核因子κB p65反义寡核苷酸对大鼠肝星状细胞增殖和I型胶原表达的影响

目的 探讨核因子κB(NF-κB)p65反义寡核苷酸(ASOND)对大鼠肝星状细胞(HSC)增殖和I型胶原表达的影响.方法 Ⅳ型胶原酶消化密度梯度离心法分离培养大鼠HSC;脂质体介导的不同浓度的NF-κB p65 ASOND(0.001、0.01、0.1和1μmol/L)进入HSC;台盼蓝染色排斥法检测NF-κBp65 ASOND对HSC的毒性实验并检测各组乳酸脱氢酶(LDH)活性;MTT法测定NF-κB p65 ASOND对1mg/LTNF-α刺激后HSC增殖影响;RT-PCR法和ELISA法检测不同浓度NF-κB p65 ASOND对1mg/LTNF-α刺激后HSC I型胶原表达的影响.结果 转染NF-κB p65 ASODN后,HSC细胞NF-κB蛋白的表达下降,不同浓度(0.001、0.01、0.1和1 μmol/L)的NF-κB p65 ASOND对于体外培养HSC的存活率和LDH无明显影响(P>0.05),0.01～μmol/L的NF-κB p65 ASOND抑制HSC的增殖,lmg/L TNF-α刺激HSC的I型胶原蛋白和mRNA的表达,且随浓度的增加作用增强(P<0.05).结论 NF-κB p65 ASOND可通过抑制NF-κB活性减少HSC活化增殖及Ⅰ型胶原生成,从而减少细胞外基质的产生.     

Antisense inhibition of urokinase reduces spread of human ovarian cancer in mice

Urokinase-type plasminogen activator (uPA) is a protease involved in the process of tissue remodelling and cell migration in vitro. To explore whether uPA is a prerequisite for human ovarian cancer spread in vivo the expression of uPA was suppressed in human ovarian cancer cells by antisense phosphorothioate oligonucleotides (PS-ODN). The suppression of uPA expression was dependent on PS-ODN concentration and only observed in the presence of liposomes. This phenomenon seemed to be due to the fact that PS-ODNs were taken up by the cancer cells only in concert with liposomes as studied by fluorescently-labeled PS-ODNs using flow cytofluorometry and laser scanning microscopy. uPA-deprived cancer cells exhibited a significantly reduced invasive capacity in vitro compared with untreated cancer cells or cells treated with control PS-ODNs (P = 0.003). The intraperitoneal spread of the cancer cells in vivo was significantly diminished when nude mice were treated with uPA antisense PS-ODNs in comparison with control mice (P = 0.009). These results suggest that uPA expression may be required for spread of human ovarian cancer and that its inhibition could provide a therapeutic approach.     

hTERT反义寡核苷酸促进紫杉醇诱导Hut78细胞凋亡

目的：探讨转染人端粒酶逆转录酶基因（hTERT)反义寡核苷酸后能否增强紫杉醇诱导皮肤T细胞淋巴瘤细胞株Hut78凋亡。方法:采用脂质体介导的基因转染技术将hTERT反义寡核苷酸（AODN)、正义寡核苷酸（SODN)导入Hut78细胞株中，应用端粒酶重复序列扩增及酶联免疫吸附方法（TRAP-PCR-ELISA)、逆转录-聚合酶链反应、四甲基偶氮唑蓝法（MTT)、台盼蓝拒染法和流式细胞仪分析测定联合应用紫杉醇后对细胞端粒酶活性、hTERT mRNA表达、细胞增殖及凋亡的影响。结果:转染hTERT基因反义寡核苷酸并联合应用紫杉醇，48 h后Hut78细胞增殖即被明显抑制，端粒酶活性下降，hTERT mRNA表达降低，分别同SODN联合紫杉醇组及单用紫杉醇组进行比较，有显著差异（P<0.05)； MTT结果显示，紫杉醇对AODN组、SODN组及空白对照组细胞的IC50值分别为(81.10 ± 1.68) nmol/L，(138.70±1.80) nmol/L和(139.40± 5.50) nmol/L，差异显著（P< 0.05)；经流式细胞仪检测，AODN联合紫杉醇组凋亡率明显高于SODN联合紫杉醇组和单用紫杉醇组。结论:hTERT反义寡核苷酸与紫杉醇有协同抗肿瘤效应，并能增加Hut78细胞对紫杉醇的敏感性，促进紫杉醇诱导Hut78细胞凋亡。     

反义人端粒酶RNA对胃癌细胞生长的抑制效应

目的 探讨反义人端粒酶ＲＮＡ（ｈＴＲ）对胃癌细胞的生长抑制作用。方法 将反义ｈＴＲ真核表达载体经脂质体介导转染入胃癌细胞系ＳＧＣ７９０１,体外培养及接种裸鼠观察其基因转染细胞的细胞周期、超微结构变化、生长速率及致瘤性。结果 反义ｈＴＲ在潮霉素筛选的阳性克隆中稳定表达,正义ｈＴＲ表达下调,并出现凋亡改变。基因转染细胞体外传代培养的生长速率大多减慢,在裸鼠皮下的致瘤性明显降低。荷瘤鼠存活时间延长。结论     

Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

Frog virus 3 (FV3) is a large DNA virus that encodes approximately 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIalpha). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIalpha triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.     

Inhibitory effects of antisense oligonucleotides on the expression of procollagen type III gene in mouse hepatic stellate cells transformed by simian virus 40

The effects of phosphorothioate antisense oligonucleotides (ASO), complementary to the AUG start region, the junctional region of the intron and exon, and to exon of the procollagen type III gene, were investigated in a mouse hepatic stellate cell (HSC) line transformed by the simian virus 40 gene, SV68c-IS cells. ASO were transfected by lipofection. Immunohistochemistry, western and northern blotting showed inhibitory effects on procollagen type III gene expression by ASO that were complementary to the AUG start region and the junctional region of the intron and exon 2. However, ASO complementary to the exon 2 and 3, junctional region of the intron and exon 3, and sense oligonucleotides complementary to each ASO did not show any inhibitory effects. The effects of ASO complementary to the AUG start region were greater than those of ASO complementary to the junctional region. The effects of ASO were transient and a large amount of ASO was required to induce inhibitory effects without lipofection. ASO were effective in inhibiting the expression of the procollagen type III gene in the HSC which is well known to play a critical role in liver fibrosis.     

u-PAR反义核酸载体的构建与高侵袭人前列腺癌细胞PC-3M亚系的转染

目的:为了特异封闭PC-3M高侵袭亚系尿激酶型纤溶酶原激活物受体(u-PAR)的表达,观察其对侵袭能力的抑制效应。方法: 应用RT-PCR获得u-PAR的cDNA片段,反向插入pcDNA3质粒载体中,构建u-PAR反义核酸载体并导入高侵袭亚系中;借助RT-PCR和免疫组化检测转染细胞u-PAR的表达。 结果:u-PAR反义核酸载体转染株的u-PAR mRNA和蛋白质水平明显低于对照组,抑制率分别为53%和73%,同时其体外侵袭能力亦明显降低,抑制率为79%。 结论: u-PAR反义核酸在PC-3M高侵袭亚系中发挥了特异封闭作用,为进一步观察u-PAR反义核酸抑制高侵袭前列腺癌细胞亚系的侵袭效应提供了细胞模型。