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基因反义寡核苷酸增强放射线诱导Raji细胞凋亡的研究

目的研究bcl-2基因反义寡核苷酸作用淋巴瘤细胞株Raji后,放射线对细胞凋亡的影响.方法bcl-2基因反义寡核苷酸作用Raji细胞后,台盼蓝拒染法计数活细胞;免疫荧光标记观测细胞Bcl-2蛋白水平;用姬姆萨染色和碘化丙啶染色法、流式细胞仪检测细胞凋亡.结果bcl-2基因反义寡核苷酸与放射线联合作用Raji细胞,显著抑制细胞的生长,并呈时间依赖性,活细胞数分别与单用放射线组及无义寡核苷酸联合放射线组相比,统计学上有显著性差异(P＜0.05).bcl-2基因反义寡核苷酸与放射线联合作用于Raji细胞后显著抑制bcl-2蛋白表达,蛋白水平分别与单用放射线组或无义寡核苷酸联用放射线组比较,差异有显著性(P＜0.05).bcl-2基因反义寡核苷酸与放射线同时作用于Raji细胞后,姬姆萨染色可见凋亡细胞,通过流式细胞仪检测的细胞凋亡率显著增加,分别与单用放射线组或无义寡核苷酸联用放射线组相比,具有显著性差异,P＜0.05.结论bcl-2反义寡核苷酸能增强放射线诱导Raji细胞凋亡.     

脂质体转染bcl-2反义寡核苷酸诱导Raji细胞凋亡的研究

目的:通过脂质体分别转染bcl-2两个靶点的反义寡核苷酸(ASODN1、ASODN2)及G3139,观察bcl-2反义寡核苷酸对淋巴瘤细胞株Raji凋亡的影响.方法:采用MTT法检测药物的半数抑制率(IC50);免疫荧光标记观察细胞bcl-2蛋白水平;用姬姆萨染色和流式细胞仪观察细胞凋亡.结果:MTT测定显示:ASODN1、ASODN2组IC50值分别与G3139组进行比较无显著差异,P＞0.05.5μmol/L ASODN1、ASODN2作用于Raji细胞72h后,姬姆萨染色均可见凋亡细胞.5μmol/L ASODN1、ASODN2和C3139作用于Raji细胞后,bcl-2蛋白阳性率均明显下降,细胞凋亡率均明显增加,P<0.05,但ASODN1、ASODN2组分别与G3139组进行比较无显著差异,P＞0.05.5μmol/L无义寡核苷酸对Raji细胞的生长活性、bcl-2蛋白水平及细胞凋亡率均无明显影响,P>0.05.结论:针对bcl-2两个靶点的反义寡核苷酸能特异性促进Raji细胞的凋亡,并且与G3139具有近似的反义效应.     

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.     

bcl-2基因的反义寡核苷酸与三氧化二砷联合对恶性淋巴瘤细胞系凋亡的影响

目的：研究bcl-2基因的反义寡核苷酸与三氧化二砷（As2O3）联合对恶性淋巴瘤细胞系凋亡的影响。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（TUNEL）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：10－40μmol/l bcl-2基因的反义寡核苷酸和0．5－2．0μmol/L三氧化二砷均能抑制恶性B淋巴瘤细胞系Raji细胞生长，诱导细胞凋亡，流式细胞仪检测Raji细胞，发现亚G1期细胞明显增多，bcl-2蛋白的表达明显降低，呈现时间剂量依赖效应，两者联合应用比单独应用抑制作用显著。结论：bcl-2基因的反义寡核苷酸与三氧化二砷联合应用，明显抑制恶性B淋巴瘤细胞系生长，促进细胞凋亡。     

单独及联合转染survivin和bcl-2反义寡核苷酸诱导胆囊癌细胞凋亡的研究

目的:探讨单独及联合转染survivin、bcl-2反义寡核苷酸(AsODN)对胆囊癌细胞系GBC-SD的凋亡促进作用.方法:设计并合成特异性靶向survivin及bcl-2的AsODN.免疫组织化学法观察胆囊癌细胞中Survivin及Bcl-2蛋白的表达情况;实验分为4组:空白对照组、survivin AsODN转染组、bcl-2 AsODN转染组、联合survivin及bcl-2 AsODN转染组.转染24 h 后,采用逆转录-聚合酶链反应(RT-PCR)检测survivin基因表达的变化,电镜下观察细胞形态变化,流式细胞仪技术检测细胞凋亡率,四甲基偶氮唑蓝(MTT)法检测AsODN对细胞的生长抑制率.结果:Survivin及Bcl-2蛋白在胆囊癌细胞中均有较高表达;单独及联合转染survivin及bcl-2 AsODN后,胆囊癌细胞内survivin基因mRNA表达下调47.8%;,电镜下胆囊癌细胞呈典型凋亡样改变;凋亡率分别为11.38%±3.91%(survivin AsODN组)、9.26%±4.15%(bcl-2 AsODN组)、28.45%±6.34%(联合转染组);分别与对照组相比差异有显著性(P＜0.01),而联合转染组同单独转染survivin AsODN组及bcl-2 AsODN组相比差异也有显著性(P＜0.05).各转染组相对于空白对照组的AsODN抑制率分别为54.3%,47.6%,76.5%.结论:Survivin及Bcl-2蛋白在胆囊癌细胞中呈高表达,单独及联合转染survivin和bcl-2反义寡核苷酸可促进胆囊癌细胞凋亡,而联合转染更具显著性.     

bcl-2反义寡核苷酸对小细胞肺癌NCI—H69细胞凋亡的影响

目的探讨bcl-2反义寡核苷酸能否增加小细胞肺癌细胞NCI—H69的凋亡。方法将NCI—H69细胞培养传代,细胞共分4组,反义寡核苷酸组、正义寡核苷酸组、无义寡核苷酸组和空白对照组,通过脂质体将不同浓度的寡核苷酸导人细胞中,WesternBlot法检测bcl-2蛋白的表达,流式细胞术检测细胞的凋亡率。结果与空白对照组比较,反义寡核苷酸组的bcl-2蛋白表达明显受到抑制,而正义寡核苷酸组和无义寡核苷酸组细胞的bcl-2蛋白表达则与空白对照组差异不明显。反义寡核苷酸组细胞在10、20、40umol／L 3个不同浓度时的凋亡率分别是（9．97±1．54）％、（15．28±1．73）％、（21．41±1．85）％,明显高于空白组和正义链、无义链组阴性对照组,且差异有统计学意义（F=7．19～15．48,q=5．21—7．98,P〈0．01）,而转染正义链和无义链组与空白组比较则差异无统计学意义。结论bcl-2反义寡核苷酸能有效封闭bcl-2基因的表达,增加小细胞肺癌细胞的凋亡。     

Bcl-2反义肽核酸诱导HL60细胞凋亡

目的　探讨不同结构的反义药物对HL6 0细胞系生物学活性的影响。方法　应用细胞计数、细胞形态观察和流式细胞术观察并比较反义肽核酸和反义寡核苷酸对白血病细胞HL6 0生物学活性的影响。结果　 10 μmol/L靶向bcl mRNA蛋白编码区的反义肽核酸能有效地抑制HL6 0细胞的生长、下调bcl 2蛋白的水平及诱导细胞凋亡。 10μmol/L同样靶点的反义肽核酸和反义寡核苷酸作用HL6 0细胞 72小时 ,细胞凋亡的百分率分别为 17.8± 1.5 3 ,13.17± 1.12 ,统计学上有显著性差异。结论　反义Bcl 肽核酸能诱导HL6 0细胞的调亡 ,比反义寡核苷酸有更好的反义作用。     

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。     

人慢性粒细胞白血病bcr-abl基因反义寡核苷酸对K562细胞株的凋亡诱导作用研究

背景与目的：随着人类基因组计划的完成,人们的研究重点已转向基因功能的研究,反义核酸技术无疑为这项宏伟工程提供了一个新的发展方向。目前,国内关于反义寡核苷酸诱导K562细胞凋亡的实验研究很少。本实验在体外构建针对人慢性粒细胞白血病(chronic myelogenou leukemia,CML)bcr-abl融合基因mRNA的反义寡核苷酸,探讨bcr-abl反义寡核苷酸对K562细胞凋亡的诱导作用。方法：以bcr-abl融合基因mRNA翻译起始点融合前区19个寡核苷酸为作用靶点,设计反义寡核苷酸,以其反义寡核苷酸序列转染人慢性粒细胞K562细胞,采用Hoechst染色法观察不同浓度寡核苷酸对K562细胞株的凋亡情况,采用蛋白[质]印迹法(Western blot)检测自噬凋亡蛋白LC3-Ⅱ的表达情况,采用流式细胞术(flow cytometry,FCM)检测细胞周期变化,采用JEM-4000EX电镜术检测细胞凋亡形态变化,通过DNA琼脂糖凝胶电泳检测K562细胞凋亡情况。结果：Hoechst染色结果显示,bcr-abl反义寡核苷酸能显著促进K562细胞的凋亡,且呈现一定的浓度依赖性。Westernblot检测结果显示,bcr-abl反义寡核苷酸各浓度组凋亡自噬蛋白LC3-Ⅱ表达水平明显高于对照组,差异有统计学意义(P<0.05)。FCM检测结果显示,bcr-abl反义寡核苷酸作用于K562细胞后,细胞周期阻滞于G₀/G₁期。各组G₀/G₁期、S期细胞数量与对照组相比,差异有统计学意义(P<0.05)。在JEM-4000EX电镜下可见明显的新月型凋亡小体。DNA琼脂糖凝胶电泳显示,10、30 μmol/mL bcr-abl反义寡核苷酸组可以明显观察到以180~200 bp碱基对整倍数出现明暗间隔的DNA梯状条带。结论：Bcr-abl反义寡核苷酸可显著诱导K562细胞凋亡,为临床上基因治疗人CML提供一定的参考。     

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.     

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.     

Antisense oligonucleotides complementary to Bax transcripts reduce the susceptibility of B-cell chronic lymphocytic leukaemia cells to apoptosis in a bcl-2 independent manner

Our previous work demonstrated that antisense oligonucleotides complementary to Bcl-2 mRNA sequences were able to reduce Bcl-2 protein expression in B-cell chronic lymphocytic leukaemia (B-CLL) cells. Furthermore, the reduction in Bcl-2 expression led to an increase in apoptotic cell death that was associated with an increase in Bax expression. In this present study antisense oligonucleotides directed towards the Bax translation initiation sequence were employed to determine whether Bcl-2 and Bax protein expression were interdependent upon one another and whether Bcl-2 antisense-induced apoptosis was mediated through a p53-dependent cell death pathway. The antisense oligonucleotide down-regulated Bax protein expression between 23.7 and 68.8% in the 20 B-CLL samples tested and significantly inhibited apoptotic cell death when compared to controls (p = 0.0001). Furthermore, these changes were independent of Bcl-2 expression suggesting that the previously observed increase in Bax expression following exposure of B-CLL cells to Bcl-2 antisense oligonucleotides was probably caused by the induction of apoptosis rather than a rheostatic response to the reduction in Bcl-2 protein expression. This notion was substantiated by the fact that p21 expression was induced in Bcl-2 antisense-treated B-CLL cells, a finding consistent with p53 activation.     

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.     

二氟甲基鸟氨酸及反义bcl-2协同诱导HL60细胞凋亡

目的　观察二氟甲基鸟氨酸 (DFMO)及反义bcl 2调控bcl 2基因表达对HL6 0细胞凋亡的诱导作用。方法　构建反义bcl 2逆转录病毒重组体 ,建立产病毒细胞系 ,转染HL6 0细胞 ,用形态观察、生长曲线、FCM分析、集落形成、DNA电泳图谱、分子杂交及免疫组化等方法研究细胞生长特性及细胞凋亡诱导。结果　成功地构建了反义bcl 2逆转录病毒重组体及产病毒细胞系。以此转染HL6 0细胞 ,引起了bcl 2mRNA及蛋白表达下降 ,但生长速率、细胞周期分布及鸟氨酸脱羧酶 (ODC)基因表达水平与亲本细胞比较差别不大 ;无明显的细胞凋亡 ,仅集落形成能力有所减弱。用小剂量DFMO处理反义bcl 2转染的细胞 ,不仅生长受到明显抑制 ,而且可诱导细胞凋亡。结论　DFMO与反义bcl 2对HL6 0细胞增殖抑制及凋亡诱导有协同作用。抑制bcl 2表达能提高肿瘤细胞对DFMO作用的敏感性。     

Bcl-x(L) antisense oligonucleotides induce apoptosis and increase sensitivity of pancreatic cancer cells to gemcitabine.

Pancreatic cancer is one of the leading causes of cancer-related death in Western countries. Bcl-x(L) is an anti-apoptotic factor of the Bcl-2 family, which is overexpressed in pancreatic cancer and its presence correlates with shorter patient survival. In this study, sequence-specific antisense oligonucleotides targeting the coding region of Bcl-x(L) were designed to examine whether apoptosis could be induced and chemosensitivity could be increased in pancreatic cancer cells. Five pancreatic cancer cell lines, Panc-1, MIA-PaCa-2, Capan-1, ASPC-1 and T3M4, were treated with Bcl-x(L) sense or antisense oligonucleotides and gemcitabine and the cell viability was examined by the SRB method. Apoptosis was determined using DAPI staining. In all examined pancreatic cancer cells, Bcl-x(L) expression was reduced after transfection of the antisense oligonucleotides. Cell death analysis using DAPI staining revealed that antisense, but not sense oligonucleotides caused apoptotic cell death. Furthermore, Bcl-x(L) antisense oligonucleotides enhanced the cytotoxic effects of gemcitabine in pancreatic cancer cells. Our results indicate that Bcl-x(L) antisense oligonucleotides effectively inhibited pancreatic cancer cell growth and caused apoptosis by reducing Bcl-x(L) protein levels. Bcl-x(L) antisense oligonucleotides also increased the chemosensitivity of pancreatic cancer cells, suggesting that Bcl-x(L) antisense therapy might be a potential future approach in this disease.