时间延长作用增强 ,呈剂量 时间效应关系 ;实验组PCNA和Ki 67表达的棕褐染色细胞数和阳性指数 (PCNA :0 .93 2± 0 .0 3 1vs 0 .3 18± 0 .0 2 3 ;Ki 67:0 .964± 0 .0 92vs 0 .2 97± 0 .0 18;n =10 )与对照组比较均显著减少 (P <0 .0 5 )。结论　NCTD能抑制人原发性胆囊癌GBC SD细胞的生长和PCNA、Ki 67增殖相关基因蛋白的表达。     

Abrogation of heat-shock protein （HSP）70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m

Aim: To investigate the effect of abrogating heat shock protein (HSP) 70 expression by antisense HSP70 oligonucleotides treatment on human androgen-independent prostate cancer cell line PC-3m growth. Methods: PC3m cells were treated with 0-16μmol/L antisense HSP70 oligomers for 0-100 hr. Cell growth inhibition was analyzed using a trypan blue dye exclusion test. Apoptotic cells were detected and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression of HSP70 and bcl-2 affected by antisense HSP70 oligomers were determined using Western blot. Results: Antisense HSP70 oligomer induced apoptosis and then inhibited proliferation of PC-3m cells in a dose- and time-dependent manner. Ladder-like patterns of DNA fragments were observed in PC-3m cells treated with 10μmol/L antisense HSP70 oligomer for 48 hr or 8μtmol/L for 72 hr on agarose gel electrophoresis. Antisense HSP70 oligomer pretreatment enhanced the subsequent induction of apoptosis by heat shock in PC-3m cells. In addition, undetectable HSP70 expression was observed at a concentration of 10μtmol/L antisense HSP70 oligomer treatment for 48 hr or 8μtmol/L for 72 hr in Western blot, which was paralleled by decreased expression levels of anti-apoptotic protein bcl-2. Conclusion: HSP70 antisense oligomer treatment abro-gates the expression of HSP70, which may disrupt HSP70-bcl-2-interactions and further down-regulate bcl-2 expression,in turn inducing apoptosis and inhibiting cell growth in PC-3m cells. (Asian JAndro12004 Dec;6:319-324)     

NF-Kappa-B downregulation strategies in head and neck cancer treatment.

OBJECTIVE: Multiple biochemical and genetic strategies were used to downregulate early response gene NF-KappaB, whose activation controls squamous cell cancer-associated pathways. STUDY DESIGN: NA cells, an oral cavity squamous cell cancer with high NF-KappaB activity, were cultured with biochemical NF-KappaB inhibitors TPCK and Calpain I inhibitor, as well as specific NF-KappaB antisense oligonucleotides. Cell proliferation was measured, as was NF-KappaB downregulation using functional luciferase reporter genes and electromobility shift assays. RESULTS: Significant downregulation of cell proliferation and NF-KappaB functional activity were demonstrated with either biochemical inhibitor, as well as the antisense oligonucleotides; however, additional nonspecific toxicities were observed with control antisense oligonucleotides. CONCLUSION AND SIGNIFICANCE: NF-KappaB is a potential target for squamous cancer treatment, as it is constitutively upregulated in vitro. Biochemical inhibition of NF-KappaB may be a viable treatment strategy for head and neck squamous cancers.     

核因子-кB/p65反义寡核苷酸对胃癌细胞增殖和凋亡的影响及其机制

目的 观察核因子(NF)-кB/p65反义寡核苷酸(ASODN)对胃癌细胞增殖和凋亡的影响.方法 设计合成NF-кB/p65 ASODN,按照0.05、0.1、0.2.0.3、0.4、0.5 μmol/L不同浓度转染胃癌SGC-7901细胞株,设正义寡核苷酸(SODN)、错义寡核苷酸(MSODN)和空白对照组进行比较.利用荧光染色观察转染效果,噻唑蓝(M1T)比色法检测细胞生长抑制率(IR),流式细胞术(FCM)检测细胞凋亡情况.结果 与SODN、MSODN及对照组比较,ASODN组细胞增殖明显受到抑制(P<0.05),72 hIR为58.3%,且这种抑制作用呈浓度依赖性.FCM结果显示ASODN可诱导细胞的凋亡.结论 NF-кB ASODN能抑制胃癌细胞生长,其机制与ASODN诱导的细胞凋亡有关,NF-кB可作为胃癌治疗的新靶点.     

Antiproliferative effects of c-myc antisense oligonucleotide in prostate cancer cells: A novel therapy in prostate cancer

Objectives

To explore the possibility of using antisense oligonucleotide therapy for prostate cancer, we investigated the effect of c-myc-antisense-oligonucleotide (c-myc-As-ODN) in human prostate cancer cell lines such as LNCaP, PC3, and DU 145.

Methods

LNCaP, PC3, and DU145 cells were incubated in the presence of c-myc-As-ODN. Dose (0 to 10 μM) and time dependent (1 to 6 days) effects on proliferation and viability were examined by [³H]thymidine incorporation and MTT assay, respectively. Flow cytometry analysis was carried out to analyze cell cycle status by determining the DNA content in LNCaP cells. Control cultures received either c-myc-sense-ODN or scrambled (nonsense) nucleotides.

Results

Time- and dose-dependent decreases in DNA synthesis and cell viability were noted for all three prostate cancer cell lines after c-myc-As-ODN treatment. Further studies using LNCaP cells indicated that these changes were accompanied by an increase in the percentage of cells with less than 2N DNA content after c-myc-As-ODN treatment. The results suggest that c-myc-As-ODN induces cell death. Comparison of a c-myc-As-ODN-treated group with a group subjected to isoleucine deprivation revealed that thymidine incorporation was almost the same in c-myc-As-ODN-treated LNCaP cells and in LNCaP cells at early S phase.

Conclusions

These results suggest that c-myc-As-ODN inhibits prostate cancer cell growth and proliferation mainly by decreasing cell viability.     

INI1基因对胃癌细胞增殖活性的影响及其机制

目的 通过改变INI1基因表达水平探讨INI1对胃癌细胞增殖活性影响的分子机制.方法 荧光定量逆转录-聚合酶链反应(RT-PCR)及Western blot方法检测15例胃癌及癌旁组织INI1 基因mRNA和蛋白质的表达水平;将INI1-GFP真核表达质粒及INI1特异性小干扰RNA(INI1-siRNA)分别转染人胃癌SGC7901细胞株,Western blot方法检测过表达及下调表达INI1的效率;噻唑蓝(MTT)比色法检测过表达及下调表达INI1后SGC7901细胞的增殖活性的改变,荧光定量RT-PCR及Western blot方法检测增殖细胞核抗原(PCNA)表达改变.结果 临床胃癌组织中INI1蛋白及mRNA表达水平明显低于癌旁组织(P＜0.01);INI1-GFP转染S1GC7901细胞可明显增加INI1表达水平,同时降低细胞增殖活性(P值均＜0.01);而INI1-siRNA转染可明显降低INI1表达水平,同时增加SGC7901细胞增殖活性(P值均＜0.01);上调INI1表达可抑制PCNA表达水平,而下调INI1表达则增加PCNA的表达(P值均＜0.01).结论 INI1具有抑制胃癌细胞增殖的作用,此作用与其负向调节PCNA表达有关.     

Effects of selected chemotherapeutic agents on PCNA expression in prostate carcinoma cell lines

Bivariate flow cytometric analysis of proliferating cell nuclear antigen (PCNA) was performed on prostate carcinoma cell lines (PC-3, DU-145). For both cell lines 100% methanol fixation provided optimal fluorescence intensity of PCNA. The ratio of PCNA/DNA increased in late G1 through early S/phase, followed by a decrease in mid- and late S and enhancement in G2/M phase. PCNA expression was increased in G2/M phase cells treated for 48 h with vinblastine. A slight decrease in PCNA expression was observed with cyclohexamide treatment. Hydroxyurea induced an increase in S-phase fraction along with enhanced PCNA expression. Methotrexate and Adriamycin had little effect on the cell cycle compartments of PC-3 or DU-145; however, methotrexate decreased PCNA expression, while Adriamycin enhanced it. Cisplatin increased S-phase in both cell lines, increasing PCNA expression in PC-3 and decreasing it in DU-145 cells. The data on the effects of drug treatment point to a dissociation between PCNA expression and S-phase fraction as calculated from the DNA distribution. In some cases, e.g., the cisplatin studies, different effects were obtained in the two different cell lines treated with the same drugs. Whether changes in PCNA expression will provide more useful information than S-phase fraction for evaluation of potential antitumor drugs is not known.Supported by NIH grants Ro-1-CA 14134     

抑制热休克蛋白70表达对Eca-109细胞生长的影响

目的：观察热休克蛋白70（HSP 70）反义寡核苷酸阻断食管癌Eca-109细胞的HSP 70表达及对细胞增殖和凋亡的影响。方法：应用逆转录聚合酶链反应（RT-PCR）和Western blot技术检测HSP 70反义寡核苷酸(10μmol/L)转染Eca-109细胞后, 其HSP 70基因mRNA和蛋白表达的变化。观察HSP 70反义寡核苷酸转染对肿瘤细胞的生长抑制效应及转染后细胞凋亡率和细胞周期分布的改变。结果：HSP 70反义寡核苷酸可阻断Eca-109细胞HSP 70 mRNA和蛋白的表达;转染后48h和72h，生长抑制率分别为25.5%和35.4%;HSP 70反义寡核苷酸组细胞凋亡率明显高于HSP 70正义寡核苷酸组和未转染组（P<0.05）。结论： HSP 70反义寡核苷酸能抑制Eca-109细胞的生长并诱导其发生凋亡。     

PCNA、VEGF反义寡核苷酸联合治疗裸鼠肝癌的研究

目的 探讨增殖细胞核抗原（PCNA）反义寡核苷酸和血管内皮生长因子（VEGF）反义寡核苷酸单用或联用对裸鼠人肝癌模型肝癌生长的抑制作用。方法 制作裸鼠皮下肝癌模型24只,分PCNA反义核酸治疗组、VEGF反义核酸治疗组、联合治疗组及对照组,接种后24h之内用PCNA反义核酸和（或）VEGF反义核酸皮下注射进行治疗。观察裸鼠瘤体变化及组织形态学改变。结果 PCNA反义核酸治疗组,VEGF反义核酸治疗组和联合治疗组裸鼠肝癌的生长均受到不同程度的抑制,抑瘤率分别为72．8％、44．9％和87．2％。以联合治疗组的疗效最佳。结论 联合应用PCNA和VEGF2种反义核酸治疗肝癌比应用单种反义核酸有更显著的疗效,2种反义核酸具有协同作用。