Microtubule-associated protein tau is required for axonal neurite elaboration by neuroblastoma cells.

NB2a/d1 neuroblastoma cells constitutively express multiple isoforms of the microtubule-associated protein tau and incorporate this protein into the axonal neurites elaborated during serum deprivation. To examine whether or not tau played an essential role in axonal outgrowth, cells cultured in serum-free medium were treated at 24 h intervals with antisense- and sense-oriented cDNA oligonucleotides (25 or 36 mers that span or are upstream of tau initiation codon) and were simultaneously serum deprived. Oligonucleotide uptake was confirmed by determination of intracellular levels of radiolabeled oligonucleotides. Treatment for 48 h with tau antisense oligonucleotides reversibly inhibited the expression of tau and the number of neurite-bearing cells compared with treatment with sense oligonucleotides. By contrast, tubulin expression was not affected. When cells were treated with antisense oligonucleotide simultaneously with serum deprivation, the initial outgrowth of neurites was unaffected, but continued neurite elongation was prevented. By contrast, neurite outgrowth at 4 h was inhibited when cells were pretreated with tau antisense 24 h before serum deprivation. Furthermore, intracellular delivery of anti-tau antiserum prevented neurite outgrowth and, in cells that had previously been deprived of serum for 24 h, induced retraction of existing neurites. These findings indicate that both the initiation and the continued outgrowth of neurites are dependent on tau and that pre-existing cytoplasmic pools of tau can mediate initial neuritogenesis.     

Improved Cellular Delivery of Antisense Oligonucleotides Using Transferrin Receptor Antibody-Oligonucleotide Conjugates

This work is dedicated to the memory of the late Dr Ian Walker.     

耐药乳腺癌MCF-7/Adr细胞c-myc的表达上调及与耐药的关系

目的观察耐药乳腺癌细胞c-myc表达及其反义寡核苷酸对耐药的逆转效应,探讨c-myc在耐药调控中的作用。方法运用流式细胞仪检测乳腺癌耐药细胞MCF-7/Adr和其药敏亲本系MCF-7的c-myc表达水平。MTT法测定阿霉素作用于上述细胞的药物半数抑制浓度(IC50)。结果MCF-7/Adr耐药细胞c-myc的表达率为70.48%,其亲本药敏细胞系MCF-7c-myc表达率仅46.02%,前者显著高于后者(P<0.05)。阿霉素单独作用于MCF-7/Adr,IC50值为(22.00±1.92)μmol/L,但与4μmol/Lc-myc反义寡核苷酸共孵育后,阿霉素的IC50值则显著下降为(9.60±1.04)μmol/L。结论与其亲本药敏细胞相比较,MCF-7/Adr的c-myc表达显著上调,抑制c-myc的过表达可部分逆转MCF-7/Adr的阿霉素抵抗,提示c-myc参与肿瘤耐药的发生。     

反义脱氧寡核苷酸对SRS病毒复制的抑制作用

用合成的反义脱氧寡核苷酸,包括修饰的和非修饰的,同聚的和异聚的脱氧寡核苷酸对SRS病毒感染细胞的增殖、细胞群落形成,XC融合细胞产生的抑制作用。结果表明,上述的寡聚物对病毒都有抑制活性。各种寡聚物的作用机制不同。     

bcl-2反义寡核苷酸对膀胱癌BIU87细胞株的影响

目的:探讨bcl-2反义寡核苷酸对培养的膀胱癌BIU87细胞株的影响.方法:采用bcl-2基因第1外显子的反义寡核苷酸,以硫代磷酸修饰(PS-ASON),序列为5′-TCTCCCAGCGTGCGCCAT-3′,与膀胱癌细胞株BIU87共同孵育作为实验组.加入正义的bcl-2寡核苷酸,序列为5′-TACCGCGTGCGACCCTCT-3′(PS-SON)作为对照组,用流式细胞仪检测细胞凋亡和坏死率,电镜观察细胞形态变化.结果:与对照组相比,实验组的细胞坏死率明显上调,电镜下细胞形态呈坏死改变.结论:bcl-2的反义寡核苷酸引起膀胱癌细胞大量坏死,可能成为膀胱癌的治疗方法之一.     

Antisense oligonucleotides and short interfering RNAs silencing the cyclin-dependent kinase inhibitor p21 improve proliferation of Duchenne muscular dystrophy patients’ primary skeletal myoblasts

Increased levels of the cyclin-dependent kinase inhibitor p21 associated with decreased myoblast proliferation may be involved in the dystrophic process in Duchenne muscular dystrophy (DMD). Therefore we are interested to improve the proliferation of primary myoblasts of DMD patients by a reduction in p21 using either antisense oligonucleotides (ASO) or short interfering RNAs (siRNA). After transient transfection of myoblasts in cell culture proliferation was analyzed using a 5-bromo-2-deoxyuridine assay comparing specific transfected cells with untransfected cells and cells transfected with scrambled ASO and luciferase siRNA, respectively. Four of five Dystrophin-deficient (Dys^–) cell culture samples revealed an increase in proliferation between 7% and 18% compared to untransfected cells and between 8% and 36% compared to cells transfected with scrambled ASO. Transfection with siRNA was performed for selected samples to determine whether siRNA is more effective in gene silencing than ASO. The increase in proliferation using luciferase siRNA as reference was comparable to or less than ASO data using scrambled ASO as reference. Using untransfected cells as reference, the increase in proliferation was higher for siRNA than ASO (20–47% vs. 7–18%), but the data must be carefully interpreted with respect to nonspecific effects on gene expression by siRNA. Our findings of transient p21 gene silencing represent a basis for viral vector-mediated drug-inducible p21 shRNA expression in Dys^– myoblasts which might enhance, prolong and regulate the proliferation effect.S. Endesfelder and A. Kliche contributed equally to this work     

Conjugation of DNA fragments to protein carriers by glutaraldehyde: immunogenicity of oligonucleotide-hemocyanin conjugates

The practical realization of the concept of specific immunotherapy for systemic lupus erythematosus (SLE) has been hampered, thus far, by an inability to link DNA fragments to carrier protein. In this paper, a novel technique is described, in which glutaraldehyde is the linking agent. A 2-stage method was used to link oligonucleotides to a soluble protein carrier, such as keyhole limpet hemocyanin (KLH) or human gamma globulin (HGG), whereas a 1-stage technique was sufficient to link oligonucleotides to sheep red cells. Both the ultraviolet absorbance spectrum and diphenylamine assay demonstrated that oligonucleotides were coupled to soluble protein. The conjugate of oligonucleotide to protein carrier appears to be recognized by anti-DNA antibody since oligonucleotide linked to either KLH or HGG inhibited the binding of anti-DNA antibody in vitro, and oligonucleotide-coupled sheep cells are agglutinating by seropositve sera from lupus patients. In addition, oligonucleotide-KLH raised hemagglutinating antibody to denatured DNA in C57BL/6, DBA/2 or NZB mice, as well as IgG antibody as detected by SPRIA in C57BL/6 and DBA/2 mice. The significance of this new method for the development of an antigen specific therapy of SLE is discussed.     

ASODN对白细胞抗原Ⅰ类基因表达的影响

目的为观察针对乙型肝炎病毒（ＨＢＶ）ｘ基因特异性反义核酸（ＡＳＯＤＮ）对２２１５细胞表面白细胞抗原Ⅰ类基因（ＨＬＡ－Ⅰ）表达的影响。方法人工合成互补于ｘ基因关键区的三段反义核酸，用流式细胞仪检测ＡＳＯＤＮ对２２１５细胞表面ＨＬＡ－Ⅰ表达的影响，细胞原位杂交观察作用细胞内ＨＬＡ－ⅠｍＲＮＡ含量变化，同时用ＰＡＰ－ＥＬＩＳＡ法检测作用细胞上清中ＨＢｘＡｇ的含量。结果三段ＡＳＯＤＮ均能抑制ＨＢｘＡｇ的表达，２２１５细胞表面ＨＬＡ－Ⅰ类抗原的表达及细胞内ＨＬＡ－ⅠｍＲＮＡ含量也降低。结论ＨＢｘＡｇ表达量的降低可能系ＡＳＯＤＮ序列特异性抑制作用所致，而ＨＬＡ－Ⅰ表达的降低可能是ＨＢｘＡｇ对ＨＬＡ－Ⅰ基因启动子的激发减少的间接结果。