以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性(英文)

目的以 hTERT 反义核酸抑制白血病细胞(HL-60和 K562)端粒酶活性,研究 CDDP 诱导凋亡敏感性的变化。方法全硫代反义核酸由上海生物化学研究所合成和纯化;端粒酶活性用试剂合测定(宝灵曼公司产品);用形态学方法和流式细胞仪检测细胞调亡。结果实验结果显示,hTERT 全硫代反义核酸,通过下调 hTERT 基因表达,显著地抑制端粒酶活性;端粒酶活性下降以后,白血病细胞对 CDDP 诱导调亡的敏感性显著升高。结论以 hTERT 基因反义核酸抑制端粒酶活性增加白血病细胞对 CDDP 诱导凋亡的敏感性。     

以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性

目的 以hTERT反义核酸抑制白血病细胞(HL-60和K562)端粒酶活性,研究CDDP诱导凋亡敏感性的变化。方法 全硫代反义核酸由上海生物化学研究所合成和纯化;端粒酶活性用试剂合测定(宝灵曼公司产品);用形态学方法和流式细胞仪检测细胞调亡。结果实验结果显示,hTERT全硫代反义核酸,通过下调hTERT基因表达,显著地抑制端粒酶活性;端粒酶活性下降以后,白血病细胞对CDDP诱导调亡的敏感性显著升高。结论 以hTERT基因反义核酸抑制端粒酶活性增加白血病细胞对CDDP诱导凋亡的敏感性。     

反义端粒酶(hTR)基因对人肝癌细胞系生物学行为的影响

背景与目的:探讨反义端粒酶RNA(hTR)对人肝癌细胞端粒酶活性的影响.材料与方法:构建反义hTR基因逆转录病毒载体,脂质体介导转染人肝癌SMMC-7721细胞,通过Southern blot、TRAP-PCR、流式细胞术检测hTR表达及端粒酶活性变化.结果:经G418筛选,转染细胞形成稳定克隆,反义hTR表达增强,细胞生长受到抑制、倍增时间延长,出现凋亡.结论:反义hTR基因表达明显抑制肝癌细胞的生长,降低肿瘤细胞的恶性度和异型性.hTR基因是肝癌基因治疗良好的靶点.     

Expression of Antisense Human Telomerase RNA Gene in SMMC-7721Cells

背景与目的 :探讨反义端粒酶RNA(hTR)对人肝癌细胞端粒酶活性的影响。材料与方法 :构建反义hTR基因逆转录病毒载体 ,脂质体介导转染人肝癌SMMC_7721细胞 ,通过Southernblot、TRAP_PCR、流式细胞术检测hTR表达及端粒酶活性变化。结果 :经G418筛选 ,转染细胞形成稳定克隆 ,反义hTR表达增强 ,细胞生长受到抑制、倍增时间延长 ,出现凋亡。 结论 :反义hTR基因表达明显抑制肝癌细胞的生长 ,降低肿瘤细胞的恶性度和异型性。hTR基因是肝癌基因治疗良好的靶点     

反义hTERT对K562细胞端粒酶活性及细胞增生的影响

目的：观察反义hTERT表达质粒转染K562细胞后是否能够抑制端粒酶活性及K562细胞的增生。方法：质粒的构建与转染；将200bphTERTcDNA片段，反向连接于pLNCX-neo质粒上得到反义hTERT基因表达质粒，在体外转染中通过脂质体法将质粒导入K562细胞中，以G418进行筛选得到阳性克隆；以MTT法和形态学法检测反义hTERT表达质粒对K562细胞增生的抑制作用；以TRAP-PCRELISA法来研究反义hTERT基因对K562细胞的端粒酶活性的抑制作用。结果：转染反义hTERT表达质粒细胞与对照组（空白对照及空质粒组）相比较。生长速率明显变慢。部分细胞出现凋亡等形态学改变。反义hTERT对K562细胞的端粒酶活性具有明显的抑制作用。结论：研究构建的端粒酶反义hTERT基因表达质粒转染到K562细胞中后，能够封闭K562细胞hTERT-mRNA的表达，在抑制端粒酶活性的同时，减慢了K562细胞的生长速度。     

反义 hTERT 基因对 K562 细胞凋亡及周期的影响简

目的：探讨反义hTERT基因抑制K562细胞端粒酶活性对其细胞周期及增殖的影响。方法：用脂质体介导反义hTERT基因和空载体质粒转染K562细胞株,绘制生长曲线,MTT法检测细胞活性,血清饥饿法同步化细胞周期,流式细胞仪检测细胞周期及细胞凋亡率变化。结果：反义hTERT基因转染后,细胞生长明显受到抑制。细胞同步化后,流式细胞仪检测显示：KA组细胞出现早期凋亡峰,细胞阻滞于S期。但正常培养48h,各组细胞无明显凋亡,周期无显著差异。结论：反义hTERT基因可明显抑制K562的增殖,细胞同步化后具有促凋亡作用。     

抑制端粒酶活性对人肾癌细胞生长的抑制作用

目的 :探讨抗端粒酶在肾细胞癌治疗中的意义。方法 :采用脂质体介导法将pBBS2 12 hTR(反义端粒酶RNA真核表达载体 )导入人肾癌GRC 1细胞系中 ,采用TRAP法测定端粒酶活性 ,通过MTT法检测细胞动力学 ,观察细胞的增殖 ,电镜下观察其对细胞凋亡的影响。结果 :端粒酶反义RNA能显著抑制细胞的端粒酶活性 ,抑制细胞增殖并促进其凋亡。结论 :以端粒酶反义RNA抑制端粒酶活性是治疗肾癌的潜在途径     

反义寡核苷酸对K562细胞增殖和端粒酶活性的影响

 目的　研究靶向封闭端粒酶反转录酶（hTERT）mRNA的反义硫代寡核苷酸（ASPSODN）对K562细胞目的基因的抑制及其对端粒酶活性及细胞增殖周期和凋亡的影响 。方法　ASPSODN转染到人类红白血病细胞株K562,采用MTT法、酶联免疫吸附（ELISA）法和流式细胞术（FCM）检测K562细胞的增殖、端粒酶活性、细胞凋亡和细胞周期的改变,RT-PCR检测hTERT mRNA的表达。结果　0.6 μmol／L的ASPSODN（0.42±0.16）能明显下调hTERT表达（P＜0.05）,端粒酶相对活性降至52 %;MTT法检测显示明显抑制K562细胞增殖活性;FCM显示细胞凋亡率为10.31 %,PI染色显示细胞被阻止在G1／G0期,S期及G2／M期的细胞减少,但无特征性的凋亡峰。结论　ASPSODN靶向 hTERT 能特异性抑制K562细胞hTERT mRNA的表达,明显下调端粒酶活性,抑制K562细胞增殖,并通过降低细胞的端粒酶活性而诱发细胞凋亡。     

端粒酶反义RNA 对人喉癌细胞系Hep-2 生长的影晌

 目的　探讨抗端粒酶在喉癌细胞治疗中的意义。方法　用脂质体介导法将 pBBS2 12 hTR(反义端粒酶RNA真核表达载体 )转染入人喉癌Hep 2细胞系中 ,采用TRAP法测定端粒酶活性 ,细胞动力学检测 ,电镜下观察等方法研究其对Hep 2细胞的端粒酶活性、细胞增殖及细胞凋亡影响。 结果　端粒酶反义RNA能显著抑制细胞的端粒酶活性 ,抑制细胞增殖并促进其凋亡。结论　以端粒酶反义RNA抑制端粒酶活性是治疗喉癌的潜在途径。     

高三尖杉酯碱抑制K562细胞端粒酶活性的实验研究

目的研究高三尖杉酯碱(HHT)对K562细胞端粒酶活性的抑制作用.方法采用端粒重复序列扩增法(TRAP)-酶联免疫吸附试验(ELISA)和聚丙烯酰胺凝胶电泳(PAGE)-银染法检测了K562细胞的端粒酶活性变化.结果 0.01～5.00μg/ml HHT处理K562细胞3～48小时,K562细胞端粒酶活性由1.203±0.078下降至0.054±0.017,并呈剂量依赖性和时间依赖性.结论 HHT能有效抑制K562细胞的端粒酶活性.     

2-5A antisense therapy directed against human telomerase RNA inhibits telomerase activity and induces apoptosis without telomere impairment in cervical cancer cells

Human telomerase RNA (hTR), an important component of telomerase, is a possible target of telomerase-based cancer gene therapy. The present study was undertaken to assess the efficacy of antisense hTR therapy using newly developed 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked oligonucleotides against cervical cancer cells. ME180 and SiHa cells were treated with 2-5A-linked antisense hTR designed to complement the region of hTR between residues 76 and 94. The hTR expression, telomerase activity, cell viability, and apoptosis were then examined. The 2-5A anti-hTR effectively degraded hTR and inhibited telomerase activity. The 2-5A mutant anti-hTR and the anti-hTR without 2-5A were not capable of inhibiting telomerase activity. Inhibition of telomerase by 2-5A anti-hTR rapidly decreased cell viability only in telomerase-positive cells within 3-6 days after the treatment, when telomere length has not yet been shortened. This inhibition was associated with apoptosis, possibly through activation of caspase family members. These findings suggest that 2-5A-linked antisense-hTR therapy has a potent telomerase-inhibitory effect associated with a cytocidal effect from caspase-induced apoptosis, and may therefore be a potential tool in telomerase-based gene therapy against cervical cancers.     

Delivering antisense telomerase RNA by a hybrid adenovirus/ adeno-associated virus significantly suppresses the malignant phenotype and enhances cell apoptosis of human breast cancer cells

Activated telomerase is frequently detected in cancer cells and is able to maintain and stabilize the integrity of telomeres; it also contributes to unlimited divisions in cancer cells. Recently, a new generation of selective anticancer strategies is under development targeting the blockage of telomerase activity either at the protein level or telomerase RNA. Here, we report suppression of the malignant phenotype by the expression of the full-length antisense human telomerase RNA (hTR) delivered by a novel hybrid vector recombining adenovirus and adeno-associated virus (vAd-AAV). The hybrid vector vAd-AAV retained the unique traits from two parental viruses, such as high efficiency of gene transfer in mammalian cells and the ability to integrate into the genomic DNA of host cells. The stable expression of antisense hTR in MCF-7 cells significantly suppressed telomerase activity and progressively shortened telomere length for 30 population doublings (PD30). Expression of antisense hTR leads to a telomere-based growth arrest and the induction of spontaneous apoptosis. Antisense hTR decreased soft agar colony formation and reduced the cell proliferation, leading to exit from the cell cycle at G1 at PD15. The expression of antisense hTR also sensitized MCF-7 cells to apoptosis induced by sodium butyrate or serum starvation. Our study demonstrates that delivering antisense hTR by the hybrid Ad/AAV vector is an effective antineoplastic gene therapeutic strategy, which significantly suppresses the malignant phenotype and enhances apoptosis of human breast cancer cells.     

Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA (siRNA) has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA (hTR) can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells (HRCCs). METHODS The siRNA duplexes for hTR were synthesized and 786-0 HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined. RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally, significant inhibition of proliferation and induction of apoptosis were observed. CONCLUSION siRNA against the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.     

端粒酶反义核酸对BEL-7402肝癌细胞生长和活性的影响

目的 研究人类端粒酶RNA（hTR)基因的反义寡核苷酸（AS－ODN）对BEL－7402肝癌细胞生长和活性的影响。方法 将AS－ODN作用于BEL－7402细胞，观察细胞生长状况，PCR－ELISA法检测端粒酶活性，流式细胞仪和细胞DNA电泳观察细胞的凋亡情况。结果 AS－ODN能抑制BEL－7402细胞的生长，降低其端粒酶活性并诱导细胞凋亡。结论 针对hTR的AS－ODN对治疗肝癌有潜在意义。     

Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR

Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.     

Apolloon反义寡核苷酸对K562细胞增殖和凋亡的影响

目的探讨Apollon反义寡核苷酸（antisense oligonucleotide,ASODN）对白血病细胞K562增殖及凋亡的影响。方法设计合成特异性的Apollon硫代磷酸ASODN及其对照错义寡核苷酸（missense oligonucleotide,MSODN）,脂质体介导转染K562细胞后,采用MTT法检测K562细胞增殖,Annexin V-FITC法检测细胞的凋亡。结果Apollon反义寡核苷酸对K562细胞的生长抑制呈浓度-时间依赖性。Apollon ASODN在终浓度为600 nmol/L作用K562细胞时,能明显抑制其增殖,K562细胞凋亡率显著增加（P<0.01）。结论Apollon 反义寡核苷酸可下调Apollon基因表达,有效抑制K562细胞的增殖,并促进K562细胞凋亡。     

hTERT基因反义核酸对K562细胞端粒酶活性的影响

探讨人类端粒酶逆转录酶（ｈｕｍａｎ ｔｅｌｏｍｅｒａｓｅ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔａｓｅ,ｈＴＥＲＴ）基因的反义寡核苷酸（ａｎｔｉｓｅｎｓ ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅ,ＡＳＯＮＤ）对Ｋ５６２细胞端粒酶活性的影响。方法：采用多聚酶链反应－酶联免疫测定（ＰＣＲ－ＥＬＩＳＡ）方法检测人工合成的反义脱氧寡核苷酸处理Ｋ５６２细胞前后端粒酶活性的改变,以逆转录ＰＣＲ（ＲＴ－ＰＣＲ）分