活性氧调控因子1在人退变椎间盘中的表达及意义

目的　探讨活性氧调控因子1（reactive oxygen species modulator 1,ROMO1）在退变椎间盘髓核中的表达。 方法　采用免疫组化和荧光探针DCFH-DA检测不同退变髓核组织中ROMO1的表达与(reactive oxygen species, ROS)含量,并分析ROMO1表达水平与ROS含量、椎间盘退变程度的相关性。 结果　各组ROMO1的平均光密度值依次为0.192±0.089、0.328±0.048、0.399±0.053、0.468±0.098;各组ROS结果分别为132.961±15.149、191.889±17.880、218.056±12.845、243.501±30.279。随着椎间盘退变程度的加重, ROMO1蛋白表达量与ROS含量逐渐升高,且差异具有显著性(P<0.05)。ROMO1的表达水平与ROS的含量及椎间盘退变程度均呈正相关关系（P＜0.05）。 结论　ROMO1的表达水平与椎间盘的退变程度呈正相关,这可能与其增加髓核组织内ROS含量而加重氧化应激损伤有关。     

AhR途径,CYP1A1、CYP1B1,雌激素代谢及作用过程中的调节

多环芳烃和多卤化烃是环境中广泛分布的有害物质,可通过与细胞芳烃受体结合,从而影响外来化合物代谢酶系如细胞色素氧化酶P450 1A1、1B1的表达,并通过这些酶的催化作用调控雌激素的代谢及作用,进而部分决定了雌激素对机体的作用效应。上述复杂的过程可受到多种因素的影响。     

Analysis of human CYP1A1 and CYP1A2 genes and their shared bidirectional promoter in eight world populations

The human CYP1A1_CYP1A2 locus comprises the CYP1A1 (5,988 bp) and CYP1A2 (7,759 bp) transcribed regions, oriented head‐to‐head, sharing a bidirectional promoter of 23,306 bp. The older CYP1A1 gene appears more conserved and responsible for critical life function(s), whereas the younger CYP1A2 gene might have evolved more rapidly due to environmental (dietary) pressures. A population genetics study might confirm this premise. We combined 60 CYP1A1_CYP1A2 SNPs found in the present study (eight New Guinea Highlanders, eight Samoans, four Dogrib, four Teribe, four Pehuenche, and one Caucasian) with those found in a previous study (six West Africans, four Han Chinese, six Germans, four Samoans, and four Dogrib), yielding a total of 106 SNPs in 106 chromosomes. Resequencing of Oceanians plus Amerindians in the present study yielded 21 New World SNPs (～20%), of which 17 are not previously reported in any SNP database. Various tests revealed selective pressures for both genes and both haploblocks; unfortunately, differences in rates of evolution between the two genes were undetectable. Fay & Wu's H test revealed a “hitchhiking event” centered around four SNPs in the CYP1A1 3′‐UTR; a study in silico identified different microRNA‐binding patterns in the hitchhiked region, when the mutations were present compared with the mutations absent. Hum Mutat 30:1–14, 2009. © 2009 Wiley‐Liss, Inc.     

Differential involvement of reactive oxygen species in apoptosis caused by the inhibition of protein phosphatase 2A in Jurkat and CCRF-CEM human T-leukemia cells

A better understanding of dysregulated signaling pathways in cancer cells may suggest novel strategies to prevent tumor development and/or progression. Here we show that Jurkat and CCRF-CEM human T-leukemia cell lines were more sensitive than normal human T cells to the cytotoxic effect of inhibiting protein phosphatase 2A (PP2A). Inhibition of PP2A by okadaic acid (OA) caused T-leukemia cells to die by apoptosis, as indicated by DNA fragmentation, caspase-3 activation, loss of mitochondrial membrane potential (DeltaPsi(m)), and changes in nuclear morphology that were consistent with apoptosis. PP2A might therefore be a useful intracellular target for the treatment of T cell-derived leukemias. We also observed that reactive oxygen species (ROS) were generated in response to PP2A inhibition in T-leukemia cells. However, loss of DeltaPsi(m) that resulted from PP2A inhibition was not prevented by exogenous antioxidants (glutathione and N-acetyl-cysteine), indicating that OA-induced changes in mitochondrial membrane permeability were not a consequence of ROS production. Moreover, exogenous antioxidants protected CCRF-CEM T-leukemia cells from apoptosis caused by PP2A inhibition but failed to prevent OA-induced apoptosis in Jurkat T-leukemia cells, indicating a differential role for ROS in apoptosis caused by PP2A inhibition in two different human T-leukemia cell lines.     

去甲肾上腺素对心肌细胞NF-κB活性的影响

目的:探讨去甲肾上腺素(NE)对心肌细胞核因子-κB(NF-κB)活性的影响及机制。方法:采用乳鼠心肌细胞培养模型,分别加入NE和哌唑嗪、普萘洛尔及维生素E等刺激,观察心肌细胞内活性氧水平和NF-κB核结合活性的改变。结果:NE刺激后,心肌细胞活性氧生成明显增加、NF-κB活性增强,维生素E、哌唑嗪和普萘洛尔可不同程度减少心肌细胞活性氧含量、降低NF-κB活性。结论:NE对心肌细胞的作用部分通过激活NF-κB实现,这与NE通过肾上腺素能受体介导活性氧过量产生有关。     

B7‐1/2, but not PD‐L1/2 molecules,are required on IL‐10‐treated tolerogenic DC and DC‐derived exosomes for in vivo function

Costimulatory molecules, such as B7‐1/2 and PD‐L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7‐1/2 and PD‐L1/2 to the function of IL‐10‐treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL‐10 treatment of DC significantly downregulated surface expression of MHC II, B7‐1, B7‐2, and decreased levels of MHC I and PD‐L2. IL‐10 treatment of DC resulted in a modified costimulatory profile of DC‐secreted exosomes with a reduction in B7‐1, PD‐L1 and PD‐L2. We further demonstrate that absence of B7‐1 or B7‐2 on donor DC results in a loss of ability of IL‐10‐treated DC and their exosomes to suppress the delayed‐type hypersensitivity response, whereas IL‐10‐treated DC deficient in PD‐L1/2 as well as their secreted exosomes retained the ability to suppress delayed‐type hypersensitivity responses. We conclude that B7‐1 and B7‐2, but not PD‐L1 and PD‐L2, on IL‐10‐treated DC and DC‐derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.     

Calcitriol prolongs recipient survival by inducing expression of zinc-finger protein A20 and inhibiting its downstream gene following rat orthotopic liver transplantation

Calcitriol has important immunomodulation action and can prolong recipient survival after organ transplantation. The data in this study demonstrated that treatment of liver allograft recipient with calcitriol can protect allograft from acute rejection and prolong recipient's survival. Calcitriol inhibited expression of proinflammatory cytokine such as Interleukin-2 and Interferon-γ intragraft. It also inhibited expression of nuclear factor κB (NF-κB) significantly as a result of enhancing its inhibitory protein I kappa B (IκB). As well, expression of zinc-finger protein A20 (A20) was enhanced significantly. The results suggest that calcitriol exerts its immunosuppression action in part through inducement of the A20, IkB, inhibition of NF-kB, and resultant proinflammatory expression pathway.     

胰淀素对离体大鼠胰岛细胞内活性氧生成的影响

目的：观察高浓度胰淀素对胰岛细胞内活性氧（ROS）生成的影响。方法：以体外培养胰岛单层细胞为模型,应用ROS特异性荧光染料2′,7′-二氯二氢荧光素双醋酸盐（H₂DCF）进行标记，以粘附细胞仪570型测定10 μmol/L胰淀素作用2 h后胰岛细胞内DCF荧光强度变化。结果：10 μmol/L胰淀素作用2 h，胰岛细胞内DCF荧光强度在16.7 mmol/L葡萄糖刺激后，迅速急骤升高进入平台期，升高速度为28×10^-3/s，而正常细胞DCF荧光强度无明显变化。光镜下可见部分细胞胞体缩小，胞质发生空泡变性，少数细胞核固缩、裂解，胞膜突起。结论：10 μmol/L胰淀素作用后的胰岛细胞内ROS生成增多，细胞形态受损，这些活性氧基的生成可以反映胰淀素对胰岛细胞损害程度，推测活性氧可作为胰岛细胞毒性损伤指标之一。     

Cytochrome P450 1A1 and manganese superoxide dismutase genes polymorphisms in reactive arthritis

To investigate the role of cytochrome P450 1A1 (CYP1A1) and manganese superoxide dismutase (Mn SOD) genes polymorphisms in the susceptibility to reactive arthritis, the polymorphisms of CYP1A1 and Mn SOD genes were determined in 43 patients with reactive arthritis following Chlamydia trachomatis infection and 92 healthy controls by polymerase chain reaction (PCR)/restriction fragment length polymorphisms (RFLP) method.

The frequencies of CYP1A1 4887C/A and C/A+A/A were significantly higher in patients with reactive arthritis than in controls. Moreover, the increased frequency of CYP1A1 4887A in patients with reactive arthritis is independent of HLA-B27. The frequency of Mn SOD 1183T/T tended to be increased in patients with reactive arthritis. We also found that the frequency of CYP1A1 4887A was significantly increased in Mn SOD 1183T/T(+) reactive arthritis patients compared with 1183T/T(+) controls.

CYP1A1 4887A may be a risk factor for the development of reactive arthritis, especially in the presence of Mn SOD 1183T/T.     

Attenuation of cytotoxicity induced by tBHP in H9C2 cells by Bacopa monniera and Bacoside A

Cardiovascular diseases are one of the major global health issues leading to morbidity and mortality across the world. In the present study Bacopa monniera and its major bioactive component, Bacoside A (Bac-A) was used to evaluate its cytoprotective property in H9C2 cardiomyocytes against tBHP (150?μM) induced ROS-mediated oxidative stress and apoptosis. Our results implicate that pre-treatment with hydroalcoholic extract of Bacopa monniera (BME) and Bac-A (125?μg/ml and 6?μg/ml respectively) significantly restored oxidative stress by scavenging the free radicals and also elevated phase II antioxidant defensive enzymes such as (SOD, CAT, GR, GPx and GSH). Membrane integrity was estimated by MMP and LDH assays and found 89 and 72% of the protective effect. Further immunoblotting studies confirmed anti-apoptotic effects by regulating protein expression like Bcl2 was up-regulated to 99 and 85% and Bax was down-regulated to 122 and 181%, iNOS by 154.38 and 183.45% compared to tBHP (277.48%) by BME and Bac-A. BME and Bac-A exerts cytoprotective efficacy by attenuation of ROS generated through oxidative stress by an increase in the concentration of antioxidant enzymes and sustain membrane integrity which leads to restoring the damage caused by tBHP.     

Systemically administered DNA and fowlpox recombinants expressing four vaccinia virus genes although immunogenic do not protect mice against the highly pathogenic IHD-J vaccinia strain

The first-generation smallpox vaccine was based on live vaccinia virus (VV) and it successfully eradicated the disease worldwide. Therefore, it was not administered any more after 1980, as smallpox no longer existed as a natural infection. However, emerging threats by terrorist organisations has prompted new programmes for second-generation vaccine development based on attenuated VV strains, which have been shown to cause rare but serious adverse events in immunocompromised patients. Considering the closely related animal poxviruses that might also be used as bioweapons, and the increasing number of unvaccinated young people and AIDS-affected immunocompromised subjects, a safer and more effective smallpox vaccine is still required. New avipoxvirus-based vectors should improve the safety of conventional vaccines, and protect from newly emerging zoonotic orthopoxvirus diseases and from the threat of deliberate release of variola or monkeypox virus in a bioterrorist attack. In this study, DNA and fowlpox recombinants expressing the L1R, A27L, A33R and B5R genes were constructed and evaluated in a pre-clinical trial in mouse, following six prime/boost immunisation regimens, to compare their immunogenicity and protective efficacy against a challenge with the lethal VV IHD-J strain. Although higher numbers of VV-specific IFNγ-producing T lymphocytes were observed in the protected mice, the cytotoxic T-lymphocyte response and the presence of neutralising antibodies did not always correlate with protection. In spite of previous successful results in mice, rabbits and monkeys, where SIV/HIV transgenes were expressed by the fowlpox vector, the immune response elicited by these recombinants was low, and most of the mice were not protected.     

中性粒细胞小GTP结合蛋白的表达与冠心病发生关系的研究

目的：探讨中性粒细胞活性氧的产生及小GTP结合蛋白Rac2和RhoGDI的差异性表达在冠心病(GHD)发生发展中的作用。方法：采用化学发光法检测活性氧，适时定量RT-PCR检测调控中性粒细胞活性氧产生的两种小GTP结合蛋白Rac2和RhoGDI mRNA表达量的变化。结果：冠心病组中性粒细胞产生活性氧显著增多，冠心病人血清中各种炎性因子有促进中性粒细胞产生活性氧的作用；Rac2mRNA在冠心病组表达量显著性高于正常对照组，而RhoGDI mRNA的表达量在两组中元显著区别，Rac2 mRNA和RhoGDImRNA表达量的比值与中性粒细胞产生活性氧的峰值呈正相关。结论：中性粒细胞通过产生活性氧参与冠心病的发生发展，冠心病病人中性粒细胞Rac2和RhoGDImRNA表达量比例失调是导致活性氧产生增多的重要因素。     

丹酚酸B抑制氧化应激引起骨髓间质干细胞凋亡的研究

目的: 观察丹酚酸B (Sal B)对氧化应激介导骨髓间质干细胞(BMSCs)凋亡的影响并探讨其作用机制。方法: 大鼠BMSCs培养鉴定后分为5组:空白对照组、氧化应激组、低浓度Sal B＋H₂O₂组、中浓度Sal B＋H₂O₂组和高浓度Sal B＋H₂O₂组。应用MTT、流式细胞仪(FCM)及Hoechst标记的方法检测Sal B对氧化应激介导的细胞活性下降以及凋亡效应的影响;通过DCF荧光染色的方法检测过氧化氢及Sal B对细胞内活性氧生成的影响;用Western blotting的方法检测p-ERK1/2表达情况。结果: MTT结果显示不同浓度的Sal B共处理BMSCs 24 h能够提高氧化应激情况下的细胞活性,其中中浓度组的作用更为明显。Hoechst标记以及FCM检测的结果表明:10 μmol/L Sal B处理能够提高细胞活性,减少凋亡数。正常组细胞的DCF阳性细胞率为28.7％±8.1％,经500 μmol/L H₂O₂刺激24 h后,阳性细胞数急剧上升,高达86.9％±12.4％。而10 μmol/L Sal B处理 组的上升不明显,仅有42.1％±10.8％。由此可见,Sal B处理可以减少细胞内活性氧的生成;细胞在氧化应激的条件下p-ERK1/2在15 min内开始上调,持续120 min。而这种上调经10 μmol/L Sal B处理可以有效降低,同时Sal B可以降低细胞基础的p-ERK1/2表达。结论: Sal B 增强BMSCs抗氧化应激能力从而减少H₂O₂刺激引起的细胞凋亡,其保护机制可能与参与调节凋亡相关信号通路MEK/ERK1/2和抑制细胞内活性氧生成有关。     

Nuclear factor κB represses the expression of latent membrane protein 1 in Epstein-Barr virus transformed cells

AIM: To investigate the role of nuclear factor κB(NF-κB) in the regulation of Epstein-Barr virus(EBV) latent membrane protein 1(LMP1) in EBV transformed cells. METHODS: LMP1 expression was examined in EBV transformed human B lymphocytes with modulation of NF-κB activity. RESULTS: EBV infection is associated with several human cancers. EBV LMP1 is required for efficient transformation of adult primary B cells in vitro, and is expressed in several pathogenic stages of EBVassociated cancers. Regulation of EBV LMP1 involves both viral and cellular factors. LMP1 activates NF-κB signaling pathway that is a part of the EBV transformation program. However, the relation between NF-κB and LMP1 expression is not well established yet. In this report, we found that blocking the NF-κB activity by Inhibitor of κB stimulated LMP1 expression, while the overexpression of NF-κB repressed LMP1 expression in EBV-transformed IB4 cells. In addition, LMP1 repressed its own promoter activities in reporter assays, and the repression was associated with the activation of NF-κB. Moreover, NF-κB alone is sufficient to repress LMP1 promoter activities. CONCLUSION: Our data suggest LMP1 may repress its own expression through NF-κB in EBV transformed cells and shed a light on LMP1 regulation during EBV transformation.     

Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.     

EZH2对胃癌细胞核因子κB靶基因的调节作用

 目的: 探讨zeste同源物增强子2 (EZH2) 蛋白的表达对胃癌细胞的影响以及可能的作用机制。方法: 用real-time PCR 和Western blot实验检测正常胃黏膜上皮细胞和不同胃癌细胞系中EZH2的mRNA和蛋白表达;采用细胞生长、细胞迁移以及软琼脂增殖实验测定EZH2对胃癌细胞系致癌能力的影响;利用萤光素酶报告基因和real-time PCR检测EZH2对核因子κB靶基因的调节作用;用免疫共沉淀法检测EZH2和p65的相互作用。结果: 与正常胃上皮细胞相比,胃癌细胞系中的EZH2过量表达(P<0.05)。EZH2特异性抑制剂腺苷类似物DZNep处理或shEZH2抑制了AGS和SNU-16细胞系的细胞活力。此外,DZNep和shEZH2 抑制了AGS细胞迁移及软琼脂细胞形成的克隆数目(P<0.05)。shEZH2 下调了核因子κB报告基因或白细胞介素8报告基因的活性以及核因子κB靶基因白细胞介素8、趋化因子配体5及趋化因子配体20的表达(P<0.05)。此外,EZH2可以特异性与p65蛋白相互作用。结论: EZH2 通过调节核因子κB靶基因介导胃癌细胞的生长。