血NAP活性联合HsCRP检测在恶性血液病发热原因鉴别中临床应用价值的探讨

目的探讨血NAP活性联合HsCRP检测在恶性血液病发热原因鉴别中的临床应用价值。方法对近两年来本科住院的300例恶性血液病伴有发热的患者分析,最后经病原微生物证实为真菌感染28例,细菌感染160例,混合感染47例,非感染者65例（无病原微生物证据,抗生素治疗无效,且给予激素以及原发病治疗有效）。对所有患者发热时常规进行的血NAP活性联合HsCRP进行回顾性分析。结果真菌感染患者的血NAP积分中位数165,阳性率82％,HsCRP水平为12mg／dl,而细菌感染组、混合感染组、非感染组患者的血NAP积分中位数分别为126、147、36,阳性率分别为68％、76％、32％,HsCRP水平分别为11．6mg／dl、10．8mg／dl、0．8mg／dl。恶性血液病患者合并真菌或者细菌抑或混合感染的患者,血NAP活性以及HsCRP水平均明显高于非感染患者,相互比较差异有统计学意义;在合并感染患者当中,真菌感染患者血NAP活性较细菌感染患者升高更为明显,混合感染介于其中,相互比较差异均有统计学意义;HsCRP真菌感染患者略高,但相互比较差异无统计学意义。结论血NAP活性联合HsCRP检测有助于恶性血液病患者发热原因分析,在未获得病原微生物以前,为临床医师治疗是否选择抗生素,以及是否抗真菌治疗提供了有力证据,有利于及早控制感染以及避免不必要的抗生素使用。     

幽门螺杆菌napA基因的克隆及生物信息学分析

[目的]获取幽门螺杆菌NCTC11639中性粒细胞激活蛋白基因（napA），预测其编码蛋白作为幽门螺杆菌疫苗候选抗原的可行性，为耶疫苗研制奠定基础。[方法]提取幽门螺杆菌标准株NCTC11639的基因组DNA，参照GeneBank中幽门螺杆菌标准株22695的序列设计引物PCR扩增napA基因，克隆入pMD18-T载体中，对重组质粒测序后进行生物信息学分析。[结果]克隆的napA基因全长为435bp，在GenBank上登录（No．DQ341279），与Gen-Bank公布的其它Hp菌株的核酸同源性为94％～98％，与国际测序模式株Hp26695、HpJ99同源性达97％。[结论]成功扩增Hp标准株NCTC 11639 napA基因，通过同源性检索分析表明Hp与人及其它哺乳动物的基因组DNA同源序列低，在不同耶菌株中高度保守，符合疫苗候选抗原的基本特征；抗原表位预测显示有4条高抗原性肽段，其中118-140肽段抗原指数最强，可能存在良好的抗原表位，结合Hp-NAP二级结构、亲水性分析认为Hp—NAP是很有前景的幽门螺杆菌疫苗候选抗原。     

Effects of hypoxia and nitric oxide on ferritin content of alveolar cells

Concentrations of ferritin in alveolar cells and on the alveolar surface are increased in patients with a variety of respiratory disorders. Ferritin synthesis by cells is modulated by iron content but is also influenced by stimuli other than iron. In this study we sought to determine whether in vitro exposure to hypoxia- or nitric oxide (NO)-induced ferritin accumulation or release by human alveolar macrophages (AMs) or a lung cancer-derived epithelial cell line (A549). Changes in cell content of iron and ferritin (L- and H-types), as well as ferritin content of cell supernatants, were determined after in vitro exposure to hypoxia (1% or 10% O(2), 18 hours) or the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.01-1.0 mmol/L, 18 hours). Exposure to 1% O(2) increased ferritin content in both cell types (>fourfold increase; P <.005) without changing iron content. Treatment with SNAP increased ferritin content of A549 cells in a dose-dependent manner, whereas treatment of AMs decreased cellular iron and ferritin content and increased supernate ferritin content. Pretreatment of cells with N-acetylcysteine (500 micromol/L) reduced hypoxia-induced ferritin accumulation in alveolar cells and completely inhibited NO-induced ferritin accumulation in A549 cells. These findings indicate that exposure to 1% O(2)can increase ferritin content in alveolar cells, whereas NO can increase ferritin content (A549 cells) or decrease ferritin content (AMs).     

可分泌表达神经保护肽的重组慢病毒对闭合性脑损伤小鼠的神经保护作用

目的：通过滴鼻给药途径,给予闭合性脑损伤小鼠可分泌表达神经保护肽NAP的重组慢病毒,观察该重组病毒经鼻-脑通路对中枢神经系统的保护作用,为携带可分泌表达NAP的重组慢病毒治疗神经系统退行性疾病提供理论依据。 方法：选用8~12周成年雄性昆明小鼠54只,随机分为4组：空白对照组10只（Control组）、重锤加害组20只（CHI组）、重组慢病毒rLent/NT4-NAP保护组20只（rLent组）和重组慢病毒rLent/GFP组4只（GFP组）。观察各组小鼠的死亡率、神经功能损伤（NSS）评分、脑水肿含量和病理改变情况。应用共聚焦显微镜观测GFP组小鼠鼻黏膜、嗅神经和脑组织内绿色荧光表达情况。 结果：rLent组小鼠死亡率（15％）明显低于CHI组小鼠（55％）;rLent组小鼠NSS评分在创伤后1、3、5和7 d均显著低于CHI组（P<0.01）; rLent组小鼠创伤后24 h的脑水肿含量明显低于CHI组（P<0.01）;HE染色显示rLent组小鼠病理改变明显轻于CHI组;GFP组仅在鼻黏膜处可见呈条索样的绿色荧光,而在嗅神经及大脑则未发现绿色荧光。 结论：分泌表达NAP的重组慢病毒可感染鼻黏膜细胞,分泌表达的短肽NAP能够改善闭合性脑损伤小鼠的神经功能;未加装分泌表达元件的重组病毒rLent/GFP仅能感染鼻黏膜细胞,不能通过血脑屏障进入中枢神经系统。     

Immunology of Helicobacter pylori: insights into the failure of the immune response and perspectives on vaccine studies

Helicobacter pylori infects the stomach of half of the human population worldwide and causes chronic active gastritis, which can lead to peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. The host immune response to the infection is ineffective, because the bacterium persists and the inflammation continues for decades. Bacterial activation of epithelial cells, dendritic cells, monocytes, macrophages, and neutrophils leads to a T helper cell 1 type of adaptive response, but this remains inadequate. The host inflammatory response has a key functional role in disrupting acid homeostasis, which impacts directly on the colonization patterns of H pylori and thus the extent of gastritis. Many potential mechanisms for the failure of the host response have been postulated, and these include apoptosis of epithelial cells and macrophages, inadequate effector functions of macrophages and dendritic cells, VacA inhibition of T-cell function, and suppressive effects of regulatory T cells. Because of the extent of the disease burden, many strategies for prophylactic or therapeutic vaccines have been investigated. The goal of enhancing the host's ability to generate protective immunity has met with some success in animal models, but the efficacy of potential vaccines in humans remains to be demonstrated. Aspects of H pylori immunopathogenesis are reviewed and perspectives on the failure of the host immune response are discussed. Understanding the mechanisms of immune evasion could lead to new opportunities for enhancing eradication and prevention of infection and associated disease.     

幽门螺杆菌儿童分离株中性粒细胞激活蛋白克隆及表达序列

目的：从幽门螺杆菌临床儿童分离株GZCH1基因组扩增中性粒细胞激活蛋白（NAP）基因,克隆入T载体,并亚克隆入表达载体pGEX-4T-1,进行测序及基因比对分析,为幽门螺杆菌疫苗研制奠定基础.方法：根据GenBank中幽门螺杆菌NAP序列,设计一对特异性引物扩增幽门螺杆菌临床儿童分离株NAP全长基因,与T载体连接,转化大肠杆菌DH5α,提取质粒进行酶切、测序鉴定,经EcoR Ⅰ、Not Ⅰ双酶切后与做相应酶切的pGEX-4T-1连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定,IPTG诱导重组蛋白表达,并对基因进行测序及比对分析.结果：以幽门螺杆菌儿童分离株GZCH1为模板,成功扩增了NAP基因,基因大小为435 bp,重组pGEX-4T-1-NAP双酶切鉴定可见目的片段,测序结果显示NAP在正确读框中,序列比对分析显示其与幽门螺杆菌J99株氨基酸一致性达99.2％,IPTG诱导后,pGEX4T-1-NAP/BL21在相应分子量（42.8 kD）可见融合蛋白的表达.幽门螺杆菌儿童分离株GZCH1 NAP序列已登录GenBank（登录号：GU301881）.结论：从幽门螺杆菌临床儿童分离株GZCH1中成功克隆了NAP基因,并获得重组蛋白的表达,为NAP幽门螺杆菌疫苗研制奠定了良好的基础.     

Reduction of aluminum ion neurotoxicity through a small peptide application – NAP treatment of Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia in late life. It is difficult to precisely diagnose AD at early stages, making biomarker search essential for further developments. The objective of this study was to identify protein biomarkers associated with aluminum ions toxicity (AD-like toxicity) in a human neuroblastoma cell model, SH-SY5Y and assess potential prevention by NAP (NAPVSIPQ). Complete proteomic techniques were implemented. Four proteins were identified as up-regulated with aluminum ion treatment, CBP80/20-dependent translation initiation factor (CTIF), Early endosome antigen 1 (EEA1), Leucine-rich repeat neuronal protein 4 (LRRN4) and Phosphatidylinositol 3-kinase regulatory subunit beta (PI3KR2). Of these four proteins, EEA1 and PI3KR2 were down-regulated after NAP-induced neuroprotective activity in neuroblastoma cells. Thus, aluminum ions may increase the risk for neurotoxicity in AD, and the use of NAP is suggested as a treatment to provide additional protection against the effects of aluminum ions, via EEA1 and PI3KR2, associated with sorting and processing of the AD amyloid precursor protein (APP) through the endosomal system.