反义RNA在基因治疗中的应用

由于反义RNA作为封闭基因表达的有效手段具有特异性强、安全性高、操作简单、靶基因范围广等特点,已被广泛应用于基因治疗肿瘤和病毒相关疾病的研究,反义RNA治疗肿瘤可以通过抑制癌基因的表达、封闭融合癌基因、抑制肿瘤细胞的耐药性、调节细胞因子的表达量等途径;反义RNA治疗病毒相关疾病多集中在艾滋病上,其手段主要是反义封闭TAR。反义RNA作为基因治疗的新途径具有良好的前景,但在设计上和应用上还存在一些急待解决的问题。     

核酶的抗病毒作用

反义核酸技术（ａｎｔｉｓｅｎｓｅｎｕｃｌｅｉｃａｃｉｄｓｔｅｃｈｎｏｌｏｇｙ）是根据核酸杂交原理设计的以选择性地抑制特定基因表达为目的的一类核酸研究新技术,它包括反义ＲＮＡ（ａｎｔｉｓｅｎｓｅＲＮＡ,ａｓＲＮＡ）、反义ＤＮＡ（ａｎｔｉｓｅｎｓｅＤＮＡ,ａｓＤＮＡ）及核酸（ｒｉｂｏｚｙｍｅ,Ｒｚ）三大技术领域,反义核酸技术同基因治疗一样已经成为一项引人瞩目研究领域。本文对核酸作用的原理和在抗病毒方面的研究进展作一综述     

反义基因治疗

除了反义核酸和核酶外,最近又发展了一种新型的反义药物———反义肽核酸(PNA)。反义治疗的经典策略是阻断异常基因的表达;随着研究的深入,又发现了以反义药物调整基因表达比例(即调控基因治疗)的反义治疗途径。本文对反义基因治疗的策略、反义药物的设计及稳定性等方面的新思路和该领域的发展与应用前景作了概括介绍。     

siRNA作用效果的靶点依赖性

小分子双链RNA(siRNA)可以高效、特异地沉默目的基因表达 ,为基因功能研究及基因治疗提供了新工具。近年来研究表明针对基因mRNA不同位点随机设计的siRNA在作用效果上存在差异 ,siRNA作用效果有序列依赖性 ,而且与其在基因mRNA上的结合部位的高级结构有关 ,与反义核酸发挥作用的靶点依赖性类似。这一性质对设计高效siRNA为基因功能和基因治疗研究提供指导作用。     

反义基因治疗

除了反义核酸和核酶外,最近又发展了一种新型的反义药物－反义肽核酸（ＲＮＡ）。反义治疗的经验策略是阻断异常基因的表达;随着研究的深入,又发现了以反义药物调整基因表达比例（即调控基因治疗）的反义治疗途径。本文对反义基因治疗的策略,反义药物的设计及稳定性等方面的新思路和该领域的发展与应用前景作了概括介绍。     

12－烷基化壳聚糖纳米粒-反义内皮素转换酶核酸表达质粒的制备及其性质的研究

摘要：目的：气道给予12－烷基化壳聚糖纳米粒（12－ACSs）包裹的反义内皮素转换酶（ECE）核酸表达质粒,观察对OVA致敏的小鼠变应性气道炎症的影响。方法：通过透射电镜观察12－ACSs/ 反义 ECE质粒复合体纳米粒的形成、形态及大小;应用凝胶阻滞、结合平衡、DNA沉淀和DNA酶消化实验等检测12－ACSs对反义ECE核酸表达质粒的结合保护作用;通过MTT实验检测12－ACSs对细胞的毒性;通过离体培养细胞及活体动物转染实验观察12－ACSs能否携带反义ECE核酸表达质粒成功转染。结果：电镜观察示纳米粒粒径在100－150 nm之间。12－ACSs与反义ECE核酸表达质粒在质量比为1：1时,全部反义ECE质粒被结合。应用DNase I消化后可见,12－ACSs可保护核酸免受破坏。MTT检测结果显示12－ACSs 对16HBE细胞在低浓度下几乎没有毒性。12－ACSs包裹的反义ECE核酸表达质粒的纳米粒能成功转染离体培养的气道上皮细胞及活体动物。结论：12－ACSs能够成功包裹反义ECE质粒并且成功转染16HBE及小鼠,其有可能作为一种基因治疗的载体选择之一。 关键词：哮喘 壳聚糖 纳米粒 内皮素转换酶 基因治疗     

CyclinD1反义核酸诱导肺腺癌细胞A549的凋亡

为了探讨CyclinD1反义核酸在肺癌基因治疗中的作用,本研究构建了含有CyclinD1反义核酸的表达载体(命名为:pcDNA3.1-CyclinD1),并将该载体转染肺腺癌细胞A549,进行G418筛选后,获得稳定表达CyclinD1反义核酸的A549细胞(命名为:anti-CyclinD1-A549)。通过MTT法检测细胞增殖,流式细胞术检测细胞的凋亡情况,结果显示稳定转染CyclinD1反义核酸后,A549细胞增殖显著被抑制,细胞凋亡增加。为探讨CyclinD1反义核酸诱导细胞凋亡的机理,应用Westernblot的方法检测了细胞内视网膜母细胞瘤蛋白质(retinoblastoma protein,pRb)、adenovirus E2 factor-1(E2F-1)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶(matrix metalloproteinase,MMP)-2和MMP-9的表达,其结果显示,稳定转染CyclinD1反义核酸后,A549细胞内pRb、E2F-1、VEGF、MMP-2和MMP-9蛋白表达均显著降低。以上结果表...     

反基因策略——基因治疗的新方向

自1980年Cline用重组β珠蛋白基因治疗两名β地中海贫血患者以来,基因治疗在取得众多成就的同时也暴露出一些问题。安全方面的风险及转移基因表达的不稳定性使基因替代疗法还远达不到人们最初所预想的效果。基因原位修复虽是基因治疗最理想的途径,但目前在技术上还很难做到。相比之下,反义治疗在现阶段可能是较好的方向,因为反义核酸具有传统药物的使用方便和容易大量生产等优点,同时它又     

癌基因与抗癌基因

本文从癌基因和抗癌基因两个领域概述了目前国内外的研究成果和方向。癌基因的研究不但可使人们从分子水平认识肿瘤多阶段发展的机理以及细胞生长、分化调节的机制,而且还有可能对肿瘤的诊断、预后判断及生物化学治疗起推动作用。抗癌基因(或称抑癌基因)则是参与维持细胞正常功能的另一组基因。虽然目前识别、克隆出的抗癌基因不多,它们的生物功能也不十分清楚,但是可以预见抗癌基因的研究必将极大地促进细胞功能调控及肿瘤基因治疗研究的发展。     

动态增强磁共振监测VEGF反义核酸对兔颌面部VX2肿瘤放疗的增敏作用

目的:研究动态增强磁共振成像(dynamic contrast enhanced magnetic resonance imaging,DCE-MRI)监测VEGF反义核酸对兔颌面部VX2肿瘤放疗后的影响。方法:24只颌面部VX2荷瘤兔模型随机分4组:放疗组(A组):给予16Gy放疗;VEGF反义核酸治疗组(B组):肿瘤局部注入VEGF反义核酸150μg;VEGF反义核酸联合放疗组(C组):16Gy放疗后立即局部肿瘤内注射VEGF反义核酸150μg;对照组(D组):肿瘤内注射300μl5%葡萄糖水溶液。治疗后第3天、14天分别行DCE-MRI检查,计算MER(Maximal enhancement ratio,最大强化率)及SLE(Slope of enhancement,强化率斜率)值,14天处死动物行病理检查和VEGF免疫组化染色。结果:C组治疗后14天肿瘤体积明显缩小,与治疗前和治疗后三天及其它组比较差别具有统计学意义(P<0.01)。MER值降低和SLE值降低,与治疗前比较差别有统计学意义(P<0.05)。病理切片显示肿瘤细胞水肿、出血,坏死,血管壁增厚闭塞,VEGF免疫阳性表达下降,经IHS评分与A...     

Silencing of human c-myc oncogene expression by poly-DNP-RNA

Deregulation of c-myc oncogene expression drives the progression of many different types of cancer. Recent experimental data suggest that even brief inhibition of c-myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Previous efforts in developing an inhibitor to silence the c-myc gene were hampered by low efficacy and lack of sequence specificity. Here, we report the synthesis of an antisense RNA inhibitor based on a new 21-nt sequence on a poly- DNP-RNA platform that can specifically inhibit cancer cell growth by silencing c-myc gene expression. Both c-myc mRNA and protein levels were significantly decreased in MCF-7 cells following treatment with this antisense DNP-RNA inhibitor. The control compounds with sense or mismatched sequence were inactive. When c-myc transgenic mice were each treated with a single dose of the antisense RNA inhibitor, in vivo silencing of c-myc gene expression was observed for up to 72 hours by real-time RT-PCR. Similar treatment of c-myc transgenic mice with unmodified (native) homologous small interfering RNA (siRNA) had no effect on the mRNA concentration of the c-myc gene. Injection of this short antisense poly-DNP-RNA into mice did not induce the synthesis of DNP-binding immunoglobulins in the host. The observed in vivo gene silencing by this antisense RNA inhibitor suggests its possible use as a therapeutic agent for cancers involving the deregulation of c-myc gene expression.     

erbB-2与胃癌细胞恶性表型及其增殖调控的关系

应用基因重组、基因转染、Ｓｏｕｔｈｅｒｎ杂交、Ｎｏｒｔｈｅｒｎ杂交、细胞生长曲线测定及裸鼠成瘤观察了反义ｅｒｂＢ－２逆转录病毒重组载体的转染对人胃癌细胞中ｅｒｂＢ－２过度表达的影响及其对ＥＧＦ的反应性．研究结果显示,ｅｒｂＢ－２的表达被其反义重组子特异抑制,伴有细胞增殖能力的下降及致瘤性的下降;在ＥＧＦ的刺激下,ｅｒｂＢ－２高表达肿瘤细胞生长速度提高的幅度显著大于ｅｒｂＢ－２反义重组载体转染细胞;ＥＧＦ促细胞增殖及促基因表达的功能在ｅｒｂＢ－２反义重组子转染后受到抑制,提示ｅｒｂＢ－２在细胞增殖调控中具有重要功能．     

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

Several lines of evidence have suggested that c-fos may act downstream from c-Ha-ras in a growth-regulatory signal transduction pathway. We used antisense RNA to inhibit c-fos gene expression and investigated the effects of diminished c-fos expression on the phenotypes induced by the EJ c-Ha-ras oncogene in NIH 3T3 cells. Immunofluorescent staining demonstrated that the antisense RNA caused a marked reduction in the amount of c-fos protein expressed following serum stimulation. EJ cells containing antisense-fos RNA continued to overexpress ras and remained capable of proliferating in vitro. However, the antisense-fos RNA caused a partial reversion of the major transformed phenotypes of EJ cells, including a restoration of both density-dependent growth arrest and the ability to be rendered quiescent by serum deprivation, a reversion to a flat morphology, inhibition of anchorage-independent growth, and inhibition of tumorigenicity in nude mice. Our results indicate that inhibition of c-fos expression, to a level still supporting in vitro proliferation, prevents the transforming effects of the ras oncogene; they thus provide additional evidence for the participation of c-fos in ras-regulated signal transduction pathways.     

Anti-tumor efficacy of a novel antisense anti-MDM2 mixed-backbone oligonucleotide in human colon cancer models: p53-dependent and p53-independent mechanisms

BACKGROUND: The MDM2 oncogene is amplified or overexpressed in many human cancers and MDM2 levels are associated with poor prognosis. MDM2 not only serves as a negative regulator of p53 but also has p53-independent activities. This study investigates the functions of the MDM2 oncogene in colon cancer growth and the potential value of MDM2 as a drug target for cancer therapy, by inhibiting MDM2 expression with an antisense anti-human-MDM2 oligonucleotide. MATERIALS AND METHODS: The selected antisense mixed-backbone oligonucleotide was evaluated for its in vitro and in vivo antitumor activity in human colon cancer models: LS174T cell line containing wild-type p53 and DLD-1 cell line containing mutant p53. The levels of MDM2, p53 and p21 proteins were quantified by Western blot analysis. RESULTS: In vitro antitumor activity was found in both cell lines, resulting from specific inhibition of MDM2 expression. In vivo antitumor activity of the oligonucleotide occurred in a dose-dependent manner in both models and synergistically or additive therapeutic effects of MDM2 inhibition and the cancer chemotherapeutic agents 10-hydroxycamptothecin and 5-fluorouracil were also observed. CONCLUSIONS: These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53- independent mechanisms. We speculate that MDM2 inhibitors have a broad spectrum of antitumor activities in human cancers regardless of p53 status. This study should provide a basis for future development of anti-MDM2 antisense oligonucleotides as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.     

Antisense oligonucleotide Elk-1 suppresses the tumorigenicity of human hepatocellular carcinoma cells

In previous studies, we showed that reducing Ets-like protein-1 (Elk-1) expression inhibited protein kinase C alpha (PKC alpha) expression and decreased cell migration and invasion in human hepatocellular carcinoma (HCC). In this study, we have investigated the role of Elk-1 in tumorigenesis. SK-Hep-1 HCC cells were transfected with the ElK-1 antisense oligonucleotide (ODN). In the pretreated cells we detected a reduction of mRNA level using RT-PCR. The inhibitory rate of cell growth was measured by MTT assay. Pretreated-SK-Hep-1 HCC cells were implanted subcutaneously into nude mice to observe the tumor growth and calculate tumor inhibitory rate. The results showed that 5 microM of the antisense ODN Elk-1 suppressed both Elk-1 and PKC alpha production by SK-Hep-1 HCC cells after cationic liposome-mediated transfection, to 8% and 1% of control values, respectively, and the growth of SK-Hep-1 HCC cells was inhibited at 2-5 microM doses of the antisense ODN Elk-1. The control reagent, sense ODN Elk-1, showed no effects. In BALB/nude mice, SK-Hep-1 HCC cells transfected with the 5 microM antisense ODN Elk-1 formed tumors much smaller than those of sense ODN Elk-1 pretreated cells. The maximum inhibitory rate of tumor growth was 80.8+/-12.6% and the tumor formation time was prolonged from 13 to 25 days. These findings suggested the usefulness of antisense ODN Elk-1 as a new reagent for liver cancer therapy.