凝血栓蛋白1基因异常甲基化与大肠腺癌关联

目的 探讨凝血栓蛋白l（THBS1）基因启动子CpG岛异常甲基化与大肠腺癌及其临床病理特征的关联。方法 THBS1基因甲基化状态用甲基化特异性PCR检测。结果 大肠腺癌、癌旁组织中，THBSI基因启动子cpG岛甲基化率的差异有显著性（X^2=5．93，P=0．025）；老年患者肿瘤组织中THBS1基因甲基化率明显商于非老年患者（X^2=5.68，P=0．017），直径≥3cm的肿瘤组织中THBSl基因甲基化率显著高于直径〈3cm的肿瘤（X^2=4．16，P=0．041），C期和D期肿瘤组织中THBS1基因甲基化率显著高于A期或B期肿瘤（X^2=8．04，u=2，P=0．018）。结论 THBS1基因甲基化与大肠腺癌的发生有关，肿瘤以老年、晚期和直径较大的肿瘤多见。     

赖型钩体flaB2与VR1012中的CpG基序分析

目的：对问号赖型钩端螺旋体（赖型钩体）DNA疫苗[包括内鞭毛蛋白基因（flaB2)和质粒DNA表达载体（VR1012）]的CpG基序（CpG motifs)进行分析，为DNA疫苗免疫机制的阐明和提高DNA疫苗的效能奠定基础。方法：以flaB2与VR1012构建重组DNA的免疫原，对flaB2及VR1012全核苷酸序列进行计算机分析（分类、计数和定位）。结果：CpG的“C”的侧翼为两个嘌呤，“G”的侧翼为两个嘧啶，在flaB2中共3个，分别为GACGCT，GACGTC和GACGCC；在VR1012中共11个，分别为GACGTC1个，GACGCT2个，GACGCC1个，GACGTT1个，GGCGTT2个，GGCGCT2个，GGCGCC1个，AACGCT1个，其中特别重要的TGACGTCA4个和TAACGCCA有1个，位于5'端456－463；509－516；592－599；778－785和486－493；4个TGACGTCA和1个TAACGCCA均位于5'端且相对集中。结论：赖型钩体flaB2与VR1012构成的DNA疫苗含有TGACGTCA等CpG，这些基序又称免疫刺激序列，构成了DNA疫苗中的佐剂。     

CpG ODN增强乙肝疫苗在老年小鼠中的免疫应答

目的：探讨CpG ODN对老年小鼠的体液和细胞免疫应答的增强作用。方法：选用老年C57BL／6小鼠，将乙肝疫苗和10μg、20μg CpG ODN同时或单独肌注到小鼠体内，两周后以同样剂量加强免疫一次，再过3周后摘除眼球取血，用EILSA方法检测抗HBs16；抗体和IL-12；无菌取脾脏作HE染色，观察脾脏淋巴细胞变化。结果：10μg和20μg CpG ODN与疫苗同时注射组产生的抗体绝对量分别是单独注射疫苗组的3倍和4倍；产生的IL-12水平较对照组有明显升高，且20μg比10μCpG组产生的IL-12水平更高。光镜下各组的脾脏淋巴细胞的变化如下：正常老年鼠组脾脏淋巴细胞较正常青年鼠组明显稀少；老年鼠 10μgCpG组脾脏淋巴细胞较正常老年鼠组有了明显增加，且细胞核也明显增大；20μgCpG组增加的更加明显。结论：CpG ODN能增强乙肝疫苗在老年小鼠中的体液和细胞免疫应答。     

肿瘤细胞中的表观遗传编码紊乱

不改变基因的DNA编码,通过改变DNA双链与组蛋白间紧密度来决定基因是否转录表达,这称为表观遗传编码机制。表观遗传编码的生理作用是通过组蛋白修饰和DNA甲基化,调控细胞在适当的时间、空间位置表达适当的基因,从而控制细胞的增殖状况和分化方向。在细胞发育过程中,细胞内DNA甲基化水平增龄性增高,基因转录活性逐渐降低,使细胞从幼稚增殖进入成熟分化。肿瘤细胞中出现表观遗传编码紊乱,致细胞增殖失控,不能进入分化成熟阶段。基因启动子出现甲基化重排,阻碍转录因子与启动子结合,导致基因转录丧失正常调控,合成成熟功能蛋白受阻。利用表观遗传机制(如,RNA干涉)可成为肿瘤治疗的新方法。     

CpG ODN序列结构特征对免疫活性影响的研究进展

CpG ODN(CpG oligonucleotide,CpG 寡脱氧核苷酸)是人工合成的含有非甲基化的胞嘧啶鸟嘌呤二核苷酸(CpG)的寡脱氧核苷酸(ODN),可模拟细菌DNA刺激多种哺乳动物包括人的免疫细胞.     

免疫调节性寡聚脱氧核苷酸筛选方法的建立、优化和应用

目的:建立一套合理而便捷的实验体系,为开发新型免疫调节性寡聚脱氧核苷酸(ODN)提供筛选方法。方法:利用含有CpG基序的免疫刺激性寡聚脱氧核苷酸(CpG ODN)作为刺激物,将人外周血单个核细胞(PBMC)作为研究对象,分别以细胞的增殖程度和刺激上清的抗病毒活性作为检测指标,优化各项实验条件,综合评定寡聚脱氧核苷酸的免疫调节活性。结果:成功建立了由阳性免疫调节性ODNA151抑制CpG ODN诱导的人PBMC增殖及抗病毒活性的筛选方法。结论:免疫调节性ODN筛选方法的成功建立,为下一步研究开发新型免疫调节性ODN奠定了基础。     

Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-alpha by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-alpha secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future.     

Systemic Administration of Immunostimulatory DNA Sequences Mediates Reversible Inhibition of Th2 Responses in a Mouse Model of Asthma

This study investigated whether immunostimulatory DNA sequences (ISS) induce a transient or sustained inhibition of Th2 responses to inhaled antigen. We sensitized mice with subcutaneous injections to develop a Th2 response to ovalbumin (ova) and then administered a dose of ISS prior to ova inhalation challenge. Mice were then rechallenged with ova by inhalation a second time at varying time points after the first ova inhalation (1 to 8 weeks later) to determine whether the ISS dose administered prior to the first ova inhalation protected against a subsequent second ova inhalation challenge. A single dose of ISS inhibited the Th2 response to the first inhalation of ova antigen, as well as 4 weeks later to the second inhalation of ova. However, ISS did not inhibit a Th2 response to the second inhalation of ova 8 weeks later. The reversible inhibition of Th2 responses at 8 weeks suggests the need for repeated ISS administration at monthly intervals.     

DNA免疫诱导小鼠产生抗人心肌肌钙蛋白T抗血清的研究

目的 通过基因免疫诱导BALB／c小鼠产生抗人心肌肌钙蛋白T(cTnT)的抗体应答，并比较不同免疫佐剂、预处理方法对免疫应答的影响。方法 构建cTnT的真核表达质粒pCI-cTnT，大量提取、PEG纯化质粒，通过肌肉注射免疫BALB／c小鼠并加强免疫，每隔2周取血，ELISA法分析抗cTnT的体液应答，免疫印迹检测抗血清的特异性。并比较布吡卡因预处理、CpG ODN核酸佐剂、弗氏不完全佐剂对抗体应答效果的影响。结果 接种pCI-cTnT质粒的小鼠血清中检测出抗cTnT特异性抗体，加强免疫使抗体滴度提高；使用CpGODN后免疫应答增强，弗氏不完全佐剂可明显增强免疫效果，而吡卡因预处理使应答水平下降；初次免疫后14周仍可测得较高的抗体应答。结论 采用含cTnT基因的质粒DNA免疫，成功地诱导小鼠产生抗cTnT的抗体；传统的弗氏不完全佐剂可作为核酸免疫的佐剂使用。