反义硫代磷酸酯寡聚核苷酸及其应用研究进展

硫代磷酸酯寡聚核苷酸(PS-ODN)是一类颇有应用前景的反义核酸,本文结合最新研究进展,就PS-ODN作为反义药物所需具备的条件和在抗病毒、抗肿瘤及遗传学分析和分子生物学研究中的应用作了较为详尽的论述。     

端粒酶反义寡聚脱氧核苷酸对人胃癌细胞的影响

目的：观察端粒酶反义寡聚脱氧核苷酸对人胃癌细胞株的作用。方法：用端粒酶反义寡聚脱氧核苷酸与人胃癌细胞株共同温育一定时间后，观察细胞形态变化，检测细胞DNA含量的分布，测定端粒酶活性。结果：端粒酶反义寡聚脱氧核苷酸能诱导人胃癌细胞凋亡，抑制端粒酶活性，在形态学上表现为细胞膜起泡、染色质固缩、核碎裂、凋亡小体形成；电泳呈凋亡特征性Ladder带；流式细胞仪分析显示，在G1期前出现亚2倍体凋亡峰；端粒酶活性抑制。结论：端粒酶反义寡聚脱氧核苷酸能诱导胃癌细胞凋亡，抑制端粒酶活性，抑制端合成，从而抑制胃癌细胞的增殖。     

端粒酶反义寡聚脱氧核苷酸诱导人胃癌细胞凋亡模型构建的研究

目的：观察端粒酶反义寡聚脱氧核苷酸对胃癌细胞株的作用。方法：用端粒酶反义寡聚脱氧核苷酸与胃癌细胞株共同孵育一定时间后,观察细胞形态变化,观察细胞DNA含量的分布,测定端粒酶活性。结果：端粒酶反义寡聚脱氧核苷酸能诱导胃癌细胞凋亡,抑制端粒酶活性,在形态学上表现为细胞膜起泡,染色质固缩,核碎裂,凋亡及小体形成;电泳呈凋亡特征性Ladder带;流式细胞仪分析显示,在D1期前出现亚2倍体凋亡峰;端粒酶活性抑制。结论：端粒酶反义寡取脱氧核苷酸能诱导胃癌细胞凋亡,抑制端粒酶活性,抑制端粒合成,从而抑制胃癌细胞的生长。     

bcl-2反义寡聚脱氧核苷酸抑制HL-60细胞生长的研究

在体外培养中研究了基因的反义寡聚脱氧核苷酸(antisense oligodeoxynucleo-tide,ASON)对HL-60细胞生长增殖的影响。根据bcl-2基因cDNA的序列分析,以bcl-2mRNA翻译起始区域前15个核苷酸为作用靶序列,人工合成了15聚ASON片段,同时合成15聚无关寡聚核苷酸(ODN)片段,以作序列特异性对照。把上述ASON和无关ODN片段与HL-60细胞共培养,同时设置空白对照组,作用一定时间后分别测定细胞动力学和~3H-TdR掺入率。实验发现:从3.5—30.0μmol/L的ASON对细胞生长有不同程度抑制作用,低于10μmol/L时抑制作用小,大于10.0μmol/L时抑制作用显著,浓度愈大,抑制程度愈大,而无关ODN作用组的细胞生长状态与空白对照组无显著差别,作用48小时后浓度3.5μmol/L抑制率为6％,而20.0μmol/L时上升为40％;作用96小时后3.5μmol/L的ASON抑制率是5.1％,30μmol/L的抑制率高达67％。相同浓度的ASON作用不同时间后,细胞生长率各不相同,当浓度达到14.0μmol/L时,48小时的细胞生长抑制率达22％,72和96小时的抑制率分别为25％和29.3％。HL-60细胞被ASON处理后,培养体系中死细胞数随时间延长而增加,浓度在10—30μmol/L范围内,死细胞率由2％增到50％,但在无关ODN及空白对照组中未观察到这种现象。同时发现由于bcl-2的ASON作用使HL-60细胞的     

鞘内注射TLR3反义寡聚核苷酸对脊神经结扎大鼠的镇痛作用

目的:探讨脊髓星形胶质细胞TLR3在神经病理性疼痛中的作用.方法:雄性SD大鼠,体重180～250 g.实验一:72只鞘内置管大鼠随机分为:生理盐水(Ns)20 μl脊神经结扎(SNL)(A 组),错义寡聚核苷酸(MM-ODN)20μg/d+SNL(B组),反义寡聚核苷酸(AS.ODN)20μg/d+SNL (C组).实验二:72只SNL后7天大鼠随机分为:SNL+NS 20μl(D组),SNL+MM-ODN 20μg/d(E组),SNL+AS-ODN 20μg/d(F组).实验一和实验二分别于SNL前1天和后7天开始鞘注,每天一次,共7天;于术后-1、1、3、5、7、10、14天和-1、7、10、12、14天行行为学实验.实验一和实验二分别于术后7天和14天每组各随机取6只大鼠利用免疫组织化学法观察脊髓背角GFAP的表达,并于术后-1、3、7、14天和术后7、10、14天每组各随机取6只大鼠断头处死,取L4～5脊髓节段采用RT-PCR法测定TLR3和IL-6 mRNA的表达.结果:实验一:与A组比较,C组PWPT升高并持续至术后14天(P<0.05),C组各时间点PWL无明显变化(P>0.05);与A组和B组比较,C组脊髓背角GFAP免疫反应阳性产物表达相对面积分别下降46.4%(P<0.01)和45.7%(P<0.01);与A组和B组比较,C组鞘内注射AS-ODN抑制了TLR3受体和IL-6 mRNA的表达.实验二D组、E组和F组在PWPT、PWL、GFAP表达和TLR3及IL-6 mRNA表达方面的差异无统计学意义(P>0.05).结论:脊髓背角星形胶质细胞TLR3可能参与了神经病理性疼痛的发生与发展.     

以阳离子脂质体为载体增强反义寡脱氧核苷酸作用的研究

反义核酸作为基因治疗的新方法,在近年来得到了迅速发展,其中反义寡脱氧核苷酸(ASODN)因制备客易而得到广泛的应用。然而,它的作用效率因细胞的低摄入及核酸酶的降解而受到影响。实验中采用以阳离子脂质体为载体的bcr-abl mRNA ASODN作用于K562细胞(CML细胞系)的方法,研究了阳离子脂质体所起的作用。结果表明,以阳离子脂质体介导转染ASODN增加了细胞对ASODN摄入量和抗核酸酶降解的能力。结论提示该方法为ASODN临床应用提供一条新的途径。     

聚乙烯亚胺包被的四氧化三铁磁性纳米颗粒对SHI-1细胞生物学特性的影响

目的 初步探讨利用聚乙烯亚胺包被的四氧化三铁(PEI-Fe3O4)磁性纳米颗粒进行磁共振细胞影像技术跟踪细胞生物学行为的可行性.方法 以人急性单核细胞白血病细胞株SHI-1为对象,用透射电镜观察PEI-Fe3O4磁性纳米颗粒能否被细胞内吞;用电感耦合等离子发射光谱仪及普鲁士蓝染色观察影响细胞磁性纳米颗粒载量的因素;用CCK-8法检测PEI-Fe3O4对SHI-1细胞增殖的影响;用流式细胞术(FCM)检测PEI-Fe3O4磁性纳米颗粒对细胞分化的影响;以甲基纤维素半固体培养基培养法检测PEI-Fe3O4对细胞集落形成能力的影响.以未经PEI-Fe3O4磁性纳米颗粒处理的SHI-1细胞作为对照组.结果 PEI-Fe3O4磁性纳米颗粒能成功被SHI-1细胞内吞,细胞内磁性纳米颗粒载量与其起始浓度(以纳米颗粒中的铁离子浓度代表PEI-Fe3O4浓度,即μg Fe/ml)及与细胞共培养的时间呈正相关,随细胞分裂传代而减少.以5～100 μg Fe/ml PEI-Fe3O4处理细胞,60 ～ 100μgFe/ml各组对细胞增殖抑制率高于5～50 μg Fe/ml各组,差异具有统计学意义(P＜0.05);FCM检测对照组细胞CD11b和CD14分子的表达分别为(78.4±18.5)％和(18.7±2.9)％,佛波醇酯(TPA)处理后增至(92.1±6.5)％和(20.8±2.3)％,PEI-Fe3O4标记后的细胞CD11b和CD14分子的表达分别为(83.3±14.2)％和(20.4±2.1)％,TPA处理后增至(95.8±3.3)％、(21.0±6.9)％,实验组和对照组间在TPA处理前后的CD11b和CD14表达差异无统计学意义(P＞0.05);0、20、50 μg Fe/ml PEI-Fe3O4处理24 h后的SHI-1细胞集落形成率分别为(25.20±7.22)％、(25.93±13.15)％和(23.37±9.33)％,各组间差异无统计学意义(P＞0.05).结论 PEI-Fe3O4磁性纳米颗粒可高效率标记SHI-1细胞,在5 ～50 μg Fe/ml范围内,与SHI-1细胞具有很好的生物相容性,不影响SHI-1细胞的生物学行为,可作为磁共振细胞影像技术的标记物.     

PLGA纳米颗粒作为基因治疗载体的实验研究

目的：探讨PLGA材料构建的纳米粒载体导入表皮生长因子受体（EGFR）反义寡核苷酸在头颈鳞癌基因治疗中的可行性,为头颈肿瘤基因治疗中载体的选择提供一个新的研究思路。方法：以PLGA为材料,采用油包水双乳化溶剂蒸发法制备载EGFR正义、反义寡核苷酸纳米颗粒;纳米颗粒转染SCCⅦ细胞株;MTT法了解纳米颗粒对细胞的毒性;通过实时荧光定量PCR检测转染后EGFR基因mRNA表达水平。结果：获得了制备载寡核苷酸PLGA纳米颗粒工艺流程,PLGA纳米颗粒平均粒径116 nm±7.57 nm。纳米颗粒体外转染SCCⅦ细胞,MTT结果显示纳米颗粒对细胞生长无明显抑制效应,同时具有明显抑制EGFR基因mRNA表达效应。结论：PLGA纳米颗粒可以有效地载入反义寡核苷酸,达到抑制靶基因的效果,同时无明显的细胞毒性。     

聚乙二醇修饰后mdr—1基因的反义寡核苷酸对K562＼AO2细胞的作用

目的：探讨聚乙二醇对反义寡核苷酸的生物利用度的影响。方法：针对多耐药基因的反义寡聚脱氧核糖核苷酸靶向第二外显子跨越起始密码子ＡＵＧ的－６－９位点，在其５’端连接聚乙二醇的ＡＳ’的５’端连接聚乙二醇；在少１个碱基的ＡＳ’的５’端连接端连接二离。从ｍｄｒ－１ｍＲＮＡ、ｐ１７０、细胞内柔红霉素浓度以及细胞对阿霉素的敏感性等各个水平上比较这几种反义寡核苷酸人耐药白血病系Ｋ５６２／ＡＯ２的作用，结果ＡＰ’能     

多药耐药基因反义寡核苷酸逆转肿瘤细胞耐药的初步研究

目的：克服肿瘤细胞的多药耐药（ＭＤＲ）。方法：用人工合成互补于ｍｄｒ１基因５′端转录起始部位的反义寡核苷酸（ＯＤＮ），直接转染耐药细胞株ＫＢ－８－５细胞，或以脂质体ｌｉｐｏｆｅｃｔｉｎ为载体进行基因转染实验，通过ＭＴＴ法检测细胞对柔红霉素（ＤＮＲ）的敏感性，流式细胞仪分析细胞内ＤＮＲ含量及免疫组化方法确定细胞表面糖蛋白（Ｐｇｐ）的表达水平。结果：ＯＤＮ可增加ＫＢ－８－５细胞内的ＤＮＲ浓度从而提高耐药细胞对ＤＮＲ的敏感性，ｌｉｐｏｆｅｃｔｉｎ进一步加强上述作用。在ＤＮＲ浓度为３．０ｍｇ／Ｌ组中，约有７４．４３％的被转染细胞对ＤＮＲ敏感而致死，基本达到药物敏感细胞株ＫＢ－３－１的水平。ＯＤＮ转染的ＫＢ－８－５细胞的Ｐｇｐ为弱阳性表达，低于阳性对照ＫＢ－８－５细胞的Ｐｇｐ表达水平。结论：ＯＤＮ的逆转作用可能与其互补结合的ｍｄｒ１ｍＲＮＡ降解或直接阻滞了Ｐｇｐ合成使其表达降低有关。     

PEGFP-C1/U6质粒载体介导的表达MDR1 shRNA的质粒构建

为了构建pEGFP-C1／U6载体介导MDR1短发卡RNA（short hairpin RNA，shRNA）表达的质粒，针对MDR1的19碱基大小的片段．分别设计2对寡核苷酸，形成双链后将其依次连入带有U6启动子的pEGFP-C1载体（命名为pEGFP-C1／U6），两对DNA双链连接后形成中间由9个碱基序列间隔的反向互补序列，构建成能产生MDR1短发卡RNA的质粒。结果表明：经酶切、连接后构建成的质粒（pEGFP-C1／U6／A和pEGFP-C1／U6／B），经酶切与测序证实构建成功，无任何碱基突变。结论：成功构建了能表达MDR1 shRNA的质粒栽体pEGFP-C1／U6／A和pEGFP-C1／U6／B，此项研究结果可能为临床上逆转肿瘤多药耐药提供一种有效的方法。     

Detection of the four sequence variations of MDR1 gene using TaqMan MGB probe based real-time PCR and haplotype analysis in healthy Japanese subjects

OBJECTIVES: P-glycoprotein (P-gp) is significant from the viewpoint of pharmacokinetics/pharmacodynamics (PK/PD). MDR1 gene encodes P-gp and has a wide variety of SNPs. As the SNPs may be one of the factors that induce pharmacogenetic individual difference, haplotype analysis is necessary to evaluate the PK/PD. DESIGN AND METHODS: The SNPs of the detected MDR1 were -129T>C, 325G>A, 2677G>T/A, and 3435C>T. For the analysis of linkage disequilibrium (LD) and haplotype analysis, and for the reconstruction of the haplotype pair, ARLEQUIN and PHASE were employed. RESULTS: The result of the chi(2) test detected significant LD between -129 and 2677, -129 and 3435, and 2677 and 3435. There were 9 haplotypes: T-G-C, T-T-C, C-T-C, T-A-C, C-A-C, T-G-T, T-T-T, C-G-T, and C-T-T. CONCLUSIONS: LD was found among the positions -129, 2677 and 3435. As a result, 9 haplotypes exists in the Japanese population. These results suggest that it would be necessary to give consideration to haplotype for the purpose of evaluating the PK/PD of the drugs transported by P-gp.     

多药耐药基因QRT-PCR检测中内标准DNA和RNA模板的构建

利用多药耐药基因(MDR1)全基因质粒和分子克隆技术,经二次克隆将戊型肝炎病毒一段238bP的核苷酸序列插入MDR1 308 bp的目的 cDNA片段,构建了插入突变型MDR1-cDNA重组体作为定量PCR的DNA竞争模板;再经第三次克隆构建了插入突变型MDR1-RNA重组体,最后经体外转录出546 nt的正链RNA作为逆转录的RNA竞争模板。所建立的定量逆转录-多聚酶链反应(QRT-PCR)技术简便、快速、灵敏,可检出1fg水平的MDR1 mRNA,为临床MDR1表达的定量检测提供了确实可行的手段。     

The effects of the human MDR1 genotype on the expression of duodenal P-glycoprotein and disposition of the probe drug talinolol

BACKGROUND AND OBJECTIVES: A single-nucleotide polymorphism (SNP) of the human multidrug-resistance gene in wobble position of exon 26 reportedly predicts expression and function of P-glycoprotein in human enterocytes and lymphocytes. Several other allelic variants of MDR1 have been identified, some of which lead to amino acid exchange with as yet unknown functional relevance. METHODS: In healthy white volunteers, we investigated the influence of the hereditary variants C3435T in exon 26 and G2677T/A (Ala893Ser/Thr) in exon 21 and the influence of 7 frequent or putative functional SNPs on duodenal MDR1 messenger ribonucleic acid (n = 32) and immunoreactive P-glycoprotein (n = 37) expression. Moreover, the disposition of the probe drug talinolol was evaluated in 55 subjects after oral administration (100 mg) and in 23 subjects after intravenous administration(30 mg). RESULTS: Duodenal MDR1 messenger ribonucleic acid and P-glycoprotein, as assessed by real-time polymerase chain reaction (TaqMan) and immunostaining, were not influenced by any MDR1 polymorphism studied. Talinolol disposition was not affected by the exon 26 mutation C3435T. In carriers of the TT/TA variants of G2677T/A, the area under the serum concentration-time curve values of oral talinolol were slightly but significantly elevated compared with those in carriers of at least 1 wild-type allele (P <.05, Kruskal-Wallis test; P =.014, Mann-Whitney U test). However, multiple comparisons with combinations of putative functional SNPs did not confirm a significant influence of the MDR1 genotype on talinolol disposition. CONCLUSIONS: We did not identify any influence of MDR1 genotypes on duodenal expression of P-glycoprotein and disposition of talinolol in humans.     

GMBP1-conjugated manganese oxide nanoplates for in vivo monitoring of gastric cancer MDR using magnetic resonance imaging

Multidrug resistance (MDR) is a huge challenge for gastric cancer chemotherapy. Therefore, MDR accurate monitoring is of great significance for the treatment of gastric cancer. GMBP1, an extracellular internalization peptide, can target MDR gastric cancer cells through specific binding to GRP78, which is an MDR-related protein that is overexpressed in gastric cancer cells. Herein, we constructed GMBP1 conjugated Mn₃O₄ nanoplates (Mn₃O₄@PEG-GMBP1 NPs) for in vivo monitoring of MDR gastric cancer through magnetic resonance imaging (MRI). The generated Mn₃O₄@PEG-GMBP1 NPs had a size of about 11 nm and exhibited a good colloidal stability in PBS and in 10% FBS medium. Serial in vivo MRI studies in mice demonstrated that the magnetic resonance signal intensity, at the tumor site, reached a peak at 3 h after tail vein injection of Mn₃O₄@PEG-GMBP1 NPs. The specific targeting ability of MDR gastric cancer cells (SGC7901/ADR) by Mn₃O₄@PEG-GMBP1 NPs was authenticated in vitro, in vivo and by immunofluorescence analysis experiments. The systematic safety evaluation indicated that the toxicity of Mn₃O₄@PEG-GMBP1 NPs in mice was negligible. Therefore, the GMBP1 conjugated Mn₃O₄ nanoplates can be clinically used for accurate imaging and monitoring of MDR gastric cancer.

GMBP1 conjugated manganese oxide nanoplates for in vivo monitoring multidrug resistance of gastric cancer through magnetic resonance imaging.     

Genetic polymorphisms in MDR1 and CYP3A5 and MDR1 haplotype in mainland Chinese Han, Uygur and Kazakh ethnic groups

BACKGROUND AND OBJECTIVE: The drug transporter MDR1 and the drug metabolizing enzyme CYP3A are the two major biological factors determining the pharmacokinetics of many drugs. The functional MDR1 single nucleotide polymorphisms (SNPs) and a prevalent CYP3A5 SNP show marked interethnic variation among Orientals, Caucasians and Africans. In this study, we investigated the distribution of MDR1 and CYP3A5 SNPs among mainland Chinese Han, Uygur and Kazakh ethnic groups. METHODS: Genotypes of the MDR1 C1236T, G2677T/A and C3435T, and CYP3A5*3, CYP3AP1*3 SNPs were determined in 434 unrelated healthy subjects (165 Chinese Han, 161 Chinese Uygur and 108 Chinese Kazakh) using polymerase chain reaction followed by restriction fragment length polymorphism analysis. RESULTS AND DISCUSSION: A significantly higher MDR1 3435T variant frequency was observed in Uygur (52.8%), than in Kazakh (39.8%) and Han (37.9%) Chinese (P < 0.01, Fisher's exact test). There was no significant difference in MDR1 1236T and 2677T/A variant frequencies between Han, Uygur and Kazakh. CYP3A5*3 (G) allele was observed at intermediate frequencies in Uygur (84.8%) and Kazakh (86.6%), relative to Han (72.7%) and values previously reported in Caucasians (91.7%). The CYP3AP1*3 (A) allele was strongly linked to CYP3A5*3 in Chinese Han, Uygur and Kazakh. CONCLUSION: Significant interethnic differences in MDR1 haplotype and CYP3A5 variant frequencies exist between mainland Chinese Han and Caucasians, and the intermediate frequencies observed in Chinese Uygur and Kazakh might be due to the genetic admixture of Eurasians and Orientals.