RT-PCR法检测HNP1基因在气管上皮细胞转染后的表达

采用脂质体转染法将人中性粒细胞防御素（ Ｈ Ｎ Ｐ１ ）的 ｃ Ｄ Ｎ Ａ 重组真核表达质粒ｐ Ｂａｂｅ Ｎｅｏ Ｈ Ｎ Ｐ１导入无血清培养的人和兔的气管粘膜上皮细胞,利用逆转录聚合酶链反应（ Ｒ Ｔ Ｐ Ｃ Ｒ）法,在核酸水平检测 Ｈ Ｎ Ｐ１ 在气管上皮细胞的表达。结果：在人和兔的转染上皮细胞中均可检测到 Ｈ Ｎ Ｐ１ｍ Ｒ Ｎ Ａ 的表达,而在未转染的上皮细胞中 Ｒ Ｔ Ｐ Ｃ Ｒ检测结果呈阴性。这一结果与作者先前用免疫组化法在蛋白质水平上检测的结果一致,并证明ｐ Ｂａｂｅ Ｎｅｏ Ｈ Ｎ Ｐ１ 转染至气管粘膜上皮细胞后能得到有效表达。     

腭扁桃体中β-防御素的表达及意义

目的 :检测人β 防御素 (hBD) 1,2mRNA在慢性扁桃体炎组织和正常对照组织中的表达 ,分析腭扁桃体在天然免疫中的作用。方法 :应用逆转录聚合酶链反应技术检测 10例扁桃体炎组和 8例正常对照组中hBD 1和hBD 2mRNA的表达。结果 :hBD 1mRNA在扁桃体炎组和正常对照组中均有表达 ,且两组间差异无统计学意义 (P >0 .0 5 )。hBD 2mRNA在正常对照组中仅有微弱表达 ,而在扁桃体炎组中表达明显增强 (P <0 .0 5 )。结论 :扁桃体通过表达hBD在天然免疫中发挥重要作用。     

The immunological benefit of higher dose N-acetyl cysteine following mechanical ventilation in critically ill patients

Background

Sepsis complication is a major cause of death in multiple trauma critically ill patients. Defensin (cysteine rich anti-microbial peptides), as an important component of immune system, might play an important role in this process. There is also rising data on immunological effects of N-acetyl-cysteine (NAC), a commonly used anti-oxidant in oxidative stress conditions and glutathione (GSH) deficiencies. The aim of the present study was to evaluate the potential beneficial effects of NAC administration on multiple trauma patients with sepsis.

Methods

In a prospective, randomized controlled study, 44 multiple trauma critically ill patients who were mechanically ventilated and met the criteria of sepsis and admitted to the intensive care unit (ICU) were randomized into two groups . Control group received all standard ICU therapies and NAC group received intravenous NAC 3 gr every 6 hours for 72 hours in addition to standard therapies. Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores, length of ICU stay, ICU mortality were recorded. Levels of serum Immunoglobulin M (IgM), Human β-Defensin 2 (HβD2) and GSH were assessed at baseline and 24, 72, 120 hours after intervention.

Results

During a period of 13-month screening, 44 patients underwent randomization but 5 patients had to be excluded. 21 patients in NAC group and 18 patients in control group completed the study. For both groups the length of ICU stay, SOFA score and systemic oxygenation were similar. Mortality rate (40% vs. 22% respectively, p = 0.209) and ventilator days (Mean ± SD 19.82 ± 19.55 days vs. 13.82 ± 11.89 days respectively, p = 0.266) were slightly higher for NAC group. IgM and GSH levels were similar between two groups (p = 0.325, 0.125 respectively), HβD2 levels were higher for NAC group (at day 3).

Conclusion

High dose of NAC administration not only did not improve patients’ outcome, but also raised the risk of inflammation and was associated with increased serum creatinine.     

小鼠β-防御素2肿瘤疫苗的构建及特性鉴定

目的：构建分泌性小鼠β-防御素2（MBD2）真核表达质粒,转染L1210淋巴细胞白血病细胞,对转染细胞进行功能及特性鉴定。方法：RT-PCR法从小鼠皮肤细胞克隆MBD2成熟部分基因片段,用overlap PCR法将小鼠Igκ信号肽与MBD2成熟片段相连,构建MBD2分泌表达载体。经测序证实序列正确后,脂质体法转染L1210细胞,筛选稳定表达细胞株。RT-PCR、Western blot检测瘤苗MBD2的表达。Transwell法检测瘤苗分泌的MBD2对不成熟树突状细胞（iDC）迁移的作用。细胞计数、PI染色及FACS观察转染MBD2对肿瘤细胞增殖、细胞周期及MHCⅠ类（H-2K^d,H-2D^d）、Ⅱ类（I-A^d）分子及共刺激分子（CD80、CD860）表达的影响。结果：转染MBD2基因的肿瘤细胞表达并分泌MBD2,并以剂量依赖方式趋化iDC,表明具有生物学活性。转染MBD2基因对肿瘤细胞生长增殖、细胞周期及细胞表面MHC分子及共刺激分子表达无明显影响。结论：本实验成功构建了MBD2白血病细胞疫苗,为进一步体内评价抗白血病效果奠定了基础。     

支气管哮喘与急性呼吸道感染患儿血浆β防御素3水平的对照研究

目的β防御素3(HBD3)除了具有天然免疫功能,还发挥调节后天适应性免疫的作用。本研究拟通过支气管哮喘患儿、急性上呼吸道感染患儿及正常儿血浆HBD3浓度的比较,探讨HBD3在哮喘发病中的作用。方法研究对象选择2009年4月到12月在哈尔滨医科大学附属第一医院儿科就诊病例共81例,其中哮喘组21例,为急性发作期支气管哮喘患儿,感染组29例,为急性上呼吸道感染患儿,正常对照组31例,为健康体检儿。采用酶联免疫吸附试验(ELISA)法进行血浆HBD3水平测定。同时测定血嗜酸性粒细胞总数和白细胞数,9例哮喘儿测定了血清IgE浓度。结果血浆HBD3浓度正常儿为(8.028±1.078)μg/ml,哮喘患儿(12.212±1.124)μg/ml感染患儿(8.976±1.110)μg/ml,哮喘患儿与急性呼吸道感染及正常儿3组总体比较,血浆HBD3浓度明显升高,差异有显著性(F=4.448,P<0.05);哮喘组与正常组比较,差异显著,P<0.01;哮喘组与感染组比较,差异有显著性P<0.05;感染组与正常组比较,差异无显著性P>0.05;同时发现血浆HBD3水平与血清总IgE,血嗜酸性粒细胞及白细胞水平无显著相关性。结论支气管哮喘发作时HBD3表达升高,HBD3可能是超越感染之外,参与哮喘发病的独立因素。HBD3是否始动因素尚待进一步探索。     

Human β‐defensin 3 increases the TLR9‐dependent response to bacterial DNA

Human β‐defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self‐DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt‐3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self‐DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9‐dependent response. In contrast to hBD3, lipofection of self‐DNA enhances inflammatory signaling, but the response is predominantly TLR9‐independent. Together, these data show that hBD3 has a role in the innate immune‐mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy‐number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA‐based vaccines.     

人类防御素的结构与功能研究进展

人类防御素是一种存在于血液、肠道及表皮细胞中具有特殊空间结构，富含精氨酸的阳离子多肽。对革兰氏阳性、阴性细菌，真菌以及被膜病毒都能产生较强的杀伤作用。是生物体天然免疫机制的重要组成部分，并与某些炎症性疾病有密切关系，是一种有前途的抗菌肽。本文就其在人体内的分布，生化特性，生物学活性及与某些疾病的关系作一概述。     

Regulation of midgut defensin production in the blood-sucking insect Stomoxys calcitrans

The Stomoxys midgut defensin (Smd) family of genes are exclusively expressed in the anterior midgut of adult flies. Their putative function is protection of the stored bloodmeal from microbial attack. Smd genes are constitutively expressed, up-regulated in response to a bloodmeal and further up-regulated by immune stimulation per os but only in the presence of a bloodmeal not a sugar meal. Smd genes are down-regulated in response to a systemic immune challenge. Smd gene constructs transfected into l(2)mbn cells undertake constitutive expression but are not up-regulated by immune challenge. Electrophoretic mobility shift assays (EMSA) suggest the rel-like sites in the proximal promoter region of Smd genes do not bind midgut factors and so are non-functional.     

Molecular and phylogenetic analysis of a novel salivary defensin cDNA from malaria vector Anopheles stephensi

Manipulating the endogenous immune responses of the mosquito such as temporal and spatial expression of antimicrobial peptides may help in the development of a refractory mosquito, unable to transmit malaria. In mosquito several small antimicrobial peptides are activated locally in the midgut and salivary glands upon Plasmodium infection. Anopheles stephensi, the major urban malaria vector in India, has been considered as an important insect model to study vector-parasite interactions; however, so far no reports are available on the antimicrobial peptides from this mosquito species. In the present study, we report identification and molecular characterization of a novel cDNA encoding defensin like peptide, isolated from the salivary gland subtractive hybridization cDNA library of mosquito A. stephensi. Defensin cDNA is 396 base pair long, bearing an open reading frame of 96 amino acids. Deduced amino acid sequence of A. stephensi defensin (Astp_def) contains a signal peptide sequence of 24 amino acids followed by 32-amino acids long putative propeptide domain and a 40-amino acid mature peptide domain carrying 23-amino acid long consensuses sequence signature of insect defensin. Mature peptide of Astp_def carries six conserved cysteine residues, with a predicted molecular weight of 4.20kDa, and isoelectric point of 8.30, characteristic features of cationic defensins. Amino acid sequence similarity and phylogenetic analysis indicated a higher variation in the pre-propeptide region, as compared to the mature defensin peptide, assuring the presence of finely tuned immune responses to counter pathogens.