Bacterial DNA activates human neutrophils by a CpG-independent pathway

Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG-dependent manner. In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L-selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL-8 secretion, and cell migration. Induction of these responses is not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG-methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs. We found that human neutrophils express Toll-like receptor (TLR) 9 mRNA. However, as expected for a CpG-independent mechanism, activation does not involve a TLR9-dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling. Of note, both single-stranded and double-stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation. Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs.     

反义寡核苷酸对脑胶质瘤细胞生长抑制作用的动态研究

目的　探讨原癌基因 c- m yb反义寡核苷酸 (AON)对 C6脑胶质瘤细胞的体内生长抑制作用。方法　将 C6脑胶质瘤细胞接种于裸鼠皮下 ,当肿瘤长至 10 mm左右时随机分成 3组 (每组 12只 ) ,分别用 c- mybAON、正义寡核苷酸 (SON)和生理盐水瘤区原位注射 ,注药后 4、8、12和 16日各组处死动物 3只 ,动态观察肿瘤的体积、重量及病理学改变 ;用流式细胞仪检测肿瘤细胞的凋亡 ;以免疫组化方法分析各组的 c- m yb、bcl- 2蛋白阳性表达率。结果　 c- myb AON组的肿瘤体积抑制率分别为第 4天 6 6 .7% ,第 8天 71.4% ,第 12天 72 % ,第 16天73% ,与对照组相比 ,其生长抑制作用具有显著性 (P<0 .0 5 ) ;AON组凋亡细胞百分率为第 4天 3.4% ,第 8天 2 7.1% ,第 12天 46 .1% ,第 16天 48.4% ,是 SON组和对照组相应时段凋亡细胞的 3.5倍以上 (P<0 .0 5 ) ;AON组 c-myb和 bcl- 2蛋白阳性表达率较对照组有显著降低 (P<0 .0 5 ) ;且 AON治疗后的脑胶质瘤中见较多炎性细胞浸润和钙化灶。结论　 c- myb癌基因有可能成为反义基因治疗恶性脑胶质瘤的优选靶点之一     

反义c-myc寡脱氧核苷酸对家兔髂总动脉损伤后内膜增生的影响

目的：观察反义c-myc寡脱氧核苷酸(ASODN)对家兔髂总动脉损伤后内膜增生的影响，探讨其防治再狭窄的潜在作用。方法：通过pluronic gel缓释系统和Lipofeclin转染导入系统，将c-myc ASODN作用于家兔髂总动脉损伤后的血管外膜，3周后对所取的血管节段进行组织切片和苏木精-伊红染色，在显微镜下进行形态学观察，并采用图像分析系统进行图像分析，计算出内膜，中膜(I/M)面积比和I／M厚度比，并进行统计学处理。结果：c-myc ASODN可明显抑制内膜增生，而正义c-myc ODN对内膜增生无影响。结论：c-myc ASODN以序列特异性方式抑制了新生内膜的形成。     

白细胞介素-18反义寡脱氧核苷酸对同种异体部分移植肝再生的促进作用

目的 探讨白细胞介素(IL)-18硫代修饰反义寡脱氧核苷酸(ASPODN)对同种异体部分移植肝再生的促进作用。 方法 将供体SD大鼠与受体LEW大鼠随机分为50％部分肝移植组(PLT组)、部分肝移植IL-18 ASPODN治疗组(IL-18 ASPODN组)及部分肝移植IL-18硫代修饰正义寡脱氧核苷酸治疗组(IL-18 SPODN组),每组供、受体大鼠各30只。检测移植肝肝细胞BrdU标记变化;检测移植肝IL-18蛋白和γ干扰素(INF-γ)mRNA表达以及受体血清IFN-γ水平。 结果 移植术后72 h,3组移植肝肝细胞再生均达到高峰。IL-18 ASPODN组移植肝肝细胞BrdU高峰标记指数(58.3％±7.5％)明显高于PLT组(31.6％±6.7％)及IL-18 SPODN组[(33.4±5.5％),t=6.503、6.558,P<0.001];移植术后48、72和96 hIL-18 ASPODN组移植肝内IL-18蛋白和IFN-γ mRNA表达量以及受体血清IFN-γ水平均明显低于PLT组(IL-18蛋白比较:t=2.950,P<0.05;t=5.916、7.947,P<0.001;INF—γ mRNA比较:t=2.558,P<0.05;t=6.292、8.925,P<0.001;IEN-γ水平比较:t=16.998、15.483、54.723,P<0.001)和IL-18SPODN组(IL-18蛋白比较:t=2.845,P<0.05;t=6.062、6.973,P<0.001;INF-γ mRNA比较:t=3.117,P<0.05;t=6.154、8.738,P<0.001;IFN-γ水平比较:t=14.531、18.139、46.924,P<0.001)。结论 IL-18 AS     

序贯顺序对反义硫代寡核苷酸联合赫赛汀抗乳腺癌活性的影响

目的:研究以HER2 mRNA为靶点的反义硫代脱氧寡核苷酸(S-ODNs)}IA6722单用及以不同的顺序与赫赛汀合用时对HER2过表达乳腺癌细胞株MDA-MB-453体外增殖的抑制作用;方法:选择HER2过表达的MDA-MB-453乳腺癌细胞, MTT法观察HA6722单用及与赫赛汀合用时对该肿瘤细胞增殖的影响;以末端转移酶介导的dUTP切口末端标记法(TUNEL)检测细胞凋亡。结果:赫赛汀及HA6722单用均可以剂量依赖方式抑制MDA-MB-453细胞的体外增殖,IC50值分别为(62．71±18．39)nmol·L-1及(86．33±14．62)mol··L-1(n=3)。联合应用的顺序直接影响二者的交互作用,如先用HA6722再用赫赛汀,则在200 nmol·L-1的浓度下联合应用对MDA-MB-453细胞增殖抑制作用增强;反之则否。结论:反义寡核苷酸HA6722与单克隆抗体赫赛汀序贯应用顺序对其抗乳腺癌细胞增殖作用有重要影响。     

Intracerebroventricular treatment with an antisense oligodeoxynucleotide to κ-opioid receptors inhibited κ-agonist-induced analgesia in rats

In vivo treatment with an antisense (AS) phosphorothioate oligodeoxynucleotide (oligo) to the rat κ-opioid receptor selectively inhibited κ-mediated analgesia in the rat cold-water tail-flick test. Intracerebroventricular (i.c.v.) AS oligo significantly inhibited the analgesic effect of i.c.v. spiradoline, but not that of μ- or δ-opioid agonists. The dose-effect curve for s.c. spiradoline was shifted to the right after AS, but not missense or sense oligo treatment. Thus, AS oligos provide another technique with which to selectively manipulate opioid receptors and further support the role of non-μ opioid receptors in mediating analgesia in rats.     

血管紧张素Ⅰ型受体反义寡核苷酸对大鼠肾脏局部肾素-血管紧张素系统的阻断作用

目的 研究血管紧张素Ⅰ型受体(AT1)反义(AS)寡核苷酸(ODN)对大鼠肾脏局部肾素-血管紧张素系统(RAS)的阻断作用。方法采用单侧肾动脉注射 原位电穿孔的方法，将AT1 AS-ODN导入到Wistat大鼠的一侧肾脏，观察以FITC荧光标记的ODN在肾脏的转移位置，以AT1放射自显影定量分析的方法观察转移基因的时间-效应曲线。在基因转移3d后以RT-PCR、Western印迹及免疫组织化学方法检测肾脏AT1的mRNA和蛋白表达与分布，并与正义(Sen)ODN转移、假注射 肾脏原位电穿孔(Sham)作为对照。结果FITC标记的ODN在基因转移10min后主要定位于转移侧肾脏的肾小球，而对侧肾脏FITC荧光阴性。AT1放射自显影定量分析显示在基因转移后的3d内，结合有血管紧张素Ⅱ(AngⅡ)的AT1较未转移的对侧肾脏减少约70％。AT1 AS-ODN基因转移后3d，转移侧肾脏AT1 mRNA水平较对侧肾脏下降约43％，蛋白水平下降约67％，而与AT1 Sen-ODN和Sham组转移侧肾脏比较，AT1的mRNA水平下降分别约47％和51％，蛋白水平下降分别约63％和71％。免疫组化检查发现AT1的阻断以系膜区的表达减少为主。结论本研究所用的单侧肾动脉注射 原位电穿孔的基因转移方法成功地抑制了单侧肾脏的肾小球AT1受体的表达，为研究全身性疾病肾损害中肾脏局部RAS的短期作用机制提供了一个比较理想的整体动物模型。     

反义寡脱氧核苷酸技术在分子影像中的应用

反义寡脱氧核苷酸(ASODN)技术是根据核酸杂交原理,设计针对特定靶序列的ASODN,从而抑制特定基因的表达.分子影像学是一门在细胞与分子水平对活体生物过程进行描述和测量的新兴交叉学科.ASODN技术以分子影像为技术平台,通过图像达到无创、实时、活体、特异、精细地直接显示细胞或分子水平的生理和病理过程.该文主要阐述ASODN的作用机制、ASODN的化学修饰、ASODN技术的进展及其在分子影像领域的应用.     

Oligodeoxynucleotide inhibition of Toll‐like receptors 3, 7, 8, and 9 suppresses cytokine production in a human rheumatoid arthritis model

Toll‐like receptors (TLRs) are innate immune receptors that respond to both exogenous and endogenous stimuli and are suggested to contribute to the perpetuation of chronic inflammation associated with rheumatoid arthritis (RA). In particular, the endosomal TLRs 3, 7, 8, and 9 have more recently been postulated to be of importance in RA pathogenesis. In this study, pan inhibition of the endosomal TLRs by a phosphorothioate‐modified inhibitory oligodeoxynucleotide (ODN) is demonstrated in primary human B cells, macrophages, and RA fibroblasts. Inhibition of TLR8 was of particular interest as TLR8 has been associated with RA pathogenesis in both human and murine arthritis models. ODN1411 competitively inhibited TLR8 signaling and was observed to directly bind to a purified TLR8 ectodomain, suggesting inhibition was through a direct interaction with the receptor. Addition of ODN1411 to human RA synovial membrane cultures significantly inhibited spontaneous cytokine production from these cultures, suggesting a potential role for one or more of the endosomal TLRs in inflammatory cytokine production in RA and the potential for inhibitory ODNs as novel therapies.     

Orphanin FQ/nociceptin analgesia in the rat

The heptadecapeptide orphanin FQ or nociceptin (OFQ/N), the endogenous ligand for the orphan opioid receptor, has a complex pharmacology in mice, eliciting either an anti-opioid/hyperalgesic action or analgesia depending upon the dose and testing paradigm. Unlike mice, orphanin FQ/nociceptin fails to elicit hyperalgesia in the rat following intracerebroventricular injection. Both OFQ/N and a truncated version, OFQ/N(1-11), produce a robust analgesic response. OFQ/N analgesia is readily antagonized by the opioid antagonists naloxone or diprenorphine, despite their very poor affinity for the cloned orphan opioid receptor. Antisense studies revealed that probes targeting the second and third coding exon of the orphan clone significantly attenuate OFQ/N analgesia, while the exon 1 probe was inactive. These results indicate that OFQ/N elicits a naloxone-sensitive analgesia in rats similar to that previously reported in mice.