杀菌/通透性增加蛋白(BPI)研究进展

杀菌/通透性增加蛋白（BPI）是人中性粒细胞中存在的一种碱性蛋白,它能与革兰氏阴性菌脂多糖（LPS）结合,具有中和内毒素和杀灭细菌作用。在革兰氏阴性菌感染的治疗方面有良好的发展前景,本文主要就BPI的生理功能,作用机理及临床研究方面的进展作一综述。     

杀菌/通透性增加蛋白基因的克隆及在CH0细胞中的表达

从一名健康中国人外周血白细胞中克隆出杀菌/通透性增加蛋白(BPI)基因,全长1452bp,编码27个氨基酸的信号肽和456个氨基酸成熟蛋白.序列比较发现,此序列与国外发表的序列有6个核苷酸的差异,编码蛋白有4个氨基酸的差异.构建真核表达质粒,在CHO细胞中实现BPI基因的稳定表达.对重组蛋白初步纯化后进行杀菌实验,结果表明重组蛋白具有与天然蛋白同样的生物活性.     

杀菌通透性增加蛋白基因的克隆及在CHO细胞中的表达

从一名健康中国人外周血白细胞中克隆出杀菌 通透性增加蛋白 (BPI)基因 ,全长1 45 2bp,编码 2 7个氨基酸的信号肽和 45 6个氨基酸成熟蛋白。序列比较发现 ,此序列与国外发表的序列有 6个核苷酸的差异 ,编码蛋白有 4个氨基酸的差异。构建真核表达质粒 ,在CHO细胞中实现BPI基因的稳定表达。对重组蛋白初步纯化后进行杀菌实验 ,结果表明重组蛋白具有与天然蛋白同样的生物活性。     

不同麻醉方法对肝癌手术患者炎性细胞因子基因表达的影响

目的:探讨不同麻醉方法对肝癌手术患者外周血炎性细胞因子基因表达的影响。方法:选择肝癌手术患者48例,随机分为单纯全麻和全麻复合硬膜外麻醉两组,在麻醉前和麻醉后4h抽取静脉血,Trizol法抽提RNA,RT-PCR检测IL-1β、IL-6、IL-8的TNF-α的mRNA表达水平。结果:麻醉前两组患者间外周血炎性细胞因子基因表达无差别,麻醉4h后全麻组外周血IL-1β、IL-6、IL-8的mRNA表达水平显著升高(P<0.05),并高于联合组(P<0.05)。结论:不同麻醉方法对细胞因子的分泌会产生不同的影响,单纯全麻将增强肝癌手术病人外周血炎性细胞因子基因的表达。     

大鼠脂多糖结合蛋白的分离纯化及对脂多糖的调节作用

经硫酸铵沉淀、Bio Rex70树脂的阳离子交换层析和MonoQ预装柱的阴离子交换层析 ,从大鼠急性期血清中分离纯化了脂多糖结合蛋白 .SDS PAGE为单一条带 ,分子量 60kD ,所纯化的大鼠脂多糖结合蛋白可明显增强脂多糖与单核巨噬细胞的结合 .脂多糖结合蛋白对脂多糖诱导肺泡巨噬细胞中肿瘤坏死因子α(TNF α)mRNA表达的调节作用具有明显的剂量依赖性 .     

Lsa蛋白对金黄色葡萄球菌介导的炎性细胞因子表达的影响

试验旨在研究家蝇Lsa(Lectin subunit alpha)蛋白在金黄色葡萄球菌(Staphylococcus aureus)刺激下的炎性反应。通过慢病毒转染的方法,将构建好的载体pLEX-Lsa及pLEX分别转染到RAW264.7细胞中,通过筛选,获得稳定表达的RAWpLEX-Lsa及RAW-pLEX细胞系,其中RAW-pLEX细胞作为阴性对照。然后通过S.aureus刺激细胞,收集未刺激和刺激后3 h,6h,12 h和24 h的细胞,且收集未刺激和刺激6 h细胞上清。提取RNA,通过实时定量PCR(RT-PCR)检测炎性细胞因子TNF-a,IL-1β,NFκB-1和NFκB-2基因的相对转录水平。通过酶联免疫吸附测定(ELISA)检测细胞上清中TNF-a和IL-1β的蛋白水平。RT-PCR结果显示,在S.aureus刺激后,与RAW-pLEX细胞相比,RAW-pLEX-Lsa细胞中TNF-a的两个转录剪接体的相对转录水平,TNF-α-tv-1在6 h显著下调(P0.05),TNF-α-tv-2在6 h和12 h显著下调(P0.05);RAW-pLEX-Lsa细胞中IL-1β的两个转录剪接体的相对转录水平,IL-1β-T在3 h,6 h和12 h显著下调(P0.05),IL-1β在3 h和6 h显著下调(P0.05);RAW-pLEX-Lsa细胞中NFκB-1和NFκB-2基因的相对转录水平无明显下降趋势。ELISA检测结果显示,在S.aureus刺激6 h后,与RAW-pLEX细胞相比,RAW-pLEX-Lsa细胞上清中,TNF-a的蛋白水平显著下调(P0.05)。结果表明,家蝇Lsa蛋白可以有效地发挥抗炎活性,显著地抑制TNF-α和IL-1β的转录和生产,而TNF-α和IL-1β的转录和生产的下调并不是通过抑制NFκB信号通路的活性引起的。     

沙眼衣原体感染诱导小鼠生殖道局部促炎性细胞因子的表达情况

建立小鼠生殖道沙眼衣原体感染模型,观察小鼠生殖道局部促炎性细胞因子的表达。将小鼠生物型沙眼衣原体C. muridarum 1＆#215;104 IFU阴道接种于C57B6背景雌性小鼠,取感染后阴道拭子做沙眼衣原体培养,计算IFU,监测小鼠感染和病原体清除情况;80 d后处死小鼠,检测子宫输卵管病理改变;ELISA检测感染过程中小鼠生殖道促炎性细胞因子IL-1α、IL-6、MIP-2和TNF-α产生情况。小鼠感染在第3至第15天维持较高水平,然后病原体被逐渐清除,整个病程约3~5周;病理检测显示子宫输卵有严重炎症、管腔扩张积水,狭窄等;于感染后第3天检测到局部IL-1α、IL-6、MIP-2分泌,第7天达高峰,然后逐渐下降至正常水平（ IL-6于11 d恢复正常,IL-1α和 MIP-2于15 d恢复正常）。 TNF-α仅在第7天检测到高水平表达。相对于TNF-α和IL-6,IL-1α和MIP-2维持时间较长。成功建立沙眼衣原体感染小鼠生殖道模型,沙眼衣原体急性感染可诱导小鼠生殖道局部分泌IL-1α、IL-6、MIP-2和TNF-α。     

香烟烟雾和脂多糖对大鼠肺组织热休克蛋白70表达的影响

目的观察香烟烟雾和脂多糖对大鼠肺组织Hsp70表达的影响。方法48只成年雄性Wistar大鼠随机分为6组:健康对照30d组、健康对照3个月组、单纯薰香烟30d组、单纯薰香烟3个月组、单纯气管注入脂多糖组、薰香烟30d加气管注入脂多糖组,每组8只。采用SABC免疫组化法观察Hsp70在各组大鼠肺组织中的表达。结果(1)健康对照30d组、健康对照3个月组、单纯薰香烟30d组、单纯薰香烟3个月组Hsp70阳性产物的平均吸光度值分别为0·194±0·027,0·189±0·025,0·339±0·049,0·147±0·033。单纯薰香烟30d组与健康对照组30d相比大鼠肺组织Hsp70水平显著升高(P<0·05);单纯薰香烟3个月组与健康对照3个月组相比大鼠肺组织Hsp70水平显著降低(P<0·05);单纯薰香烟3个月组与单纯薰香烟30d组相比大鼠肺组织Hsp70水平显著降低(P<0·05)。(2)健康对照30d组、单纯气管注入脂多糖组、薰香烟30d加气管注入脂多糖组Hsp70阳性产物的平均吸光度值分别为0·194±0·027,0·285±0·059,0·143±0·024。单纯气管注入脂多糖组与健康对照30d组相比大鼠肺组织Hsp70水平显著升高(P<0·05);薰香烟30d加气管注入脂多糖组与健康对照30d组相比大鼠肺组织Hsp70水平显著降低,差异具有显著性(P<0·05);薰香烟30d加气管注入脂多糖组与单纯气管注入脂多糖组相比大鼠肺组织Hsp70水平显著降低(P<0·05)。结论香烟烟雾或脂多糖短期暴露使大鼠肺组织Hsp70表达升高;香烟烟雾长期暴露或香烟烟雾与脂多糖短期联合暴露使大鼠肺组织Hsp70表达降低。提示Hsp70可能与慢性阻塞性肺疾病的发病有一定关系。     

补体C3与BPI活性区融合蛋白（CB）的构建与表达

补体C3和杀菌通透性增加蛋白（BPI）对血液中的病原体均有黏附、促吞噬甚至杀灭作用,但两者的作用机制不同,制备两者活性区融合蛋白,可能具有更好的清除血液病原的作用。通过重叠延伸PCR融合人补体C3的补体受体Ⅰ、Ⅲ两个结合区,同时调取了杀菌通透性增加蛋白（BPI）活性区段rBPI,先后将补体C3活性区与BPI蛋白功能区基因克隆入原核表达载体pET28a中,获得融合蛋白（CB）表达载体pET28-CB,在大肠杆菌中进行了高表达产量、可溶性表达等条件的摸索,CB融合蛋白主要以包涵体形式表达,Western印迹证明CB具有C3的抗原活性,将包涵体蛋白变性与复性后,利用Ni2+固相化的螯合Sepharose Fast Flow亲和层析柱进行浓缩和纯化,最后得到了纯度较高的CB原核表达蛋白。CB融合蛋白的构建和高效表达、纯化为下步探讨其在促进血液病原清除上的功能鉴定和应用奠定了基础。     

重组BPI-(23)-Fcγ1融合蛋白在CHO细胞中的表达

杀菌 通透性增加蛋白 (Ｂａｃｔｅｒｉｃｉｄａｌ ｐｅｒｍｅａｂｉｌｉｔｙ ｉｎｃｒｅａｓｉｎｇｐｒｏｔｅｉｎ ,ＢＰＩ)是人和许多哺乳类动物白细胞中存在的一种碱性蛋白 ,它能与革兰氏阴性菌脂多糖 (ＬＰＳ)结合 ,具有中和内毒素和杀灭细菌作用[1 ] 。人ＢＰＩ基因位于第 2 0号染色体上 ,完整蛋白由 45 6个氨基酸组成 ,其活性部位主要在蛋白氨基端约 2 0 0个氨基酸 (ＢＰＩ2 3) [2 ,3 ] 。实验室研究和临床试验表明重组ＢＰＩ(ｒＢＰＩ)及其衍生物在革兰氏阴性菌感染的治疗方面有良好的发展前景[4] 。目前ＢＰＩ在应用上存在的主要问…     

Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability-increasing protein share a similar tertiary structure

Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability-increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand-binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti-CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215-219, 219-223, 223-227, and 444-450, respectively, are predicted to be situated on the external surface of the central beta-sheet and a fifth epitope (residues 225-258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317-331, 360-366, and 393-410, would form part of the putative carboxy-terminal beta-barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies.     

Autoantibodies against bactericidal/permeability-increasing protein (BPI) in children with acute pneumonia

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein (BPI), an inhibitor of a lipopolysaccharide of gram-negative bacteria, are a common feature of chronic neutrophilic inflammatory processes such as cystic fibrosis. We investigated whether serum and salivary anti-BPI autoantibodies also appear in the course of acute pneumonia in 24 otherwise healthy children. Nine (38%) and four (17%) patients had detectable serum anti-BPI immunoglobulin G (IgG) (≥4 IU mL⁻¹) and IgA (ratio≥1.2), respectively, on the day of hospital admission (day 0). There was no increase in the rate of occurrence or the concentration of these antibodies in the convalescent sera obtained on day 30. The presence of anti-BPI IgG on admission did not correlate with inflammatory markers (peripheral white blood cell count, C-reactive protein) or temperature on admission. Also, salivary anti-BPI IgA, determined on days 0, 3–5 and 30, did not appear during the course of acute pneumonia. In summary, a substantial proportion of previously healthy children have pre-existing anti-BPI IgG autoantibodies. Acute neutrophilic infection, i.e. pneumonia, however, neither triggered the appearance of new antibodies nor boosted the concentrations of pre-existing ones. Thus, in typical acute pneumonia in children, autoantibodies directed against BPI may not have clinical significance.     

白头翁汤对炎症性肠病中促炎细胞因子表达的影响

目的：探讨白头翁汤治疗炎症性肠病的分子机制。方法：40只Wistar雄性大鼠随机分为5组（n=8）：正常对照组、模型组、模型＋阳性药物对照组（美沙拉嗪）、模型＋白头翁中、高剂量治疗组。阳性对照组、中药治疗组分别灌胃给药。疗程结束后取大鼠结肠组织提取RNA,利用realtimePCR的方法检测白介素-1β（IL-1β）、白介素-6（IL-6）YL肿瘤坏死因子-α（TNF-α）在各组中的表达变化。结果：相对于模型组,阳性药物及白头翁汤治疗组尤其是高剂量组可有效抑制IL-1β、IL-6及TNF-α的表达。结论：白头翁汤可通过抑制促炎因子的表达从而发挥了在炎症性肠病中的治疗作用。     

Effects of inflammatory cytokines on bone/cartilage repair

Many inflammatory factors can affect cell behaviors and work as a form of inter-regulatory networks through the inflammatory pathway. Inflammatory cytokines are critical for triggering bone regeneration after fracture or bone injury. Also, inflammatory cytokines play an important role in cartilage repair. The synergistic or antagonistic effects of both proinflammatory and anti-inflammatory cytokines have a great influence on fracture healing. This review discusses key inflammatory cytokines and signaling pathways involved in bone or cartilage repair.     

杀菌/渗透性增强蛋白功能及应用前景

杀菌/通透性增强蛋白(BPI)是存在于中性粒细胞中的阳离子蛋白,约为55 kD.BPI能与革兰氏阴性菌外膜上的脂多糖结合,增加外膜对抗菌药的通透性,具有特异性杀灭革兰氏阴性菌及结合/中和内毒素的生物学功能,在革兰氏阴性菌感染的治疗方面有良好的发展前景.近年来,国内外对BPI研究颇多,并逐渐发现BPI还具有调理 作用、抗真菌、抗原虫和抑制血管生成等许多功能,被学者称为未来的“超级抗生素 ”.本文主要就BPI的结构、分布、生理功能、作用机理及临床研究方面的研究进展作一综述.     

Peripheral injection of lipopolysaccharide enhances expression of inflammatory cytokines in murine locus coeruleus: possible role of increased norepinephrine turnover

Cytokines and catecholamines are known to constitute a significant portion of the regulatory neuroimmune networks involved in maintaining homeostasis in the central nervous system (CNS). Although we have already reported an increase in norepinephrine (NE) turnover within the locus coeruleus (LC) at 2 and 4 h after the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), the implication of this increase remains unclear. In view of evidence that norepinephrine (NE) acts in an anti-inflammatory manner by way of negatively regulating pro-inflammatory cytokine expression, we examined the inflammatory cytokine expression levels in the LC of C3H/HeN mice (male, 8 weeks old) after an i.p. LPS injection. The mRNA expression levels of the genes encoding IL-1beta and TNF-alpha within the LC increased during the first 2 h, and showed two peaks, the first at 4 h and the second lesser one at 15 h after the LPS injection. Microglia, which are one of the major cell types that produce pro-inflammatory cytokines in the CNS, were isolated from mouse neonate brains in order to clarify more precisely the relationship between the changes in NE content and the up-regulation of inflammatory cytokines in the LC. Simultaneous incubation of microglia with LPS and NE enhanced the expression of IL-1beta at both mRNA and protein levels, but reduced the mRNA and protein levels of TNF-alpha. These data support the hypothesis that NE negatively regulates the expression of pro-inflammatory cytokine expression, at least in the case of TNF-alpha, which action could contribute to the observed anti-inflammatory properties of NE. This report, based on the results of both in vivo and in vitro experiments, is the first to suggest a relationship between NE content and cytokine expression levels in the CNS.