bFGF反义寡核苷酸增强鼠黑色素瘤B16细胞化疗药物敏感性研究

目的:研究bFGF反义硫代寡核苷酸增强肿瘤细胞对化疗药物敏感性作用。方法:设计、合成bFGF寡核苷酸,用聚乙烯亚胺(polyemyleneimine,PEI)介导bFGF反义硫代寡核苷酸转染入黑色素瘤B16细胞,MTT法检测bFGF反义硫代寡核苷酸及其与化疗药物联合处理后的细胞增殖率;半定量RT-PCR测定bFGF反义硫代寡核苷酸转染后细胞中bFGF mRNA水平;流式细胞仪分析bFGF反义硫代寡核苷酸诱导的细胞凋亡。结果:bFGF反义硫代寡核苷酸对B16细胞增殖的抑制率为64.8%,且呈剂量依赖效应。B16细胞中bFGF mRNA被bFGF反义硫代寡核苷酸显著降低,为对照细胞的57.9%,且bFGF反义硫代寡核苷酸诱导B16细胞凋亡,凋亡率为41.8%。bFGF反义硫代寡核苷酸转染能显著增强B16细胞对阿霉素、5-氟脲嘧啶及顺铂的敏感性,非特异性硫代寡核苷酸不影响阿霉素、5-氟脲嘧啶及顺铂抑制B16细胞增殖。结论:bFGF反义硫代寡核苷酸显著增强B16细胞的化疗敏感性,表明其可协同化疗药物用于治疗肿瘤。     

靶向Survivin的反义寡核苷酸对肿瘤细胞增殖的抑制作用

 Survivin是新近克隆的一种凋亡抑制蛋白 (IAP)家族成员 ,在几乎所有肿瘤组织中特异性表达 ,而在正常成年终末分化组织中低表达甚至不表达 .采用四唑盐 (MTT)比色实验法比较 2 0条抗人survivin反义寡核苷酸对HeLa细胞增殖的抑制效果 ,并从中筛选效果显著的反义寡核苷酸 ,在体外水平进一步验证其抑制survivin表达的能力 .在用 4 0 0nmol L反义寡核苷酸转染HeLa细胞 4 8h后 ,有 4条反义寡核苷酸对细胞增殖的抑制率超过 4 0 %,其中 4 5号反义寡核苷酸的抑制率可达5 9%,而阳性对照序列ISIS2 372 2的抑制率仅达 30 %.Northern和Western印迹分析证明 :4 5号反义寡核苷酸可明显降低细胞中survivin基因的mRNA含量和蛋白水平 .4 5号反义寡核苷酸还可在较低浓度 (2 0 0nmol L)显著增强HeLa细胞对化疗药三尖杉酯碱的敏感性 .因此 ,4 5号反义寡核苷酸有望应用于survivin高表达肿瘤的辅助治疗之中     

MNP- hTR 复合物对K562 细胞增殖影响的实验研究

探讨MNP-端粒酶反义寡核苷酸复合物,对K562细胞凋亡和增殖的影响。应用荧光显微镜检测法和集落形成法,分别观察了凋亡细胞的形态学变化和细胞增殖能力。MNP-端粒酶反义核酸复合物可诱导K562细胞凋亡和抑制细胞增殖,两实验组数值经统计学处理,具有非常显著性差异（p分别〈O．001）。由此认为,MNP-端粒酶反义核酸复合物对K562白血病细胞具有促进凋亡的作用,此效果与剂量呈依赖性关系（r=0．992）。     

氧化苦参碱对K562肿瘤细胞增殖的影响

目的:研究氧化苦参碱(OM)对人白血痛细胞系K562生长增殖的影响.方法:运用MTT比色法、活细胞计数法、集落形成法以及透射电镜观察检测OM对人白血病细胞系K562增殖抑制作用.结果:MTT实验、生长曲线及集落形成实验显示OM能明显抑制K562细胞的增殖.随着OM浓度的增加,K562细胞存活细胞显著降低,呈现明显的刺量依赖性,经相关分析,细胞抑制率与OM浓度呈正相关(r=0.9010),其半数抑制浓度(IC50)为0.33 mg/ml.透射电镜下显示在低浓度即有明显的诱导细胞凋亡的作用,出现核固缩、核碎裂、凋亡小体等典型的凋亡形态.结论:OM具有抑制K562白血病细胞增殖诱导肿瘤细胞凋亡的作用.     

VEGF反义寡核苷酸抑制小鼠Lewis肺癌生长的研究

目的探讨血管内皮生长因子(VEGF)反义寡核苷酸对C57BL/6小鼠Lewis肺癌肿瘤生长的抑制作用。方法制备C57BL/6小鼠皮下肺癌模型30只,随机分为3组,每组10只VEGF反义寡核苷酸(ASPODN)治疗组、VEGF正义寡核苷酸(SPODN)治疗组及对照组。接种Lewis肺癌细胞后24h内,分别皮下注射ASPODN及SPODN进行治疗,对照组只注射生理盐水,每周2次,连续4周;观察各组小鼠肿瘤的生长情况、游标卡尺测量肿瘤体积大小。所有C57BL/6小鼠于接种后第25天先用二维超声观察肿瘤的实时图象,利用脉冲多普勒获取血流频谱,获得收缩期峰值速度(PS),阻力指数(RI)。断颈处死小鼠,光镜及电镜下观察肿瘤组织形态学改变及超微结构变化,免疫组化法检测微血管密度(MVD)。结果与对照组瘤重比较(7.83±0.78)g、VEGF-ASPODN组(4.49±0.43)g能明显抑制小鼠肿瘤生长(P<0.01)、VEGF-SPODN组(7.73±0.69)g则无明显作用(P>0.05)。VEGF-ASPODN组、VEGF-SPODN组抑瘤率分别为42.7%、5.9%。组织形态学及超微结构观察,VEGF-ASPODN对肿瘤生长具有抑制作用。VEGF-ASPODN组与对照组相比较PS及RI有明显差异(P<0.01);VEGF-SPODN组与对照组相比无明显差异(P>0.05)。结论肿瘤原位注射VEGF反义寡核苷酸能抑制小鼠肺癌生长。     

X连锁凋亡抑制蛋白反义寡核苷酸逆转K562细胞及难治性癫痫大鼠对卡马西平和苯妥英钠耐药性

凋亡在癫痫发生机制中起重要作用,但其在难治性癫痫耐药机制中的作用尚不清楚．为研究X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)反义寡核苷酸对K562/Dox(阿霉素诱导)耐药细胞及难治性癫痫大鼠耐药性的影响,首先建立耐药的K562/Dox细胞株,比较XIAP在耐药细胞株和正常K562细胞株的表达情况,观察转染XIAP反义寡核苷酸后,线粒体膜电位变化以及对卡马西平和苯妥英钠耐药性的影响．另外,建立慢性杏仁核点燃癫痫模型,筛选出耐药组和药物敏感组,通过侧脑室注射XIAP反义寡核苷酸,对照组注射生理盐水．观察其对各组大鼠后放电阈值(after discharge threshold,ADT)、后放电时程(after discharge duration,ADD)等电生理指标的影响．结果发现,XIAP在K562/Dox耐药细胞上的表达明显高于正常K562细胞,XIAP反义寡核苷酸转染K562/Dox耐药细胞后,XIAP的表达明显下降．导致了K562/Dox细胞线粒体跨膜电位的下降,而且对苯妥英钠和卡马西平的耐药性明显下降,IC₅₀分别由(1 978.2 ± 90.3) mg/L和(1 875.6 ± 83.2) mg/L,降低到(1 123.5 ± 54.2) mg/L和(1 084.5 ± 60.6) mg/L,逆转倍数分别为1.76和1.73．同时动物实验发现,耐药组大鼠在给予XIAP反义寡核苷酸后,ADT明显高于对照组(P < 0.05), ADD时程也明显缩短．上述结果证明,XIAP在耐药的K562/Dox细胞株存在高表达,下调XIAP在K562/Dox细胞株表达可以改善K562/Dox对卡马西平和苯妥英钠的耐药性．而且下调XIAP表达可以协助AEDs改善耐药大鼠的电生理活动,提示XIAP参与了难治性癫痫的耐药．     

脂质体介导反义核酸对乳腺癌MCF-7细胞增殖的影响

目的:探讨脂质体LipfectAMINETM介导c-myc反义寡核苷酸(ASODN)对MCF-7细胞增殖的影响.方法:MCF-7细胞分五组处理:c-mycSODNs组、LR/c-mycSODNs组、c-mycASODNs组、LR/c-mycASODNs组、LR组和空白对照组.以MTT法检测72h各处理组细胞增殖的情况;以免疫细胞化学ABC法检测LR/c-mycASODNs组转染前后细胞中c-myc蛋白的表达.结果:c-mycASODNs组(0.383±0.015)和LR/c-mycASODNs组(0.178±0.015)均能明显抑制细胞生长,差异具有显著性(P＜0.01),且后者对细胞的生长抑制率(73.13%)明显高于前者(17.47%):LR/c-mycASODNs组免疫细胞化学显示c-myc蛋白表达明显降低.结论:LR介导的c-mycASODN能明显抑制MCF-7细胞生长和c-myc蛋白表达.     

MDM2反义寡核苷酸联合紫杉醇对乳腺癌MCF-7细胞株的作用

目的:探讨靶向MDM2反义寡核苷酸(ASON)联合紫杉醇对乳腺癌MCF-7细胞株的影响。方法:合成一段与MDM2 mRNA特异性结合的反义寡核苷酸和与反义寡核苷酸有4个碱基不同的的错义寡核苷酸(MON),脂质体2000介导不同浓度的MDM2ASON转染MCF-7乳腺癌细胞系,转染的乳腺癌细胞通过1μmol/L紫杉醇药物处理后,采用RT-PCR和Western Blot方法检测MDM2 ASON联合紫杉醇的协同作用及对乳腺癌MCF-7细胞株的抑制效率,MTT观察给药后MCF-7细胞的增殖能力和药物敏感性。结果:MDM2反义寡核苷酸联合紫杉醇明显下调MDM2 mRNA及MDM2蛋白表达水平,抑制MCF-7细胞的生长,随着MDM2 ASON浓度的增加,MDM2表达越来越低,协同作用越来越强,呈剂量依赖关系,A500联合紫杉醇的协同作用最明显,MTT显示紫杉醇处理的转染MCF-7细胞增殖抑制率明显增高,A500抑制增殖作用最明显,抑制率达(13.0±0.84)%。结论:不同浓度MDM2 ASON转染后的乳腺癌MCF-7细胞,等浓度紫杉醇处理后,乳腺癌MCF-7细胞MDM2表达明显降低,细胞凋亡增加,,MDM2 ASON联合紫杉醇对MCF-7细胞有协同作用,提高了乳腺癌MCF-7细胞对紫杉醇的药物敏感性。     

TnI-fast基因表达抑制卵巢癌移植瘤生长

为探讨利用TnI-fast 基因进行卵巢癌基因治疗的有效性及其机制, 将TnI-fast基因 cDNA转染人卵巢癌细胞系SKOV3. 采用MTT法和流式细胞技术分别检测TnI-fast基因转染、空载体转染和未转染的SKOV3细胞体外生长状态. 收集3种细胞培养上清液, 检测3种培养上清液对人脐静脉内皮细胞增殖抑制效应. 3种细胞分别接种到裸鼠, 观察肿瘤生长、细胞凋亡、肿瘤血管生成和TnI-fast基因局部表达. 体外试验发现, 与空载体转染和未转染的SKOV3细胞比较, TnI-fast基因表达对肿瘤细胞自身的生长无抑制作用, 但可抑制人脐静脉内皮细胞增殖. 动物实验中, TnI-fast基因表达可显著抑制肿瘤生长, 生长抑制率达73%. 其肿瘤细胞增殖率与对照组相当, 但微血管密度显著降低, 细胞凋亡显著增加. 提示, 肿瘤自身血管生成抑制可显著延缓卵巢癌生长. 利用血管生成特异性抑制基因TnI-fast进行抗肿瘤血管生成基因治疗可作为肿瘤治疗的新策略之一.     

Prediction of VEGF mRNA antisense oligodeoxynucleotides by RNA structure software and their effects on HL60 and K562 cells

Seven antisense oligodeoxynucleotides were selected using RNA structure 3.7 software and the principle of low overall DeltaG (free energy). Their effects on cell growth, VEGF protein expression and apoptosis in HL60 and K562 leukemic cells were examined, and cell numbers and viability were assessed using trypan blue dye exclusion, MTT, ELISA and flow cytometry. The results showed that six of the seven antisense sequences inhibited the cell growth and down-regulated VEGF protein expression significantly. Endogenous VEGF plays an important role in the proliferation of HL60 and K562 leukemic cells. RNA structure software provides a rapid and efficient way of identifying effective antisense oligodeoxynucleotides.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense bcl-2 oligodeoxynucleotides enhanced the sensitivity of leukemia cells to arsenic trioxide. A combination of arsenic trioxide with antisense bcl-2 oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced bcl-2 protein expression in K562 and NB4 leukemic cells more significantly than either arsenic trioxide or the oligodeoxynucleotides on their own (P<0.01). Thus, bcl-2 antisense oligodeoxynucleotides increase the sensitivity of leukemic cells to arsenic trioxide. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.     

Imatinib mesylate (STI-571) reduces Bcr-Abl-mediated vascular endothelial growth factor secretion in chronic myelogenous leukemia

A large and diverse spectrum of oncogenes has been implicated as a contributor to angiogenesis in solid tumors based, in part, on its ability to induce proangiogenic growth factors such as vascular endothelial growth factor (VEGF), and the fact that various anti-oncogenic signaling inhibitor drugs have been shown to reverse such proangiogenic effects both in vitro and in vivo. Because leukemias are now also considered to be angiogenesis-dependent malignancies, we asked whether a similar paradigm might exist for the BCR-ABL oncogene and the Bcr-Abl targeting drug, STI-571 (imatinib mesylate), in the context of chronic myelogenous leukemia (CML) cells. We found that levels of VEGF expression in BCR-ABL-positive K562 cells were reduced in vitro by treatment with STI-571 in a dose-dependent fashion. Transfection of BCR-ABL into murine myeloid 32D and human megakaryocyte MO7e hematopoietic cells resulted in enhanced VEGF expression, which could be further elevated by the exposure to cytokines such as interleukin 3 and granulocyte macrophage colony-stimulating factor. We also found that conditioned media taken from 32D-p210-transfected cells could stimulate human umbilical vein endothelial cells by increasing phosphorylation of VEGF-R2/KDR and the downstream serine/threonine kinase PKB/Akt, an important regulator of endothelial cell survival. Moreover, amplification of BCR-ABL in STI-571-resistant cells was associated with elevated VEGF expression levels which could be reversed by treatment with higher concentrations of STI-571. Taken together, our results implicate BCR-ABL as a possible regulator of CML angiogenesis and raise the possibility that STI-571 could mediate some of its anti-CML properties in vivo through an angiogenesis-dependent mechanism.     

低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响;CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率;AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率;PI单染流式细胞仪检测药物作用后细胞的周期分布改变;Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用;可明显抑制K562细胞的生长,呈时间一剂量依赖性;尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞,但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后,K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％,与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）;低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度,长时间作用于人白血病K562细胞后,具有较强的集落形成抑制和生长抑制作用,此作用可能与诱导细胞周期相关分子p16高表达,导致细胞G0／G1期阻滞有关。     

5'-,3'-inverted thymidine-modified antisense oligodeoxynucleotide targeting midkine. Its design and application for cancer therapy

Oligodeoxynucleotides modified at both 5'- and 3'-ends with inverted thymidine (5'-,3'-inverted T) were introduced as new reagents for antisense strategies. These modifications were performed to make the oligodeoxynucleotides resistant to nucleases. The effectiveness of these oligodeoxynucleotides was evaluated in terms of inhibition of synthesis of midkine (MK), a heparin-binding growth factor, and consequent inhibition of growth of CMT-93 mouse rectal carcinoma cells. 5'-,3'-Inverted T antisense MK suppressed synthesis of MK by CMT-93 cells and their growth in culture. Furthermore, 5'-,3'-inverted T oligodeoxynucleotides exhibited less cytotoxicity and better stability than phosphorothioate oligodeoxynucleotides. When 5'-,3'-inverted T antisense MK was mixed with atelocollagen, and injected into CMT-93 tumors pregrown in nude mice, tumor growth was markedly suppressed as compared with tumors injected with sense controls. The suppressive effect of 5'-,3'-inverted T antisense MK on tumor growth was stronger than that of phosphorothioate antisense MK. These findings indicated the usefulness of inverted thymidine-modified antisense oligodeoxynucleotides as a new reagent instead of phosphorothioate-modified oligodeoxynucleotides.     

Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein-based assay. Discovery of selective and potent inhibitors of glutathione S-transferase Mu expression.

Antisense oligodeoxynucleotides (AS-ODNs) are frequently used for the down-regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness, fast screening methods for the selection of effective AS-ODNs are needed. We describe a new cellular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of the gene of interest and the gene encoding the enhanced green fluorescent protein (EGFP) are cotransfected with AS-ODNs to COS-7 cells. Subsequently, cells are analysed for expression of the EGFP fusion protein by flow cytometry. With the assay, we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S-transferase Mu1 (GSTM1) and/or Mu2 (GSTM2). We found several AS-ODNs that demonstrated potent, sequence-specific, and concentration-dependent inhibition of fusion protein expression. At 0.5 microm, AS-6 and AS-8 inhibited EGFP-GSTM1 expression by 95 +/- 4% and 81 +/- 6%, respectively. AS-5 and AS-10 were selective for GSTM2 (82 +/- 4% and 85 +/- 0.4% decrease, respectively). AS-2 and AS-3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77-95% decrease). Other AS-ODNs were not effective or displayed non-target-specific inhibition of protein expression. The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity. As isoform-selective, chemical inhibitors of GSTM and GSTM knock-out mice are presently unavailable, the selected AS-ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protection against chemical-induced carcinogenesis.     

TAT-CC fusion protein depresses the oncogenicity of BCR-ABL in vitro and in vivo through interrupting its oligomerization

Chronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the BCR-ABL protein. BCR-ABL is a constitutively active tyrosine kinase and plays a critical role in the pathogenesis of CML. Imatinib mesylate, a selective tyrosine kinase inhibitor, is effective in CML, but drug resistance and relapse occur. The coiled-coil (CC) domain located in BCR_1–72 mediates BCR-ABL tetramerization, which is essential for the activation of tyrosine kinase and transformation potential of BCR-ABL. CC domain is supposed to be a therapeutic target for CML. We purified a TAT-CC protein competively binding with the endogenous CC domain to reduce BCR-ABL kinase activity. We found that TAT-CC co-located and interacted with BCR-ABL in Ba/F3-p210 and K562 cells. It induced apoptosis and inhibited proliferation in these cells. It increased the sensitivity of these cells to imatinib and reduced the phosphorylation of BCR-ABL, CRKL and STAT5. We confirmed that TAT-CC could attenuate the oncogenicity of Ba/F3-p210 cells and diminish the volume of K562 solid tumor in mice. We conclude targeting the CC may provide a complementary therapy to inhibit BCR-ABL oncogenicity.     

多表位肽抗原诱导特异性CTL抗慢性髓性白血病细胞的体外实验研究

目的：在应用基因工程技术人工表达获得多表位BCR-ABL融合蛋白的基础上,对该融合抗原在体外诱导对自血病细胞的特异性杀伤效应进行检测,探索慢性髓性自血病（CML）免疫治疗的新途径。方法：从外周血单个核细胞培养树突细胞（DC）,以BCR-ABL融合抗原脉冲刺激DC,诱导特异性细胞毒T淋巴细胞（CTL）产生;MTT法检测CTL对白血病靶细胞的特异性杀伤活性。结果：以BCR-ABL融合抗原刺激产生的CTL能特异性抑制b3a2＋的靶细胞生长,包括K562细胞（P〈0．01）和HIJA-A2＋／b3a2＋的CML原代细胞（P〈0．05）,而对HIA-A2-或b2a2＋靶细胞无明显抑制作用。结论：设计表达的多表位BCR-ABL融合抗原能在体外诱导特异性抗CML免疫反应,抑制b3a2＋自血病细胞生长,有望为进一步的体内实验奠定基础。