C-myc反义寡脱氧核苷酸抑制类风湿滑膜细胞增生诱导滑膜细胞凋亡

目的 研究硫代磷酸化修饰的c—myc反义寡脱氧核苷酸(antisens phosphorothioate oligodeoxynucleotides，ASP-ODN)在血清存在条件下对培养的人类风湿关节炎(rheumatoid arthritis，RA)滑膜B型细胞增生的影响及诱导凋亡的作用。方法 培养RA滑膜B型细胞，合成c—myc AS P-ODN，以阳离子脂质体。lipofectin为载体转染滑膜B型细胞，用显微镜观察和四唑盐(MTT)法检测对细胞增生的抑制作用，用荧光染色、荧光显微镜、流式细胞仪检测诱导细胞凋亡和c—myc蛋白表达的变化。结果c—myc AS P-ODN呈序列特异性抑制滑膜B型细胞增生并下调c—myc蛋白表达，诱导滑膜B型细胞凋亡。结论 c—myc AS P-ODN可能对RA的滑膜增生性炎症具有潜在的治疗作用，c—myc原癌基因可能成为反义核酸技术治疗RA的靶基因。     

Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma

BACKGROUND: Kirsten ras (Ki-ras) mutations are common in gastrointestinal cancer and one codon 12 mutation, glycine to valine, is particularly aggressive in colorectal cancer. AIMS: To investigate if this valine point mutation could be targeted with antisense oligonucleotides and to determine the efficacy of any antisense/mRNA interaction. METHODS: Twenty nine antisense oligonucleotides were screened against target and control Ki-ras RNA in a cell free system and against target and control cell lines in culture. RESULTS: The activity and specificity of the oligonucleotides varied. Results for the individual oligonucleotides were consistent in a cell free model and in cell culture using two different uptake promoters. Only one oligonucleotide was specific in its cleavage of target Ki-ras mRNA in the cell free system and appeared specific in cell culture, although changes in Ki-ras mRNA and protein expression following a single treatment could not be detected. Experiments in the cell free system showed that the point mutation is relatively inaccessible to oligonucleotides. Other sites on the Ki-ras RNA molecule, away from the point mutation, can be targeted more effectively. CONCLUSIONS: Successful targeting of the clinically relevant Ki-ras point mutation with antisense oligonucleotides is difficult because of RNA structure at the mutated site and is inefficient compared with other sites on the Ki-ras mRNA.     

MYH9 spectrum of autosomal-dominant giant platelet syndromes: unexpected association with fibulin-1 variant-D inactivation

The autosomal-dominant giant platelet syndromes (Fechtner, Epstein, and Sebastian platelet syndromes and May-Hegglin anomaly) represent a group of disorders characterized by variable degrees of macrothrombocytopenia with further combinations of neutrophil inclusion bodies and Alport-like syndrome manifestations, namely, deafness, renal disease, and eye abnormalities. The disease-causing gene of these giant platelet syndromes was previously mapped by us to chromosome 22. Following their successful mapping, these syndromes were shown to represent a broad phenotypic spectrum of disorders caused by different mutations in the nonmuscle myosin heavy chain 9 gene (MYH9). In this study, we examined the potential role of another gene, fibulin-1, encoding an extracellular matrix protein as a disease modifier. Eight unrelated families with autosomal-dominant giant platelet syndromes were studied for DNA sequence mutations and expression of the four fibulin-1 splice variants (A-D). A mutation in the splice acceptor site of fibulin-1 exon 19 was found in affected individuals of the Israeli Fechtner family, whereas no MYH9 mutations were identified. Unexpectedly, fibulin-1 variant D expression was absent in affected individuals from all eight families and coupled with expression of a putative antisense RNA. Transfection of the putative antisense RNA into H1299 cells abolished variant D expression. Based on the observation that only affected individuals lack variant D expression and demonstrate antisense RNA overexpression, we suggest that these autosomal-dominant giant platelet syndromes are associated, and may be modified, by aberrant antisense gene regulation of the fibulin-1 gene.     

心肌营养素-1对血管紧张素Ⅱ诱导心肌细胞肥大的影响

目的:研究心肌营养素-1(CT-1)对血管紧张素Ⅱ(AngⅡ)诱导的心肌细胞肥大的影响。方法:利用AngⅡ刺激心肌细胞,导致细胞肥大,应用CT-1反义脱氧寡核苷酸进行干预。检测心肌细胞大小及3H-亮氨酸掺入率的变化;以逆转录聚合酶链反应(RT-PCR)检测CT-1 mRNA及β-肌球蛋白重链(β-MHC)mRNA表达,免疫组织化学法检测心肌细胞CT-1蛋白的表达。结果:经AngⅡ刺激后,在相差显微镜下可见心肌细胞面积变大,3H-亮氨酸掺入率、CT-1蛋白的表达增高;CT-1和β-MHC mRNA水平的表达增加。利用Fugene6可将CT-1反义脱氧寡核苷酸转染入心肌细胞。CT-1反义脱氧寡核苷酸组心肌细胞面积较肥大组明显减小[(81.257±3.995)mm2∶(127.214±5.693)mm2],3H-亮氨酸掺入量[(1653.33±17.91)cpm∶(1971.50±42.16)cpm]及CT-1蛋白[(3.16±0.17)%∶(3.51±0.29)%]明显减少;CT-1[(0.2137±0.0227)∶(0.4023±0.0160)]和β-MHC mRNA[(0.5032±0.0261)∶(0.773 4±0.0486)]表达亦显著减少(P0.05~0.01)。各指标Fugene 6组、错义组与肥大组无明显差异。结论:AngⅡ致心肌细胞肥大过程中伴有CT-1表达增加,应用CT-1反义脱氧寡核苷酸后可使之下降,说明CT-1参与了心肌细胞的肥大机制。     

黏着斑激酶对血小板源性生长因子刺激的血管平滑肌细胞迁移和黏附的调控

目的探讨黏着斑激酶(FAK)信号分子在雷帕霉素抑制血小板源性生长因子(PDGF)诱导血管平滑肌细胞(VSMC)迁移、黏附中的调控作用。方法将培养的大鼠VSMC分为对照组、PDGF组、雷帕霉素+PDGF组(雷帕霉素组)和FAK反义寡核苷酸+PDGF组(FAK组)。用PDGF诱导VSMC的迁移和黏附,计数贴壁细胞;采用Boyden检测细胞迁移;采用RT-PCR、Western blot、免疫沉淀方法分别检测FAK基因、蛋白及蛋白磷酸化表达量。将FAK反义寡核苷酸经脂质体转染VSMC,观察FAK mRNA及蛋白磷酸化、细胞迁移和黏附的变化。结果与对照组比较,PDGF组明显诱导细胞迁移和黏附,上调FAK mRNA的表达,提高FAK蛋白和磷酸化FAK表达,差异有统计学意义(P0.05),与PDGF组比较,FAK组和雷帕霉素组细胞迁移、黏附能力减弱,FAK mRNA、FAK蛋白和磷酸化FAK表达水平明显降低,差异有统计学意义(P0.05)。结论 PDGF诱导细胞迁移和黏附可能是FAK介导的,雷帕霉素可能是通过抑制FAK蛋白和磷酸化FAK来抑制VSMC的迁移和黏附。     

Progress in therapeutic antisense applications for neuromuscular disorders

Neuromuscular disorders are a frequent cause of chronic disability in man. They often result from mutations in single genes and are thus, in principle, well suited for gene therapy. However, the tissues involved (muscle and the central nervous system) are post-mitotic, which poses a challenge for most viral vectors. In some cases, alternative approaches may use small molecules, for example, antisense oligonucleotides (AONs). These do not deliver a new gene, but rather modulate existing gene products or alter the utilization of pathways. For Duchenne muscular dystrophy, this approach is in early phase clinical trials, and for two other common neuromuscular disorders (spinal muscular atrophy and myotonic dystrophy), significant preclinical advances have recently been made.     

磷脂酰肌醇3-激酶调控白介素-18诱导核因子-кB活化(英文)

目的:研究磷脂酰肌醇(PI)3-激酶在白介素18(IL-18)诱导核因子-кB(NF-кB)活化中的作用。方法:Lipofectin介导反义PI 3-激酶寡核苷酸转染HepG2细胞。用逆转录PCR法检测PI 3-激酶mRNA表达水平,以Sandwich ELISA法检测NF-кB的活化。结果:(1)反义PI 3-激酶寡核苷酸抑制PI 3-激酶mRNA表达。(2)IL-18诱导NF-кB活化。(3)反义PI3-激酶寡核苷酸呈时间(5-24h)和浓度(1-8mg/L)依赖性地抑制IL-18诱导的NF-кB活化。结论:PI 3-激酶调控白介素-18诱导的NF-кB活化。     

Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice.

Angiopoietin-1 (Ang1) is an angiogenic growth factor that functions through activation of its endothelium-specific tyrosine kinase receptor Tie2; it mediates the interaction between endothelial and surrounding cells to promote the remodeling, maturation and stabilization of blood vessels. Although Ang1 is expressed constitutively in many adult tissues, its role in tumor growth and metastasis is not clear. Here we describe experiments in which Ang1 expression was inhibited in HeLa cells by an antisense RNA approach. The modified HeLa cells produced significantly less Ang1 protein both in cultured cells and in tumors formed when these cells were injected into immunodeficient mice. The Ang1 antisense tumors grew much more slowly, with significantly reduced tumor angiogenesis compared with control tumors. Furthermore, they also had substantially increased tumor cell apoptosis and decreased tumor necrosis. Our results indicate that the perturbation of Ang1 expression in tumors could be an effective method to control tumor growth by inhibiting tumor angiogenesis and that antisense RNA is an efficient way to inhibit Ang1 protein production in tumor cells.