用计算机软件辅助设计和实验分析筛选有效的反义核酸(英文)

目的用 RNAstructure 软件设计和实验分析来筛选有效的反义核酸。方法选择 Bcl-2基因为靶基因,利用 RNAstructure 软件,模拟 Bcl-2 mRNA的二级结构,根据自由能不稳定区域或者无折叠的区域,设计5条反义核酸序列,研究它们对白血病细胞的细胞生长作用,用免疫组织化学和流式细胞技术研究蛋白的表达,RT-PCR 检测 mRNA 的含量,有细胞形态学方法、电泳和流式细胞技术检测细胞凋亡。结果 5条中有两条反义核酸能显著抑制白血病细胞的生长,降低 Bcl-2 mRNA 和蛋白水平,显著诱导细胞凋亡。结论计算机软件预测有效反义比其他方法快而有效,这种方法能有效加速研究和临床反义序列的设计。     

p53和Bcl-2 mRNA表达在三七皂苷R1诱导HL-60细胞凋亡中的作用

目的：探讨三七皂苷R1诱导HL-60细胞凋亡的作用机制.方法：采用MTT比色法观察三七皂苷R1对人白血病细胞株HL-60细胞增殖的抑制作用.采用流式细胞术检测细胞凋亡改变,并以RT-PCR检测凋亡调节基因p53、Bcl-2的表达.结果：三七皂苷R1能明显抑制人白血病细胞株HL-60细胞的生长,且呈时间和浓度依赖性.三七皂苷R1作用后人白血病细胞株HL-60细胞呈现凋亡特征,流式细胞术显示凋亡细胞比例升高.RT-PCR检测可见p53 mRNA表达显著增加,而Bcl-2 mRNA表达减少.结论：三七皂苷R1能诱导人白血病细胞株HL-60细胞凋亡,其作用可能与凋亡调节基因p53的上调和Bcl-2的下调有关.     

survivin 反义核酸增强PANC-1 细胞对5-Fu敏感性的研究

 目的 研究survivin反义核酸诱导胰腺癌细胞株PANC-1的凋亡和增强对5-Fu敏感性的影响。方法 我们应用构建的survivin反义核酸真核表达栽体电转染PAND-1细胞，RT-PCR检测survivin mRNA表达，细胞计数和MTT实验测定转染后细胞对5-Fu敏感性，流式细胞仪检测细胞凋亡。结果 survivin反叉核酸可抑制PANC-1细胞中survivin mRNA的表达，并能抑制PANC-1细胞生长，提高PANC-1对5-Fu敏感性，流式细胞仪检测凋亡率明显增加。结论 survivin反义核酸能增强5-Fu诱导的PANC-1细胞凋亡，联合survivin反义核酸和5-Fu可以改善胰腺癌的治疗效果。     

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。     

bcl-2反义核酸提高γ线对恶性淋巴瘤细胞凋亡的作用

目的研究bcl-2反义核酸能否增加^60Coγ线对恶性淋巴瘤细胞凋亡的作用。方法采用细胞染色方法观察细胞凋亡的形态，原位末端标记法检测凋亡细胞，流式细胞仪检测细胞的DNA含量和bcl-2蛋白的表达。结果1～8Gyγ线和10～40μmol/L bcl-2基因反义核酸均能抑制B淋巴瘤细胞株Raji细胞生长，诱导细胞凋亡。流式细胞仪检测亚G1期细胞增多，bcl-2蛋白表达降低，呈现时间剂量依赖效应，联合应用比单独应用抑制作用显著。结论bcl-2反义核酸能够增加^60Coγ线对恶性淋巴瘤细胞凋亡的作用。     

hTR基因反义核酸对K562细胞端粒酶活性和凋亡的影响

目的: 研究人类端粒酶(hTR)基因的反义核酸对K562细胞端粒酶活性和细胞凋亡的影响.方法:采用TRAP法检测K562细胞在反义核酸处理前后端粒酶活性的变化,以及采用TUNEL法观察反义核酸处理前后细胞凋亡的变化.结果:hTR基因反义核酸能有效抑制K562细胞的端粒酶活性,并且诱导K562细胞发生凋亡.结论:hTR基因反义核酸能显著抑制肿瘤细胞的端粒酶活性,为恶性肿瘤治疗提供了一条新的有效的途径.     

Bcl-2反义寡核苷酸增强阿糖胞苷诱导原代白血病细胞凋亡的研究

背景与目的:有研究证实,针对bcl-2mRNA翻译起始区和蛋白编码区的2个有效反义作用靶点的反义寡核苷酸能增强HL-60和K562细胞对阿糖胞苷(Ara-C)、柔红霉素、足叶乙甙的敏感性。本研究观察这两个新靶点的反义寡核苷酸对Ara-C诱导原代培养的急性白血病(AL)、慢性淋巴细胞白血病(CLL)细胞凋亡的影响。方法:用细胞记数法观察细胞的生存情况;免疫荧光标记法通过流式细胞仪检测细胞Bcl-2蛋白水平;用姬姆萨染色法观察并用流式细胞仪检测细胞凋亡。结果:靶向bcl-2mRNA翻译起始区与靶向蛋白编码区的两个反义寡核苷酸分别与Ara-C联合作用AL、CLL细胞48h,细胞的活性受到明显的抑制,分别同无关寡核苷酸(NS-ODN)联合Ara-C组、单用Ara-C组进行比较,差异有显著性(P<0.05)。这两个不同靶点的反义寡核苷酸分别与Ara-C联用均能明显下调AL、CLL细胞Bcl-2蛋白的表达,并且联合作用AL、CLL细胞48h的凋亡细胞百分率分别与NS-ODN联合Ara-C组、单用Ara-C组进行比较,差异有显著性(P<0.05)。与靶向bcl-2mRNA翻译起始区的反义寡核苷酸比较,靶向蛋白编码区的反义寡核苷酸提高AL细胞对Ara-C的敏感性作用要强些(P<0.05)。结论:针对bcl-2mRNA翻译起始区和蛋白编码区两个靶点的反义寡核苷酸能增强Ara-C诱导AL、CLL细胞的凋亡。     

Bcl-2表达受抑可增加单核白血病细胞系对TNFα的敏感性

 目的　探讨Bcl-2反义基因在单核白血病细胞系U937细胞中的作用,以期丰富Bcl-2及其反义核酸调控肿瘤细胞凋亡的资料。方法　将反义Bcl-2基因转染于U937细胞,建立U937/as-bcl2细胞模型,经流式细胞技术检测模型细胞Bcl-2的的表达水平,通过细胞计数观察模型细胞在正常培养条件下和有重组人肿瘤坏死因子α(rhTNFα)存在时的生长和存活特性变化。结果　① U937/as-bcl2模型细胞的Bcl-2蛋白表达水平明显下降,但其在正常培养条件下的生长和存活能力无明显变化。② 在rhTNFα的作用下,模型细胞的存活率较对照细胞者明显下降。结论　① 单独反义bcl-2基因转染可明显抑制U937细胞Bcl-2的表达,但对细胞在正常培养条件下的生长和存活能力无明显影响。② 反义Bcl-2基因转染可增加U937细胞对TNFα的敏感性。     

反义寡核苷酸抑制survivin基因表达及肝癌细胞生长的研究

目的:采用反义寡核苷酸抑制肝癌细胞中survivin基因的表达,研究其对肝癌细胞生长的作用.方法:采用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞,Western blot及原位杂交方法检测survivin蛋白及mRNA表达,流式细胞仪检测凋亡细胞比率,检测转染前后细胞贴壁率变化,并绘制细胞生长曲线.结果:survivin反义寡核苷酸转染后,肝癌细胞survivin蛋白及mRNA表达均显著降低,细胞贴壁率显著降低,细胞增殖活性明显受抑,细胞凋亡率显著增加.结论:survivin反义寡核苷酸转染可以有效降低细胞内survivin基因的表达,诱导细胞发生凋亡,抑制肝癌细胞生长.     

不同途径免疫的AFP基因修饰DC瘤苗体内抗肿瘤效应的研究

目的:评价bcl-2反义寡核苷酸对肾细胞癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用.方法:合成与已知人类基因无同源性的bcl-2反义寡核苷酸(AS1与翻译起始端互补,AS2与编码区互补),以阳离子脂质体Lipofectin为转染载体,MTS比色实验测定细胞活率,反转录-聚合酶链反应(RT-PCR)测定bcl-2 mRNA的表达,Western杂交法测定Bcl-2蛋白表达,碘化丙啶(PI)染色流式细胞术测定凋亡细胞.结果:所测定的5个肾癌细胞株均有稳定的bcl-2 mRNA表达;转染的反义寡核苷酸对ACHN细胞Bcl-2蛋白的表达有明显抑制作用,而正义寡核苷酸无明显影响,AS2的抑制作用大于AS1;反义寡核苷酸可诱导ACHN细胞的凋亡,AS1和AS2的诱导率分别为32.1%和43.2%.结论:bcl-2反义寡核苷酸可抑制肾癌细胞Bcl-2蛋白的表达,并进而诱导细胞的调亡.     

Effect of two new bcl-2 antisenses on drug-sensitivity of cells fromn leukemia patients

Objective: To investigate the effect of two antisense oligonucleotides on cell surviving, bcl-2 expression and apoptosis of leukemia cells. Methods: The experimental assays were performed with cell culture, immunochemistry and flowcytometry. Results: The two antisense oligodeoxynucleotides, combined with Vpl6 or Ara-c or DNR, were able to decline the survival rate of myeleukemic cells, downregulate bcl-2 gene expression and induce apoptosis of leukemic cells significantly, as compared with Vp16 or Ara-c or DNR alone. Conclusion: It is possible for the two new bcl-2 antisenses to be developed into clinical trials for leukemia and tumor with bcl-2 gene overexpression.     

Antisense-mediated downregulation of anti-apoptotic proteins induces apoptosis and sensitizes head and neck squamous cell carcinoma cells to chemotherapy

We have earlier reported that the inhibition of apoptosis in head and neck squamous cell carcinomas (HNSCC) is because of upregulated expression of Bcl-2, Bcl-X(L) and Survivin. Hence, we addressed the question whether antisense approach towards these inhibitors of apoptosis could restore the apoptosis in HNSCC. Further, we wanted to see whether chemotherapeutic efficacy of Cisplatin and Etoposide could be enhanced by using these drugs in combination with antisense oligonucleotides in human laryngeal carcinoma HeP2 and tongue carcinoma Cal27 cells. The effect of these antisense oligonucleotides was examined on the mRNA expression by RT-PCR and on protein expression by Western blotting. Apoptosis was measured by flowcytometry, TUNEL assay and caspase-3 activity assay. Treatment of HeP2 and Cal27 cells with 400 nM antisense oligonucleotides against Bcl-2, Bcl-X(L) and Survivin for 48 hrs decreased their expression both at the mRNA as well as at the protein level, resulting in the induction of apoptosis. Treatment of HeP2 and Cal27 cells with these antisense oligonucleotides augmented Cisplatin and Etoposide induced apoptosis. Our findings emphasize the importance of Bcl-2, Bcl-X(L) and Survivin as survival factors in HNSCC cells. Antisense treatment against these survival factors in combination with lower doses of chemotherapy offers potential as a less toxic chemoadjuvant therapy.     

Chemosensitization and delayed androgen-independent recurrence of prostate cancer with the use of antisense Bcl-2 oligodeoxynucleotides

BACKGROUND: Increased expression of the bcl-2 gene has been observed in prostate cancer cells after androgen withdrawal and has been associated with the development of androgen independence and chemoresistance. The objective of this study was to determine whether antisense Bcl-2 oligodeoxynucleotides could enhance paclitaxel cytotoxicity and delay androgen-independent progression. METHODS: Northern and western blot analyses were used to measure changes in Bcl-2 expression in mouse Shionogi tumor cells after treatment with antisense Bcl-2 oligodeoxynucleotides and/or paclitaxel. Growth inhibition and induction of apoptotic cell death were assessed with the use of standard methods. All P values are two-sided. RESULTS: Treatment of Shionogi tumor cells with 500 nM antisense Bcl-2 oligodeoxynucleotides decreased expression of Bcl-2 messenger RNA (mRNA) by approximately 85%. Paclitaxel treatment induced Bcl-2 protein phosphorylation but did not alter Bcl-2 mRNA expression. Antisense Bcl-2 oligodeoxynucleotide treatment substantially enhanced paclitaxel chemosensitivity in a dose-dependent manner. Characteristic apoptotic DNA laddering and cleavage of poly(adenosine diphosphate-ribose) polymerase were demonstrated only after combined treatment. Adjuvant in vivo administration of antisense Bcl-2 oligodeoxynucleotides and micellar paclitaxel following castration resulted in a statistically significant delay of androgen-independent, recurrent tumors compared with administration of either agent alone (P<.001, Mantel-Cox log-rank test). Combination therapy also statistically significantly inhibited the growth of established hormone-refractory tumors compared with treatment with either agent alone (P<.001, Student's t test). CONCLUSIONS. Combined treatment with antisense Bcl-2 oligodeoxynucleotides and paclitaxel could be a novel and attractive strategy to inhibit progression to androgen-independent disease as well as growth of hormone-refractory prostate cancer through deprivation of Bcl-2 function.     

bcl-2反义寡核苷酸对肾癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用

目的:经Beagle犬肝脏血管注射重组腺病毒介导反义c-myc基因(Ad-ASmyc)载体,观测其体内分布及毒副作用,以评价Ad-ASmyc治疗肝癌的临床前期安全性.方法:选择4只体重8～10kg健康Beagle犬,全麻后开腹,经肝动脉或门静脉注射Ad-ASmyc,注射Ad-ASmyc前及注射后第3,7,14,21天取肝组织及抽取静脉血化验,PCR检测Ad-ASmyc在各器官组织中的分布,常规切片观察各器官病理变化,ELISA方法检测血中抗腺病毒抗体的产生.结果:经Beagle犬肝脏血管注射后,Ad-ASmyc可持续转导正常肝细胞达3周,实验犬一般情况好,血、肝、肾功能无明显异常,在肝脏、脾脏、肾脏、胃、心脏、皮肤中可检测到重组腺病毒的分布,镜下可见Ad-ASmyc剂量依赖性的肝组织轻微的炎症反应,注射后7d血中有抗腺病毒载体的抗体产生,14d达高峰,21d开始下降.结论:经Beagle犬肝动脉或门静脉途径注射Ad-ASmyc均可转导至肝细胞,Ad-ASmyc对实验犬的毒副作用较轻.     

Bcl-2反义寡核苷酸诱导HL-60细胞凋亡的研究

目的：探讨bcl-2反义寡核苷酸诱导ＨＬ－６０细胞的凋亡。方法：将人工合成的bcl-2反义寡聚脱氧苷酸片段与ＨＬ－６０,细胞共培养,在作用的第０天、３天、５天通过免疫组化法（ＡＰＡＡＰ法）检测细胞内bcl-2蛋白表达水平的变化,通过流式细胞仪分析细胞凋亡的情况。结果：bcl-2反这寡核苷酸可下调ＨＬ－６０细胞内bcl-2的蛋白表达水平,其作用与其浓度和时间呈正相关,同时可诱导细胞凋亡,而bcl-2反义寡核苷酸及对照无此作用。     

Livin反义寡核苷酸对人乳腺癌细胞株MCF-7增殖和凋亡影响的研究

目的：探讨Livin mRNA反义寡核苷酸（ASODN）对人乳腺癌MCF-7细胞抑制增殖及诱导凋亡的影响。方法：设计合成特异性的Livin硫代磷酸ASODN及其对照错义寡核苷酸（MSODN）,脂质体转染至培养的MCF-7细胞。采用MTT法检测Livin ASODN对MCF-7细胞的增殖抑制作用,RT-PCR检测Livin mRNA的表达水平,电镜、流式细胞仪与吖啶橙／溴化乙锭（AO／EB）细胞染色法检测细胞凋亡水平和形态学改变。结果：Livin ASODN在终浓度为600nmol／L作用MCF-7细胞48h时,能明显地抑制其增殖（IC50=604.4）,降低Livin mRNA的表达;电镜、流式细胞仪与A0／EB细胞染色法则表明MCF-7细胞在形态学上出现明显的凋亡改变,细胞凋亡率显著增加,而对照组寡核苷酸未见抑制效应。结论：Livin ASODN能够特异性下调MCF-7细胞中Livin基因表达,在诱导肿瘤细胞凋亡、抑制增殖中发挥重要作用。     

Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-xL antisense treatment

Our study was designed to investigate the role of the anti-apoptotic proteins Bcl-2 and Bcl-xL in the chemoresistance of cells derived from malignant pleural mesothelioma. First, we determined the basal expression levels of Bcl-2 and Bcl-xL in mesothelioma cells and examined the effect of their downregulation by antisense oligonucleotides. Bcl-xL mRNA and protein could be readily detected in mesothelioma cell lines, whereas only low levels of Bcl-2 mRNA and protein were found. Preferential downregulation of either Bcl-xL alone or of Bcl-xL and Bcl-2 simultaneously was achieved by treatment with antisense oligonucleotides 4259 and 4625, respectively, whereas the expression of other apoptosis-relevant genes remained unaffected. Treatment with oligonucleotides 4259 or 4625 lowered the apoptosis threshold in ZL34 mesothelioma cells, as indicated by an increase in cell death accompanied by increased caspase-3-like activity, a decrease of the mitochondrial transmembrane potential and the cleavage of procaspase-7 and ICAD. In addition to the direct induction of apoptosis, antisense treatment sensitized ZL34 cells to the cytostatic effect of cisplatin and gemcitabine, with the combination of 4625 and cisplatin being the most effective. Our results demonstrate that Bcl-2 and Bcl-xL antisense treatment facilitates apoptosis in mesothelioma cells and suggest the use of Bcl-2/Bcl-xL bispecific antisense treatment in combination with cisplatin or gemcitabine for therapy of malignant pleural mesothelioma.     

PTEN/PI3K/Akt信号通路对K562细胞凋亡调控的研究

 目的 探讨PTEN/PI3K/Akt信号传导通路对人慢性粒细胞白血病细胞系K562的增殖、凋亡调控的研究及可能的分子作用机制。 方法 将携带有野生型PTEN及绿色荧光蛋白的腺病毒（Ad-PTEN-GFP）及空载体（Ad-GFP）腺病毒,转染人慢性粒细胞白血病细胞系K562。通过MTT检测细胞生长曲线,流式细胞术检测细胞凋亡率和细胞增殖指数,同时用细胞光镜、电镜形态等方法检测细胞凋亡,荧光定量PCR（FQ-PCR）检测PTEN及凋亡相关基因Bcl-2、Bcl-_xL、Bax mRNA水平变化,Western blot检测PTEN及Akt、p-Akt蛋白水平变化。 结果 与Ad-GFP组相比,Ad-PTEN-GFP 转染K562细胞后,细胞增殖受抑,增殖指数降低,凋亡率增加,p-Akt表达降低,抗凋亡相关基因Bcl-2、Bcl-_xL mRNA表达降低,促凋亡基因Bax mRNA表达增加。 结论 过表达PTEN可能通过抑制PI3K/Akt通路抑制K562细胞系增殖,促进细胞凋亡。