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     

反义RNA寡核苷酸及其与脂质体的复合物对于乙型肝炎病毒的抑制

我们设计合成了特异性靶向乙型肝炎病毒(HBV)mRNA的反义RNA寡核苷酸P-2987、X-60和X-519.在瞬时转染pHBV1.3质粒(含有1.3拷贝的HBV基因组)的HepG2细胞和整合了HBV基因组的HepG2.2.15细胞中,转染2μmol/L的反义RNA寡核苷酸,ELISA和实时定量PCR结果表明,这3条寡核苷酸可以明显抑制HBV的复制和抗原表达.在HBV转基因鼠中,尾静脉注射反义RNA寡核苷酸,结果表明,肝脏中HBV的复制得到了抑制,但是血清中抗原含量和HBV DNA拷贝数没有明显变化.反义RNA寡核苷酸X-519与脂质体的复合物可以增强其对于HBV在肝脏中复制的抑制作用.在通过高压尾静脉注射pHBV1.3质粒建立的HBV急性感染模型中,反义RNA寡核苷酸X-519可以显著地抑制HBV在肝脏中的复制以及降低血清中病毒抗原水平和DNA拷贝数.上述实验结果说明,X-519及其与脂质体的复合物对于HBV的复制和抗原表达起到明显的抑制作用,可能作为一种潜在的针对HBV的基因治疗药物.     

反义RNA寡核苷酸及其与脂质体的复合物对于乙型肝炎病毒的抑制（英文）

我们设计合成了特异性靶向乙型肝炎病毒(HBV)mRNA的反义RNA寡核苷酸P-2987、X-60和X-519.在瞬时转染pHBV1.3质粒(含有1.3拷贝的HBV基因组)的HepG2细胞和整合了HBV基因组的HepG2.2.15细胞中,转染2μmol/L的反义RNA寡核苷酸,ELISA和实时定量PCR结果表明,这3条寡核苷酸可以明显抑制HBV的复制和抗原表达.在HBV转基因鼠中,尾静脉注射反义RNA寡核苷酸,结果表明,肝脏中HBV的复制得到了抑制,但是血清中抗原含量和HBV DNA拷贝数没有明显变化.反义RNA寡核苷酸X-519与脂质体的复合物可以增强其对于HBV在肝脏中复制的抑制作用.在通过高压尾静脉注射pHBV1.3质粒建立的HBV急性感染模型中,反义RNA寡核苷酸X-519可以显著地抑制HBV在肝脏中的复制以及降低血清中病毒抗原水平和DNA拷贝数.上述实验结果说明,X-519及其与脂质体的复合物对于HBV的复制和抗原表达起到明显的抑制作用,可能作为一种潜在的针对HBV的基因治疗药物.     

HSP70 高表达保护低氧诱导的大鼠海马DG 区神经细胞凋亡

目的:研究诱导HSP70高表达对低氧引起的大鼠海马DG区神经细胞凋亡的保护作用。方法:大鼠海马DG区神经细胞分别在41℃温浴2h和加入砷酸钠诱导HSP70高表达。对照低氧而不预热,并用HSP70反义寡核苷酸链抑制HSP70合成,观察HSP70与低氧大鼠海马DG区神经细胞凋亡的关系。DNA碎片法检测细胞凋亡,Western Blotting检测HSP70。结果:预热和砷酸钠都可以诱导细胞HSP70高表达;HSP70高表达可以明显减少低氧诱导的细胞凋亡。在预热前导入HSP70反义核酸,可以降低HSP70抑制细胞凋亡的作用。结论:HSP70高表达可以保护细胞由于低氧引起的细胞凋亡。     

抑制核转录因子PPARα的反义寡核苷酸的抗乙型肝炎病毒活性研究

目的:研究靶向过氧化物酶体增殖活化受体α(PPARα)的寡核苷酸是否具有抑制乙型肝炎病毒(HBV)复制的活性作用。方法:反义寡核苷酸作用于能稳定表达HBV丹氏颗粒的HepG2.2.15细胞,ELISA检测细胞上清中HBV表面抗原(HBsAg)及e抗原(HBeAg)的分泌;实时荧光定量PCR考察反义寡核苷酸对HBV DNA复制水平的影响;通过反转录PCR和Western印迹考察反义寡核苷酸作用于细胞后靶基因及靶蛋白的差异表达情况。结果:抑制PPARα表达的反义序列PPARα-2可剂量依赖且特异性地抑制肝癌细胞中HBsAg和HBeAg的表达,且显著降低细胞中PPARαmRNA水平和蛋白水平。结论:通过筛选初步确定了基于PPARα设计的反义寡核苷酸有较好的抗HBV活性,同时也验证确定了PPARα可能成为抗HBV药物的新型作用靶点。     

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。     

I组mGluRs反义寡核苷酸抗谷氨酸对小鼠大脑皮层神经元的兴奋毒作用

目的:研究I组代谢型谷氨酸受体(mGluRs)反义寡核苷酸对谷氨酸钠(Glu)引起的小鼠大脑皮层神经元损伤的保护作用。方法:以细胞乳酸脱氢酶(LDH)漏出、光镜下细胞形态变化为指标,观察培养液中加入Glu引起的神经元损伤及mGluRl反义寡核苷酸或mGluR5反义寡核苷酸的保护作用;用免疫细胞化学检测神经元mGlulα仪和mGluR5表达。结果:实验显示0.1mmol.L-1的谷氨酸钠可明显造成神经元损伤,使LDH漏出增加(P<0.01),mGluRl反义寡核苷酸或mGluR5反义寡核苷酸6μmol.L-1和8μmol.L-1可明显拮抗Glu引起的神经元损伤,使LDH漏出显著减少(P<0.01);免疫组化实验证实体外培养神经元mGluRlα和mGluR5阳性表达。结论:mGluRl反义寡核苷酸和mGluR5反义寡核苷酸可对抗Glu引起的皮层神经元损伤。     

胰腺癌原位种植瘤中VEGF-C的器官差异表达和作用

目的 研究胰腺癌裸鼠原位种植瘤自发性淋巴结转移模型中VEGF-C表达的器官差异,以及VEGF-C反义寡核苷酸对不同生长部位胰腺癌细胞生长、凋亡能力的影响。方法 建立人胰腺癌细胞株PANC-1裸鼠原位种植瘤自发性淋巴结转移模型,分离、原代培养原发灶和自发性淋巴结转移灶中的胰腺癌细胞,并应用荧光定量PCR、MTT、流式细胞术检测VEGF-C反义寡核苷酸转染对原发胰腺癌细胞和淋巴结转移的胰腺癌细胞各自生长特性、凋亡能力的影响。结果 淋巴结转移胰腺癌细胞VEGF-C的mRNA表达水平显著高于原发灶胰腺癌细胞（P〈0.05）。VEGF-C反义核苷酸抑制胰腺癌细胞VEGF-C的表达后,淋巴结转移灶中胰腺癌细胞生长抑制率、凋亡率均显著提高（P〈0.01）,而原发灶中胰腺癌细胞无明显影响（P〉0.05）。结论 VEGF-C反义寡核苷酸能显著抑制淋巴结转移灶中胰腺癌细胞生长、促进凋亡,但对原发灶胰腺癌细胞无影响;VEGF-C基因的表达和作用存在器官差异性。     

Ⅰ组mGluRs反义寡核苷酸抗谷氨酸对小鼠大脑皮层神经元的兴奋毒作用

目的研究Ⅰ组代谢型谷氨酸受体(mGluRs)反义寡核苷酸对谷氨酸钠(Glu)引起的小鼠大脑皮层神经元损伤的保护作用.方法以细胞乳酸脱氢酶(LDH)漏出、光镜下细胞形态变化为指标,观察培养液中加入Glu引起的神经元损伤及mGluRl反义寡核苷酸或mGluR5反义寡核苷酸的保护作用;用免疫细胞化学检测神经元mGlulα仪和mGluR5表达.结果实验显示0.1 mmol·L-1的谷氨酸钠可明显造成神经元损伤,使LDH漏出增加(P＜0.01),mGluRl反义寡核苷酸或mGluR5反义寡核苷酸6 μmol·L-1和8μmol·L-1可明显拮抗Glu引起的神经元损伤,使LDH漏出显著减少(P＜0.01);免疫组化实验证实体外培养神经元mGluRlα和mGluR5阳性表达.结论mGluRl反义寡核苷酸和mGluR5反义寡核苷酸可对抗Glu引起的皮层神经元损伤.     

HSP70在放线菌素D所致肺腺癌细胞增殖抑制和凋亡中的作用

为探讨热休克蛋白70(heatshockprotein70,HSP70)在肺腺癌中的表达及其作用,首先采用免疫印迹检测经临床支纤镜确诊并行手术切除的肺腺癌组织标本中HSP70的表达,结果显示在癌旁正常组织中HSP70的表达量显著低于其在癌组织中的表达量.其次,采用HSP70反义寡核苷酸阻断A549细胞中HSP70表达后,经MTT和Hoechst33258检测发现,HSP70下调能显著促进放线菌素D(actinomycinD,ActD)所致的A549细胞增殖抑制及凋亡,反义寡核苷酸处理组与正义或随机寡核苷酸处理组比较P值均小于0.05.进一步构建HSP70真核重组质粒并瞬时转染A549细胞后,能显著增加A549细胞中HSP70表达,同时采用MTT和流式细胞术及基因组DNA琼脂糖凝胶电泳检测发现,HSP70过表达能显著抵消ActD所致细胞增殖抑制及凋亡,转染HSP70重组质粒组与转染空载体组相比P值小于0.05.上述结果提示,HSP70在肺腺癌组织中高表达,高表达的HSP70在降低肺腺癌细胞对ActD的敏感性及促进癌细胞增殖与抑制凋亡等方面发挥了重要作用.     

Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis

Aberrant apoptosis-mediated cell death is believed to result in a number of different human diseases. For example, excessive apoptosis in the liver can result in fulminant and autoimmune forms of hepatitis. We have explored the possibility that inhibition of Fas expression in mice would reduce the severity of fulminant hepatitis. To do this, we have developed a chemically modified 2'-O-(2-methoxy)ethyl antisense oligonucleotide (ISIS 22023) inhibitor of mouse Fas expression. In tissue culture, this oligonucleotide induced a reduction in Fas mRNA expression that was both concentration- and sequence-specific. In Balb/c mice, dosing with ISIS 22023 reduced Fas mRNA and protein expressions in liver by 90%. The ID50 for this response was 8-10 mg kg-1 daily dosing, and the reduction was highly dependent on oligonucleotide sequence, oligonucleotide concentration in liver, and treatment time. Pretreatment with ISIS 22023 completely protected mice from fulminant hepatitis induced by agonistic Fas antibody, by a mechanism entirely consistent with an oligonucleotide antisense mechanism of action. In addition, oligonucleotide-mediated suppression of Fas expression reduced the severity of acetaminophen-mediated fulminant hepatitis, but was without effect on concanavalin A-mediated hepatitis. Our results demonstrate that 2'-O-(2-methoxy)ethyl containing antisense oligonucleotides targeting Fas can exert in vivo pharmacological activity in liver, and suggest that oligonucleotide inhibitors of Fas may be useful in the treatment of human liver disease.     

Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression.

Following transection of the optic nerve (ON), retinal ganglion cells (RGCs) upregulate Bax protein expression and undergo apoptosis. The present study aimed at reducing Bax expression in order to test whether Bax plays a causative role in the induction of secondary RGC apoptosis. Following injection into the vitreous, fluoresceinated oligonucleotides transfected RGCs in vivo at the injection site in the temporal superior retina. Following ON lesion, and repeated injections of a partially phosphorothioated Bax antisense oligonucleotide, but not following injection of control oligonucleotides, expression of Bax protein was locally inhibited, and the number of surviving RGCs was increased in Bax antisense treated rats 8 days after axotomy. Our results indicate that Bax induction is a prerequisite for the execution of RGC apoptosis following ON axotomy. While the Bax antisense strategy offers an exciting perspective to inhibit secondary neuronal degeneration in vivo, both limited transfection efficacy, and the temporal restriction of this effect currently limit the use of this approach with respect to clinical applications for the treatment of neurodegeneration.     

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.     

IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death

Fas ligand (FasL), perforin, TNF-alpha, IL-1, and NO have been considered as effector molecule(s) leading to beta cell death in autoimmune diabetes. However, the real culprit(s) in beta cell destruction have long been elusive, despite intense investigation. We and others have demonstrated that FasL is not a major effector molecule in autoimmune diabetes, and previous inability to transfer diabetes to Fas-deficient nonobese diabetic (NOD)-lpr mice was due to constitutive FasL expression on lymphocytes from these mice. Here, we identified IFN-gamma/TNF-alpha synergism as the final effector molecules in autoimmune diabetes of NOD mice. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, induced classical caspase-dependent apoptosis in insulinoma and pancreatic islet cells. IFN-gamma treatment conferred susceptibility to TNF-alpha-induced apoptosis on otherwise resistant insulinoma cells by STAT1 activation followed by IFN regulatory factor (IRF)-1 induction. IRF-1 played a central role in IFN-gamma/TNF-alpha-induced cytotoxicity because inhibition of IRF-1 induction by antisense oligonucleotides blocked IFN-gamma/TNF-alpha-induced cytotoxicity, and transfection of IRF-1 rendered insulinoma cells susceptible to TNF-alpha-induced cytotoxicity. STAT1 and IRF-1 were expressed in pancreatic islets of diabetic NOD mice and colocalized with apoptotic cells. Moreover, anti-TNF-alpha Ab inhibited the development of diabetes after adoptive transfer. Taken together, our results indicate that IFN-gamma/TNF-alpha synergism is responsible for autoimmune diabetes in vivo as well as beta cell apoptosis in vitro and suggest a novel signal transduction in IFN-gamma/TNF-alpha synergism that may have relevance in other autoimmune diseases and synergistic anti-tumor effects of the two cytokines.     

Antisense inhibition of membrane-bound human interleukin-5 receptor-alpha chain does not affect soluble receptor expression and induces apoptosis in TF-1 cells

Binding of human interleukin-5 (HuIL-5) to its membrane-anchored receptor (IL-5R) triggers multiple signaling pathways, cellular proliferation, and maturational responses, as well as protection from apoptosis. In contrast, soluble forms of the HuIL-5R have been shown to inhibit IL-5 signaling and, therefore, may represent naturally occurring negative regulators of IL-5 function. Because of the central role of IL-5 in promoting eosinophilia and airway hyperresponsiveness in animal models of asthma, antisense oligonucleotides specific either for the membrane form alone or for sequences shared between both the membrane and soluble forms of the HuIL-5Ralpha ligand binding chain were designed. The activities of these oligonucleotides were characterized in IL-5R-expressing erythroleukemic TF-1 cells. Herein we report that an antisense oligonucleotide targeted to a sequence unique to the alternatively spliced membrane-bound form of the HuIL-5Ralpha chain has been developed that selectively inhibits membrane, but not soluble, mRNA isoform expression. Both this membrane-specific oligonucleotide and an antisense oligonucleotide targeted to sequence common to both membrane and soluble isoforms were found to potently suppress cell surface IL-5Ralpha levels and IL-5-mediated cell survival by inducing apoptosis similar to IL-5 withdrawal. Thus, these oligonucleotides represent unique genetic agents with therapeutic potential for diseases with an eosinophilic component.     

Antisense oligonucleotides as therapeutic agents.

Antisense oligonucleotides can block the expression of specific target genes involved in the development of human diseases. Therapeutic applications of antisense techniques are currently under investigation in many different fields. The use of antisense molecules to modify gene expression is variable in its efficacy and reliability, raising objections about their use as therapeutic agents. However, preliminary results of several clinical studies demonstrated the safety and to some extent the efficacy of antisense oligodeoxynucleotides (ODNs) in patients with malignant diseases. Clinical response was observed in some patients suffering from ovarian cancer who were treated with antisense targeted against the gene encoding for the protein kinase C-alpha. Some hematological diseases treated with antisense oligos targeted against the bcr/abl and the bcl2 mRNAs have shown promising clinical response. Antisense therapy has been useful in the treatment of cardiovascular disorders such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy. Antisense oligonucleotides also have shown promise as antiviral agents. Several investigators are performing trials with oligonucleotides targeted against the human immunodeficiency virus-1 (HIV-1) and hepatitis viruses. Phosphorothioate ODNs now have reached phase I and II in clinical trials for the treatment of cancer and viral infections, so far demonstrating an acceptable safety and pharmacokinetic profile for continuing their development. The new drug Vitravene, based on a phosphorothioate oligonucleotide designed to inhibit the human cytomegalovirus (CMV), promises that some substantial successes can be reached with the antisense technique.     

Fas and Fas ligand in gut and liver

Apoptosis (programmed cell death) has been shown to play a major role in development and in the pathogenesis of numerous diseases. A principal mechanism of apoptosis is molecular interaction between surface molecules known as the "death receptors" and their ligands. Perhaps the best-studied death receptor and ligand system is the Fas/Fas ligand (FasL) system, in which FasL, a member of the tumor necrosis factor (TNF) family of death-inducing ligands, signals death through the death receptor Fas, thereby resulting in the apoptotic death of the cell. Numerous cells in the liver and gastrointestinal tract have been shown to express Fas/FasL, and there is a growing body of evidence that the Fas/FasL system plays a major role in the pathogenesis of many liver and gastrointestinal diseases, such as inflammatory bowel disease, graft vs. host disease, and hepatitis. Here we review the Fas/FasL system and the evidence that it is involved in the pathogenesis of liver and gastrointestinal diseases.     

The inhibition of apoptosis in myositis and in normal muscle cells

The mechanism of injury and death of muscle cells in the inflammatory myopathies (dermatomyositis, polymyositis, and inclusion body myositis) remains obscure. We and others have not detected apoptosis in the muscle biopsies from patients with myositis despite clear evidence of cell damage and loss. We provide evidence in this study that Fas ligand (FasL) as well as Fas is present on muscle cells and inflammatory cells in myositis biopsies: Fas is present on most muscle cells and lymphocytes, and FasL is present on degenerating muscle cells and many infiltrating mononuclear cells. The expression of both Fas and FasL in the inflamed tissue makes the absence of apoptosis more striking. To address the mechanisms of this resistance to classical apoptosis in muscle cells, we have investigated the expression of the antiapoptotic molecule FLICE (Fas-associated death domain-like IL-1-converting enzyme)-inhibitory protein (FLIP) in muscle biopsies of myositis patients and in cultured human skeletal muscle cells. Using laser capture microscopy, we have shown that FLIP is expressed in the muscle fibers and on infiltrating lymphocytes of myositis biopsies. Furthermore, we have shown that FLIP, but not Bcl-2, is expressed in cultured human skeletal muscle cells stimulated with proinflammatory cytokines, and inhibition of FLIP with antisense oligonucleotides promotes significant cleavage of poly(ADP-ribose) polymerase autoantigen, a sensitive indicator of apoptosis. These studies strongly suggest that the resistance of muscle to Fas-mediated apoptosis is due to the expression of FLIP in muscle cells in the inflammatory environment in myositis.     

Transforming growth factor alpha protects against Fas-mediated liver apoptosis in mice

The Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors have recently been found to function in preventing apoptosis. In this study, we demonstrated that overexpression of transforming growth factor alpha (TGFalpha) has a dramatic protective effect on Fas-mediated hepatic apoptosis at the biochemical and histological levels. Moreover, 85.7% (six out of seven) of TGFalpha transgenic mice survived the lethal liver damage, whereas all wild-type mice died. Expression of Bcl-xL, an anti-apoptotic protein, was greatly increased in the transgenic mice. Taken together, our findings suggest that TGFalpha protects against Fas-mediated liver apoptosis in vivo and up-regulation of Bcl-xL may participate in protective effect of TGFalpha.