肝细胞生长因子受体反义寡核苷酸影响胶质瘤细胞对丝裂霉素C敏感性的研究

目的探讨肝细胞生长因子受体(c-Met)反义寡核苷酸(ASODN)增强胶质瘤细胞系 U251细胞对丝裂霉素C(MMC)敏感性的作用。方法用5 μmol／L c-Met ASODN封闭U251细胞 c-Met mRNA,将50μg/L的MMC与其共培养,采用逆转录-聚合酶链反应(RT-PCR)技术检测c- Met mRNA表达,噻唑蓝(MTT)试验检测U251细胞的生长情况,免疫组织化学法检测细胞PCNA 蛋白的表达,体外检测细胞黏附率。以无义寡核苷酸处理及未处理U251细胞为对照。结果经 c-Met ASODN处理的U251细胞 ,c-Met mRNA的表达(吸光度值为62±21)明显低于经无义寡核苷酸处理U251细胞(吸光度值为150±25,P<0．05);对MMC的敏感性,细胞生长抑制率、细胞黏附率及PCNA指数分别为(53．84±12．21)％、(14．61±3．82)％和(0．35±0．02)％,明显高于相应的无义[(9．86±3．42)％、(24．84±5．90)％和(0．55±0．04)％,P<0．05]。结论 c-Met ASODN能增强胶质瘤细胞系U251细胞对MMC的敏感性,其分子机制可能与c-Met反义寡核苷酸下调了c- Met基因和PCNA蛋白的表达有关。     

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。     

脂质体介导的聚集素反义寡核苷酸对膀胱癌EJ细胞的作用

目的探讨clusterin对膀胱癌EJ细胞生物学特征的影响. 方法应用脂质体包裹的clusterin反义寡核苷酸转染EJ细胞,阻断其表达,通过3′端末端标记、MTT试验、伊红排斥实验等方法观察clusterin反义基因对EJ细胞增殖、凋亡、坏死等生物学行为的影响. 结果转染clusterin反义寡核苷酸组(反义组)EJ细胞凋亡增加,第5天达峰,凋亡率80%,与转染clusterin正义寡核苷酸组(正义组)比较,差异有显著性意义(P<0.05).反义组细胞死亡率增加,以转染后1～4 d明显(13.8%～33.3%),显著高于相应正义组(P<0.05).反义组细胞增殖活性降低,以转染后第6、7天明显,显著高于相应正义组(P<0.05). 结论 clusterin是重要抗凋亡因子,其表达与膀胱癌细胞的生存、增殖、抗凋亡发展密切相关.     

脂质体介导的端粒酶反义寡核苷酸体外转染率及对人膀胱癌EJ细胞的影响

目的通过以脂质体为载体介导端粒酶反义寡核苷酸(antisense oligodeoxynucleotide,asODN)转染人膀胱癌EJ细胞,促进反义寡核苷酸对膀胱癌EJ细胞的生长抑制。方法利用脂质体为载体将针对端粒酶RNA模板区的asODN转染膀胱癌EJ细胞;荧光显微镜观察细胞转染率;PCR-ELISA法测定端粒酶活性;MTT法检测反义寡核苷酸对膀胱癌细胞的抑制。结果脂质体介导的asODN在膀胱癌EJ细胞中的转染率1/2h、4h、8h分别为15%、56%、80%,并且细胞的端粒酶活性和细胞生长明显被抑制,与对照组比较,具有显著性差异(p(0.05)。结论脂质体介导的端粒酶asODN能够有效抑制膀胱癌EJ细胞端粒酶活性和生长,可应用于实验性肿瘤基因治疗研究和端粒酶与膀胱癌关系的进一步研究。     

半胱氨酸蛋白酶32在丝裂霉素C诱导膀胱癌细胞凋亡中的作用

目的　探讨半胱氨酸蛋白酶 32 (caspase 3)在丝裂霉素C(MMC)诱导膀胱癌EJ细胞凋亡中的作用。　方法　采用末端脱氧核苷酸转移酶介导生物素标记法 (TUNEL)和流式细胞术 (FCM)研究EJ细胞凋亡及细胞周期变化 ,分析caspase 3抗体对低剂量MMC诱导EJ细胞凋亡的影响。　结果　TUNEL法显示MMC组EJ细胞出现细胞凋亡特有的形态学特征 ,凋亡指数 (6 2 .9± 2 .2 ) % ,较cas pase 3抗体加MMC组 (4.9± 0 .3) %和空白组 (2 .7± 0 .7) %显著增高 (P <0 .0 0 1)。FCM检测结果明 ,MMC主要诱导G0 /G1期细胞凋亡而出现凋亡峰 ,凋亡率 2 6 .0 6 % ;caspase 3抗体能特异性抑制MMC诱导EJ细胞凋亡 ,凋亡率仅为 4.6 5 %。　结论　低剂量MMC对caspase 3的活化在诱导膀胱癌细胞凋亡中有重要作用。     

阻滞真核细胞起始因子-4E对膀胱癌细胞乙酰肝素酶表达的影响

目的 探讨膀胱癌细胞真核细胞起始因子-4E( eIF-4E)与乙酰肝素酶表达的关系.方法 脂质体包裹eIF-4E于mRNA翻译起始点互补的反义寡核苷酸(asODN)及相应对照的正义寡核苷酸(sODN)以100、200 pmol/L两种浓度分别转染膀胱癌EJ细胞,逆转录-聚合酶链反应(RT-PCR)检测eIF-4E转录水平的改变.荧光定量PCR和Western blot分别检测乙酰肝素酶mRNA及蛋白表达.结果 asODN转染显著抑制eIF-4E基因水平.eIF-4E被阻滞后,乙酰肝素酶mRNA水平及蛋白表达量:asODN1组(0.276±0.050、0.255±0.028)及asODN2组(0.195±0.022、0.160±0.032),均显著低于空白对照组(0.896±0.057、0.603±0.029)及sODN1组(0.867±0.099、0.572±0.029)和sODN2组(0.879±0.087、0.560±0.035,P＜0.01).结论 asODN转染膀胱癌EJ细胞可以阻滞eIF-4E基因表达,阻滞eIF-4E后,膀胱癌乙酰肝素酶mRNA水平及蛋白表达均明显降低.     

热休克蛋白70 mRNA在膀胱癌中的表达及与细胞凋亡的关系

目的　探讨热休克蛋白 70 (HSP 70 )mRNA在膀胱移行细胞癌中的表达及与细胞凋亡的关系。　方法　应用原位杂交法对 6 0例膀胱癌组织中HSP 70mRNA进行检测 ,原位DNA末端标记法 (TUNEL)检测细胞凋亡情况。　结果　 6 0例标本中 ,HSP 70mRNA阳性表达率为 5 6 .7% (34/ 6 0 )。HSP 70mRNA表达随肿瘤分级增高而增加 (P <0 .0 5 ) ;G1,G2 和G3 各级间两两比较 ,HSP 70mRNA表达差异也有显著性意义 (P <0 .0 5 )。随着肿瘤分期增高 ,HSP 70mRNA表达随之增加 (P <0 .0 5 ) ;Ⅰ ,Ⅱ和Ⅲ各期间两两比较 ,差异有显著性意义 (P <0 .0 5 )。肿瘤细胞凋亡指数 (AIs)随恶性程度的增高显著降低 (P <0 .0 5 ) ;HSP 70mRNA表达率和AIs呈负相关 (rs=- 0 .6 85 ,P <0 .0 5 )。　结论　HSP 70mRNA表达随膀胱癌恶性程度增加显著增高 ,细胞凋亡则显著降低 ,提示HSP 70表达能够抑制膀胱癌细胞的凋亡。     

丝裂霉素对膀胱癌EJ细胞作用机制的研究

目的　从诱导坏死和凋亡角度探讨丝裂霉素 (MMC)对膀胱癌EJ细胞的作用机制。　方法　将不同浓度的MMC作用于膀胱癌EJ细胞 ,在不同时间内采用流式细胞术 (FCM )和 3’末端脱氧核苷酸转移介导生物素 (TUNEL)及伊红排斥实验检测EJ细胞的凋亡情况和对其的杀伤作用。　结果　不同浓度的MMC在 2 4h内可以持续诱导EJ细胞凋亡 ,MMC 0 .1mg/ml组 2 4h时凋亡率为 71% ,对EJ细胞的杀伤作用与剂量呈正比。　结论　MMC对膀胱癌EJ细胞的抑制作用是通过促凋亡机制和促坏死机制完成的     

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     

ets-l反义寡核苷酸对胃癌细胞株SGC-7901体外侵袭力的影响

目的 :研究ets l反义寡核苷酸对胃癌细胞株SGC 790 1体外侵袭力的影响。方法 :应用ets l反义、正义寡核苷酸转染SGC 790 1细胞 ,并设PBS对照组。转染后应用逆转录聚合酶链式反应检测ets lmRNA的变化 ,应用克隆形成试验检测细胞增殖活性的变化 ,Transwell小室检测侵袭能力的变化。结果 :转染ets l反义寡核苷酸后 ,ets lmRNA表达降低 ,反义组形成克隆数目 (2 4 2± 4 8)较正义组、对照组减少 (47 6± 8 1,4 4 3± 7 6 ,P <0 0 1) ,侵过小室滤膜细胞数目减少 (97 1± 11 1,198 7± 18 3,2 0 5 8± 2 2 2 ,P <0 0 1)。结论 :ets l反义寡核苷酸能够抑制胃癌细胞株SGC 790 1的体外侵袭力。     

Vascular endothelial growth factor antisense pretreatment of bladder cancer cells significantly enhances the cytotoxicity of mitomycin C, gemcitabine and Cisplatin

PURPOSE: Due to unsatisfactory success in the treatment of local and systemic bladder cancer and the low response rates to commonly used chemotherapy (CT) alternative and additive approaches must be found. The function of vascular endothelial growth factor (VEGF) in neo-angiogenesis and, therefore, in solid tumors makes it a promising target for a specific antitumor therapy. We investigated the possibility of sensitizing transitional bladder cancer cell lines to CT by pretreatment with VEGF antisense (AS) oligodeoxynucleotides (AS-ODNs). MATERIALS AND METHODS: The human bladder cancer cell lines EJ28 and 5637 were transiently transfected with 3 antiVEGF AS-ODNs, followed by incubation with 3 doses of mitomycin C, gemcitabine or cisplatin CT. WST-1 (a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay (Roche, Mannheim, Germany) was performed to assess effects on cell viability. Apoptosis was examined by Annexin V staining. In all experiments a nonsense ODN served as a control. RESULTS: Each cell line responded in a dose dependent manner to all CTs. Combined treatment with VEGF AS-ODNs and CT resulted in decreased viability compared with isolated CT. VEGF857 plus CT significantly decreased the viability of the 2 cell lines compared with nonsense ODN plus CT for all 3 CT agents (p <0.007). This detected chemosensitization was based on an AS mediated increase in apoptosis. CONCLUSIONS: One of the 3 AS-ODNs tested (VEGF857) significantly sensitizes human transitional cell carcinoma cells to CT. We suggest VEGF as an additional putative target to enhance the therapeutic benefit of, for example mitomycin C and gemcitabine instillation treatment schedules.     

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)     

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。     

HIPK3在膀胱癌细胞中的表达及其意义

目的：探讨HIPK3在膀胱癌发生、发展中的作用及其意义。方法：培养膀胱癌细胞系EJ,以丝裂霉素诱导细胞凋亡,利用细胞爬片免疫组化的方法测定HIPK3在被诱导凋亡的EJ细胞中的表达情况,并进一步经由流式细胞仪研究HIPK3抗体对细胞凋亡的影响。结果：被诱导凋亡的EJ细胞中,HIPK3表达呈阳性,其阳性率随着诱导凋亡的时间增加,但不同剂量的丝裂霉素作用,其表达没有统计学意义;HIPK3抗体可对抗丝裂霉素诱导的细胞凋亡作用,减少凋亡的EJ细胞。结论：HIPK3在被诱导凋亡的膀胱癌细胞系EJ中表达增加,揭示膀胱癌细胞凋亡过程同HIPK3有密切关系。     

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。     

低剂量丝裂霉素短时加药诱导膀胱癌细胞凋亡的研究

目的探讨低剂量丝裂霉素（MMC）短时处理,诱导膀胱癌细胞凋亡的可能性。方法应用细胞和分子生物学技术检测各MMC处理条件对靶细胞生长和凋亡的影响。结果低剂量MMC处理EJ细胞1小时或2小时,均出现典型凋亡特征．其中,100mg／L,和20mg／L,MMC对凋亡的诱导作用最佳且处理时间的长短,对抑癌结果无明显影响。结论以一半的临床MMC用药剂量和时间即可有效诱导膀胱癌EJ细胞凋亡,为拟定更合理的膀胱癌腔内化疗方案提供可靠的实验佐证。     

The effect of antisense Bcl-2 oligonucleotides on Bcl-2 protein expression and apoptosis in human bladder transitional cell carcinoma

PURPOSE: Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model. RESULTS: Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death. CONCLUSIONS: Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.