COPD患者气道中性粒细胞弹力酶活性与肺组织Elafin表达的关系及意义

目的　探讨慢性阻塞性肺疾病 (COPD)患者气道中性粒细胞弹力酶与肺组织 Elafin表达的关系及意义。方法　通过底物检测法、EL ISA法及 Dot- Blot印迹杂交分别检测正常肺组织和 COPD患者肺组织支气管肺泡灌洗液 (BAL F)中性粒细胞弹力酶 (NE)活力、Elafin含量及肺组织 Elafin m RNA水平。结果　 COPD组 BAL F中 NE活力单位与正常组相比较明显增高 (P<0 .0 5 ) ,COPD组 Elafin m RNA与正常组相比较亦见明显增高 (P<0 .0 1) ,NE活力单位与 Elafin m RNA呈显著正相关 (r=0 .6 1,P<0 .0 5 )。但 COPD组 BAL F中 Elafin含量与正常组相比并未见明显升高 (P>0 .0 5 )。结论　 NE可引起 COPD患者肺组织中 Elafin转录水平增加 ,由于 NE可与游离的 Elafin结合 ,这种消耗使得 BAL F中 Elafin蛋白水平未见明显提高。     

重组人Elafin转染气道上皮对炎性损伤因子攻击的保护作用

目的探讨重组人Elafin对炎症损伤因子中性粒细胞弹性蛋白酶(NE)攻击气道上皮保护作用的分子机制。方法通过构建人中性粒细胞弹性蛋白酶抑制剂Elafin真核表达载体pEGFP-C1-Elafin,并将其转染入NCI-H292细胞中,再用中性粒细胞弹性蛋白酶(NE)刺激24h后,用底物法测定培养上清中NE的活性,Westernbolt检测细胞中ZO-1表达。结果转染Ela-fin NE组培养上清液中NE活性与转染空载体的细胞相比较明显降低,而细胞内ZO-1蛋白含量明显增高。结论NE是引起气道慢性炎症的终效因子,通过转染重组人Elafin可对抗NE破坏气道上皮完整性的作用,增强气道抗感染能力。     

内生多肽Elafin对气道黏蛋白5AC表达的影响

目的 探讨内生多肽Elafin调节气道黏液高分泌的分子机制.方法 构建人 Elafin 重组质粒,原代培养正常人支气管上皮细胞 HBE16,分为对照组、香烟抽提物(CSE)刺激组、CSE 刺激+转染重组质粒、CSE 刺激+空质粒组、单纯转染重组质粒以及空质粒组6组.四甲基偶氮唑盐法检测各组细胞活力,Western blot法检测p-JNK、p-ERK、p-P38和IκBα蛋白含量,荧光素酶报告基因系统测定激活蛋白-1(AP-1)及核转录因子-κB(NF-κB)活性,RT-PCR检测各组黏蛋白(MUC)5AC mRNA 表达水平,ELISA 法分析各组细胞 MUC5AC 蛋白的相对含量.结果 CSE刺激组的p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC和MUC5AC mRNA 含量分别为(0.55±0.03)μg/mg、(0.64±0.06)μg/mg、(0.60±0.07)μg/mg、(0.27±0.03)μg/mg、7.49±0.31、4.42±0.22、(0.71±0.04)mg/L和0.81±0.04,与对照组的(0.26±0.02)μg/mg、(0.30±0.05)μg/mg、(0.19±0.04)μg/mg(0.61±0.04)μg/mg、2.54±0.22、2.37±0.16、(0.23±0.02)mg/L和0.32±0.03比较差异有统计学意义(均P＜0.05).转染重组Elafin后再给予CSE刺激,p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC和MUC5AC mRNA含量分别为(0.38±0.04)μg/mg、(0.31±0.04)μg/mg、(0.14±0.03)μg/mg、(0.54±0.03)μg/mg、2.60±0.19、2.55±0.21、(0.28±0.03)mg/L、0.35±0.05,与CSE组相比差异有统计学意义(均P＜0.05);p-P38蛋白含量在CSE刺激及转染Elafin前后无明显变化.结论 内生多肽Elafin可降低JNK和ERK磷酸化水平并抑制IκBα蛋白降解,从而降低转录激活蛋白-1和核因子-κB的活性,下调黏蛋白5AC的高表达,而p38MAPK在其中的作用并不明显.     

成骨生长肽在骨与骨髓相互作用中的角色

成骨生长肽(OGP)是近年在大鼠胫骨再生骨髓中发现的一种致有丝分裂的多肽类生长因子,以微摩尔浓度广泛存在于哺乳动物血清中,体内可以促进骨形成与造血,体外试验可以刺激成骨细胞的增殖,调节碱性磷酸酶活性。OGP C端5肽[OGP（10-14）]是OGP的生理活性形式,OP和OGP（10-14）在骨代谢和造血系统方面的作用已引起广泛关注。     

α-突触核蛋白功能片段对大鼠原代培养神经元的促生长作用

目的 研究α-突触核蛋白(α-Syn)对Wistar大鼠原代培养神经元的促生长作用.方法 培养新生24h的Wistar大鼠大脑皮质神经元,以一定密度接种至培养皿中.设计分组,在各组别培养基中加入α-Syn和它的功能片段NAC段、N端和C端.培养至1、2、4h固定观察,计数存活神经元的数目.Western印迹法、免疫荧光法进行特异性鉴定.结果 当原代神经元培养至第1、2小时,α-Syn组和C端组的神经元数目比对照组多(P＜0.05),N端组和NAC段组与对照组无差异(P＞0.05);培养至第4小时,α-Syn组和C端组的存活神经元数目比对照组明显增多(P＜0.01),而N端组和NAC段组与对照组无差异(P＞0.05).增加C端浓度,可以提高神经元的存活率(P＜0.05).Western印迹法和免疫荧光法证实蛋白质片段N端和C端可由培养基进入到原代神经元中,而NAC片段不能进入神经元.结论 α-Syn在原代神经元培养初期对其生长有促进作用,具有这一功能的蛋白片段为C端,其可能的机制是进入到神经元内,促进胞内代谢活动,从而利于神经元的生长.     

血管内皮生长因子和内皮抑素在肺纤维化中的作用

血管内皮牛长因子是一种生长因子,在正常肺组织和多种肺部疾病中均有表达.通过血管内皮牛长因子受体发挥生理作用,具有促进内皮细胞分裂增殖、促血管生成、增加血管通透性及抗凋亡等作用.内皮抑素是一种内源性血管生成抑制剂,可以抑制血管内皮细胞增殖、迁移并诱导其调亡,从而达到抑制血管生成作用.在肺纤维化中二者均有异常表达,但具体作...     

支气管哮喘和变应性鼻炎的舌下特异性免疫疗法进展

舌下特异性免疫疗法在支气管哮喘和变应性鼻炎的治疗方面发挥了重要作用,它提供了一种可以替代传统皮下免疫疗法的变应原免疫治疗方法.目前已经有两种制剂(滴剂和片剂)可以用于舌下特异性免疫疗法.研究表明舌下特异性免疫疗法是安全、有效的.     

瘦素启动青春发育的研究进展

瘦素 (LEP)是由脂肪细胞分泌的一种多肽激素 ,LEP作为脂肪组织和中枢神经网络联系的代谢信号参与哺乳动物青春发育启动的调节机制。研究证实青春发育前期血LEP水平升高可能是一种允许信号。LEP启动青春发育可能的机制 :(1)神经内分泌作用机制 ,其中神经肽Y、N端 原阿片黑皮素、一氧化氮可能是LEP重要的介导因子。 (2 )LEP可以部分解除吗啡对促性腺激素释放激素脉冲发生器电释放的抑制。 (3)LEP亦可能通过LEP受体直接作用于促性腺激素释放激素神经元。目前LEP在青春发育启动中的意义及作用机制仍有待进一步探讨     

髋关节腔内注射重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白治疗强直性脊柱炎髋关节受累一例

强直性脊柱炎(ankylosing spondylitis,AS)是一种以骶髂关节炎、肌腱端炎和脊柱炎为特点的慢性炎症性进行性疾病,严重者可发生脊柱强直和关节强直.25%AS患者累及髋关节,髋关节受累是本病致残最关键的病变.目前对于AS髋关节病变的治疗尚缺乏有效手段.肿瘤坏死因子(TNF)-α是炎症级联反应中重要的促炎症细胞因子之一,目前研究已证实TNF-α在AS发病机制中发挥重要作用.     

ZAP-70的表达与慢性淋巴细胞白血病预后的关系

慢性淋巴细胞白血病(CLL)是一种主要累及B淋巴细胞的恶性肿瘤,具有显著的临床预后异质性,预后的参数比较多。ZAP-70是T细胞相关的相对分子量为70000的一种Syk家族蛋白酪氨酸激酶,目前研究证实可以作为B-CLL独立的预后指标。1ZAP-70的生物学特征ZAP-70是T细胞相关的分子量为70000的一种Syk家族蛋白酪氨酸激酶,为一种细胞质内具有PTK活性的信号蛋白,N端含有2个SH-2(SRC homology region2,SH-2)结构域以及一个与猪脾中PTK、SYK相关的激酶结构域,C端有一个SH1激酶结构域。2个SH2结构域可以与多种激活型免疫受体细胞质区的磷酸…     

Elafin in human endometrium: an antiprotease and antimicrobial molecule expressed during menstruation

Elafin is an antiproteinase and antimicrobial molecule that is expressed at epithelial sites (for example, cervix). This study details the expression and regulation of elafin in the human endometrium. Elafin mRNA and protein expression were examined in endometrium throughout the menstrual cycle and in first-trimester decidua. Real-time quantitative PCR showed that expression of elafin mRNA peaked during menstruation. Elafin protein was localized to leukocytes scattered in the endometrial stroma during the late secretory and menstrual phases. Faint immunostaining was also present in glandular epithelium at these cycle stages. Immunofluorescent colocalization of elafin with neutrophil elastase confirmed that elafin was expressed by endometrial neutrophils around the time of menstruation. This is consistent with the expression profile observed from immunohistochemical studies. Primary endometrial epithelial cells were treated with proinflammatory molecules, and elafin mRNA was studied. A combination of the proinflammatory mediators, IL-1 beta and TNFalpha, increased elafin mRNA levels by 4.6-fold. These results show that endometrium expresses elafin in a menstruation-dependent manner. This is attributable to the presence of infiltrating leukocytes and increased inflammatory signaling. Elafin will regulate proteolytic enzymes during menstruation and will contribute to the innate defense against uterine infection.     

Regulation of protein function by native metastability

In common globular proteins, the native form is in its most stable state. In contrast, each native form exists in a metastable state in inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins. Metastability in these proteins is critical to their biological functions. Mutational analyses and structural examination have previously revealed unusual interactions, such as side-chain overpacking, buried polar groups, and cavities as the structural basis of the native metastability. However, the mechanism by which these structural defects regulate protein functions has not been elucidated. We report here characterization of cavity-filling mutations of alpha(1)-antitrypsin, a prototype serpin. Conformational stability of the molecule increased linearly with the van der Waals volume of the side chains. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. These results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions.     

Inhibition of natural killer cell cytotoxicity and interferon gamma production by the envelope protein of HIV and prevention by vasoactive intestinal peptide

Natural killer (NK) cell dysfunction is common in human immunodeficiency virus (HIV)-infected subjects, although its mechanisms are poorly understood. A direct effect of HIV envelope glycoprotein gp120 may be involved. We investigated the in vitro effects of gp120 on the major NK cell effector functions, natural cytotoxicity and cytokine production. In addition, the ability of the vasoactive intestinal peptide (VIP) to modulate these effects was investigated. Our results indicated that gp120 inhibits NK natural cytotoxicity and showed, for the first time, that the inhibition affects also the production of the proinflammatory cytokine interferon-gamma (IFN-gamma). Interestingly, the inhibitory effect on NK cell functions was obtained with gp120 at concentrations within the range measured in the serum of HIV-infected subjects. Furthermore, we showed that the inhibitory activity of gp120 can be prevented by coincubation with VIP, even if VIP has no stimulatory activity by itself. Taken together these data suggest that (1) an inhibitory effect of gp120 may account for the NK cell dysfunction in HIV-infected subjects; (2) the gp120-mediated decrease in IFN-gamma production by NK cells may contribute to the cytokine imbalance observed in HIV infection; and (3) VIP counteracts the inhibitory effect of gp120 on NK cell functions.     

Haemophilia A: effects of inhibitory antibodies on factor VIII functional interactions and approaches to prevent their action

Factor VIII (FVIII) is an essential component of the intrinsic pathway of blood coagulation. Normal functioning of FVIII requires its interactions with other components of the coagulation cascade. In the circulation, it exists as a complex with von Willebrand factor (vWF). Upon activation by thrombin or activated factor X (FXa), activated FVIII (FVIIIa) functions as a cofactor for the serine protease factor IXa. Their complex assembled on the phospholipid surface activates FX to FXa, which consequently participates in formation of thrombin, the key protease of the coagulation cascade. Genetic deficiency in FVIII results in a coagulation disorder haemophilia A, which is treated by infusions of FVIII products. Approximately 25-30% of patients develop antibodies inhibiting FVIII activity (FVIII inhibitors). The major epitopes of inhibitors are located within the A2, C2 and A3 domains of the FVIII molecule. The inhibitory effects of antibodies are manifested at various stages of the FVIII functional pathway, including FVIII binding to vWF, activation of FVIII by thrombin, and FVIIIa incorporation into the Xase complex. We summarize the current knowledge of the FVIII sites involved in interaction with its physiological ligands and different classes of inhibitory antibodies and describe their inhibitory mechanisms. We outline the strategies aimed to overcome the effects of inhibitory antibodies such as development of human/porcine FVIII molecules, resistant to inhibitors. We also discuss approaches to modulate the antibody response, as well as efforts to develop a long-term immunotolerance to FVIII protein.     

Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.     

Serotonin modulates the oxidative burst of human phagocytes via various mechanisms

Serotonin, the major secretory product of activated platelets, has been widely reported as regulating various constituents of the immune system and immune functions. This modulation is complex and the data available are rather controversial. The aim of the present study was to clarify the mechanisms of serotonin action on human phagocytes. The effect of serotonin in a concentration range of 10(-7) M-10(-3) M on various parameters of oxidative burst of phagocytes was studied using various luminol-enhanced chemiluminescence methods. Serotonin inhibited the chemiluminescence response of the cells in a dose dependent manner. The effect of serotonin on the activity of myeloperoxidase was studied in further experiments. In this case, serotonin again exerted a dose dependent inhibition of the myeloperoxidase activity. The hypothesis that the inhibitory activity of serotonin might be also receptor mediated was evaluated using various serotonin receptor agonists and antagonists. None of the agonists studied exerted any direct antioxidative properties. Only (+/-)-DOI hydrochloride, a selective 5-HTR(2) agonist, exerted similar effects on phagocytic cells as serotonin. It can be concluded that serotonin could affect the oxidative burst of phagocytes. Responsibility for its inhibitory effects lies with both the decrease in the generation of reactive oxygen species (due to the inhibition of myeloperoxidase activity) and with direct scavenging of reactive oxygen species. The effect of serotonin on phagocytes is also partially mediated by 5-HTR(2) receptor.