血管内皮生长因子反义寡核苷酸抑制甲状腺癌血管生成的效应

目的探讨反义寡核苷酸(ASODN)抑制甲状腺癌细胞血管内皮生长因子(VEGF)表达及内皮细胞生长的效应。方法设计合成靶向VEGF的ASODN转染人髓状甲状腺癌细胞系(TT)细胞,并制备相应条件培养基作用内皮细胞ECV304,设正义寡核苷酸(SODN)和空白对照组进行比较。观察细胞生长状态,RT PCR、免疫细胞化学法检测TT细胞VEGFmRNA和蛋白表达,四氮唑蓝法检测TT和ECV304细胞生长抑制率(IR),流式细胞仪、吖啶橙/溴化乙锭染色法检测ECV304细胞凋亡状态。结果ASODN组TT细胞VEGFmRNA和蛋白表达显著低于SODN和对照组(P<0.01),但IR差异无统计学意义(P>0.05);各转染组ECV304细胞IR差异亦无统计学意义(P>0.05);而经各ASODN组TT细胞条件培养基作用的ECV304细胞生长明显受抑,IR(分别为0.21±0.03、0.31±0.01、0.42±0.22)显著高于SODN组(0.05±0.03,P<0.01),并伴明显细胞凋亡,上述效应呈浓度依赖性。结论ASODN可通过特异性封闭甲状腺癌细胞VEGF表达,抑制内皮细胞生长,干扰肿瘤血管生成。     

靶向抑制存活素表达对卵巢癌细胞株生物学行为的影响和意义

目的：通过存活素（ Survivin）反义寡核苷酸（ ASODN）转染人卵巢癌SKOV3细胞株抑制存活素Survivin蛋白表达,分析靶向抑制 Survivin 表达对卵巢癌细胞生长、细胞周期改变、凋亡、侵袭迁移能力的影响和意义。方法用脂质体（LipofectamineTM2000）介导Survivin ASODN转染人卵巢癌细胞株SKOV3,通过四唑盐比色法（MTT法）分析细胞生长活性改变,通过流式细胞仪检测Survivin蛋白表达改变、细胞周期分布和细胞凋亡率,通过Transwell小室检测细胞侵袭迁移能力的改变。结果转染Survivin ASODN后卵巢癌细胞株中Survivin蛋白表达明显下降（t＝7．82,P＜0．01）。细胞生长明显减慢（F＝3．75,P＜0．01）,600nm/L ASODN转染48小时细胞存活率为49．50±20．76％,接近IC50值。细胞凋亡明显增加（t＝6．37,P＜0．05）,细胞增殖明显下降（t＝-10．35,P＜0．01）,更多的细胞停留在G0/G1期（t＝10．38,P＜0．01）。细胞迁移和侵袭能力下降（t值分别为19．26、27．42,均P＜0．01）。结论 Survivin ASODN通过靶向抑制卵巢癌细胞存活素表达,可抑制细胞生长,促进细胞凋亡,减少进入有丝分裂的细胞数,降低细胞的侵袭迁移能力,从而达到治疗卵巢癌的作用。靶向抑制存活素的治疗将成为卵巢癌治疗的重要手段。     

端粒酶反义寡核苷酸抑制端粒酶活性和诱导肺腺癌A549细胞凋亡的研究

目的 研究端粒酶反义寡核苷酸在肺腺癌A549细胞中抑制端粒酶活性和诱导细胞凋亡的能力.方法 用脂质体将荧光标记的端粒酶反义寡核苷酸转染入肺腺癌A549细胞后,TRAP-PCR-ELISA检测端粒酶活性变化,凋亡细胞用透射电镜(TEM)观察,TUNEL法检测各组细胞凋亡率.结果 转染72h后,端粒酶反义寡核苷酸进入A549细胞内,端粒酶活性显著降低.透射电镜下,端粒酶反义寡核苷酸转染组细胞凋亡增加,细胞凋亡率比其他组明显增高,差异有统计学意义(P＜0.01).结论 端粒酶反义寡核苷酸能靶向端粒酶,抑制其活性,并诱导肺腺癌A549细胞凋亡.     

Ezrin蛋白mRNA的一、二级结构分析和反义技术作用靶点设计

目的 分析肿瘤远处转移相关蛋白Ezrin Mrna的一、二级结构,寻找其各种反义技术作用的靶点.方法 利用RNAdraw和RNAstructure两种计算机程序对Ezrin Mrna进行分析计算,得到其一、二级结构信息,选择两种程序计算出的共同的连续4个以上碱基未配对的单链成环区作为反义技术的靶区域,设计反义寡核苷酸(AsODNs)、核酶(Ribozyme)、脱氧核酶(DNAzyme or Deoxyribozyme)和小干扰RNA(SiRNA)等4种常用反义技术的作用靶点,再通过计算机程序OligoWalk利用最低自由能原则进行筛选,最后得到各反义技术的作用靶点.结果 各反义技术的靶点数目分别为:AsODNs17个,Ribozyme5个,DNAzymel5个,SiRNA10个;其中AsODNs靶点中较为理想的为G1749-A1771和C1777-G1794,Ribozyme为A2359-G2360,DNAzyme为A1789-U1790、A1756-G1757及A1784-G1785,SiRNA为G802-G808、G1749-A1771及A2354-G2360,并且A2354-G2360是所有4种反义技术共同的作用区域,可以作为一个通用靶点.结论 正确的靶点设计为应用反义技术抑制Ezrin表达从而控制恶性肿瘤的远处转移提供了有效的指导作用.     

KDR反义寡核苷酸对人前列腺癌PC-3细胞增殖调控的研究

目的探讨血管内皮生长因子受体2(kinase insert domain-containing receptor,KDR)反义寡核苷酸(antisense oligode-oxynucleotides,ASODN)对人前列腺癌PC-3细胞的增殖调控作用。方法设计并合成KDR的正义、反义寡核苷酸,经脂质体介导转染人前列腺癌PC-3细胞。用噻唑盐法观察不同浓度(0.01,0.05,0.1,0.2,0.4μmol/L)正义、反义寡核苷酸及阳离子脂质体对肿瘤细胞增殖的抑制作用,RT-PCR方法检测不同浓度(0.01,0.05,0.1,0.2,0.4μmol/L)ASODN转染后KDRmRNA的表达情况,流式细胞仪分析细胞周期分布和细胞凋亡。结果转染ASODN后48 h达抑制高峰,低浓度(0.01μmol/L)即发生抑制,抑制强度与反义寡核苷酸浓度有相关性,而正义寡核苷酸和阳离子脂质体对PC-3细胞的增殖无显著影响。转染KDR反义寡核苷酸各浓度组细胞中KDR mRNA的表达都不同程度地降低,各浓度组与空白对照组相比KDR mRNA表达的差异有统计学意义。各组均出现不同程度的细胞凋亡,但各浓度组间细胞周期分布无显著性差异。结论 KDR基因在促进人前列腺癌PC-3细胞增殖中发挥着一定的作用,有望成为治疗雄激素非依赖性前列腺癌的分子靶点。     

特定寡核苷酸对大鼠实验性牙移动的影响

目的：探讨特定寡核苷酸(ODN)对大鼠实验性牙移动的影响,阐明其在牙周组织改建中的作用特点。方法：40只雄性Wistar大鼠,随机分为实验组（n=20）和对照组（n=20）,建立大鼠牙移动模型。加力当天于实验组大鼠两侧上颌第一磨牙颊侧牙龈黏膜下各注射50μL特定ODN(20 mg· L-1),对照组则于相同部位注射等量...     

端锚聚合酶1反义寡核苷酸对人肺癌细胞增殖的影响

目的　探讨端锚聚合酶 1(tankyrase 1,TANK1)反义寡核苷酸对人肺癌细胞的影响 ,为肺癌治疗的靶向研究探索新途径。方法　合成TANK1正义寡核苷酸 (TANK1 SODN)、反义寡核苷酸(TANK1 ASODN) ,待人肺癌细胞株CALU在裸鼠蹊部皮下接种成瘤后 ,将成瘤裸鼠随机分成 3组 :对照组 4只 (每天上午于瘤体内多位点注射生理盐水 2 0 0 μl/只 ) ,正义组 (每天同法注射含TANK SODN10 0 μg的生理盐水 2 0 0 μl/只 )和反义组 (每天同法注射含TANK ASODN 10 0 μg的生理盐水 2 0 0 μl/只 )各 5只 ,观察肿瘤的生长情况及组织学改变。用链霉亲和素 生物素 酶复合物 (SABC)法检测肿瘤细胞Ki6 7及端粒酶反转录酶 (hTERT)的阳性表达率。结果　在裸鼠成瘤部位连续注射相应液体 16d后 ,反义组移植瘤体积为 (1 4 6± 0 70 )cm3 ,明显小于对照组的 (3 2 9± 0 4 7)cm3 及正义组的 (3 14±0 70 )cm3 ,差异有显著性 (P均 <0 0 1) ;肿瘤细胞Ki6 7标记指数及hTERT细胞阳性率均明显低于对照组及正义组 (P均 <0 0 1)。结论　人肺癌细胞株端粒酶呈高度表达 ,TANK1 ASODN可降低瘤细胞端粒酶活性 ,抑制肺癌细胞增殖     

Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

A functional RNase P ribozyme (M1GS RNA) was constructed to target the overlapping mRNA region of two murine cytomegalovirus (MCMV) capsid proteins essential for viral replication: the assembly protein (mAP) and M80. The customized ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in cells that either did not express the ribozyme or produced a “disabled” ribozyme carrying mutations that abolished its catalytic activity. When the ribozyme-expressing constructs were delivered into MCMV-infected SCID mice via a modified “hydrodynamic transfection” procedure, expression of ribozymes was observed in the livers and spleens. Compared with the control animals that did not receive any M1GS constructs or received the disabled ribozyme construct, animals receiving the functional ribozyme construct exhibited a significant reduction of viral gene expression and infection. Viral titers in the spleens, livers, lungs, and salivary glands of the functional ribozyme-treated SCID mice at 21 days after infection were 200- to 2,000-fold lower than those in the control animals. Moreover, survival of the infected animals significantly improved upon receiving the functional ribozyme construct. Our study examines the use of M1GS ribozymes for inhibition of gene expression in animals and demonstrates the utility of RNase P ribozymes for gene targeting applications in vivo.     

Low expression of long non-coding RNA ARAP1-AS1 can inhibit lung cancer proliferation by inducing G0/G1 cell cycle organization

BackgroundThis paper examines the expression, function, and molecular mechanism of long non-coding ribonucleic acid (lncRNA) ARAP1 antisense RNA 1 (ARAP1-AS1) in lung cancer. Specifically, it aims to clarify the molecular mechanism of lncRNA ARAP1-AS1 that affects the occurrence and development of lung cancer, and provide a theoretical basis and molecular targets for targeted therapy or early diagnosis of lung cancer.MethodsFluorescence quantitative detection of lncRNA ARAP1-AS1 expression in lung cancer tissues and cell lines, and methylthiazolyldiphenyl-tetrazolium (MTT), plate cloning experiment, and flow cytometry were used to detect the effect of knockdown of lncRNA ARAP1-AS1 on cell proliferation, clone formation, and the cell cycle, respectively. Western blotting was used to detect the expression of cell cycle-related proteins as well as the effect of knockdown of lncRNA ARAP1-AS1 on lung cancer. Cell proliferation was assessed by a nude mouse subcutaneous tumor formation experiment.ResultsLncRNA ARAP1-AS1 is highly expressed in lung cancer tissues and cells. Knockdown of LncRNA ARAP1-AS1 can significantly inhibit the proliferation and clonal formation of lung cancer cells and induce G0/G1 cell cycle arrest. Knockdown of ARAP1-AS1 can markedly inhibit the expression of cell cycle-related protein cyclin D1, but has no significant effect on the expression of cyclin-dependent kinase (CDK)4 and CDK6. Furthermore, knockdown of ARAP1-AS1 can also notably inhibit the growth of lung cancer cells and substantially reduce the expression of Ki-67 in tumor-bearing tissues in nude mice.ConclusionsLncRNA ARAP1-AS1 is highly expressed in lung cancer. Knocking down of this gene can significantly inhibit cell proliferation in vitro and in vivo, and can also cause G0/G1 cell cycle arrest by inhibiting the expression of cyclin D1.     

Nano-MK-ASODN对人肝癌裸鼠原位移植模型的影响

目的 探讨中期因子反义寡核苷酸纳米脂质体（Nano-MK-ASODN）对人肝癌裸鼠原位移植模型的抑制作用。方法 利用人肝癌裸鼠原位移植模型,观察受试药品中期因子反义寡核苷酸（MK-ASODN）和Nano-MK-ASODN对荷瘤裸鼠体重、瘤重、以及肿瘤生长的抑制率的影响。检测荷瘤裸鼠血常规和血浆甲胎蛋白水平。结果 MK-ASODN高剂量组25 mg.kg-1组的瘤重抑制率为53.72%,12.5 mg.kg-1组的瘤重抑制率为48.76%,6.25 mg.kg-1组的瘤重抑制率为42.98%,Nano-MK-ASODN 25 mg.kg-1剂量组抑制率为66.94%,12.5 mg.kg-1组的瘤重抑制率为56.20%,6.25 mg.kg-1组的瘤重抑制率为52.07%,所有试验组肿瘤重量与溶剂对照组比较均有显著差异,P<0.01。Nano-MK-ASODN组肿瘤重量明显小于同等剂量MK-ASODN,P<0.01。Nano-MK-ASODN治疗组肿瘤体积以及AFP水平明显小于对照组,P<0.01。治疗组瘤重明显小于生理盐水对照组,P<0.05。Nano-MK-ASODN各治疗组及用药后体重、血常规与对照组比较指标差异均无显著性意义,P>0.05。同时,解剖学和病理组织学检查显示,Nano-MK-ASODN治疗组荷瘤裸鼠的肝内肿瘤体积缩小,并可见液化,能使部分肝癌细胞变性坏死。结论 Nano-MK-ASODN对人肝癌裸鼠原位移植瘤具有显著的抑制作用,且效果显著强于MK-ASODN。