甲型流感病毒的变异及其跨物种传播

甲型流感病毒基因组由8个节段组成,其众多的血清型构成其变异的基础,不同病毒株之间的重配导致其能够跨越物种屏障传播。因此给人类造成极大的威胁。本文对甲型流感病毒的基本特性、变异机制、跨物种传播机制及在世界范围内的分子流行学进行综述。     

双黄连抑制甲1型流感病毒诱导细胞凋亡的机制研究

目的:研究双黄连抑制甲1型流感病毒诱导细胞凋亡的机制。方法:将宫颈癌(Hela)细胞分成4组:病毒对照组、实验组、细胞对照组、药物对照组,采用流式细胞术计算细胞凋亡的百分率。结果:实验组细胞凋亡的百分率与病毒对照组比较有非常显著性差异(P<0.01)。结论:双黄连可抑制甲1型流感病毒诱导Hela细胞凋亡。     

通窍止咳液体外抗甲型人流感病毒作用研究

目的:研究通窍止咳液体外抗甲型人流感病毒的作用。方法:以利巴韦林作阳性对照药物,采用存留细胞结晶紫染色法测定通窍止咳液不同加药方式对甲型人流感病毒H3N2的体外抑制作用及其时效关系。结果:通窍止咳液对甲型人流感病毒具有良好的综合抑制作用及抑制病毒吸附后的复制增殖作用;通窍止咳液对病毒吸附的预防作用弱,无直接杀伤病毒的作用;随药物作用时间延长,通窍止咳液在低浓度时的抗病毒有效率呈降低趋势,在高浓度时的抗病毒能力基本不变。结论:通窍止咳液具有较好的体外抗甲型人流感病毒的作用。     

中西医结合体外抗甲型流感病毒的实验研究

目的研究中西医结合药物体外对甲型流感病毒的抑制作用。方法体外接种甲型流感病毒鼠肺适应株H1N1于狗肾细胞（MDCK）,观察各组药物体外抗病毒作用。结果根据实验,测得各组药物对甲型流感病毒的选择指数,利巴韦林注射液组为4.68;痰热清注射液组为5.50;达菲组为5.53;中西医结合组为14.48。结论中西医结合组具有明显的体外抗甲型流感病毒的作用。     

知母宁体外抗甲型流感病毒作用研究

目的:考察知母宁体外抗甲型流感病毒作用.方法:以利巴韦林作阳性对照药物,用中性红染色法测定知母宁不同加药方式对甲型人流感病毒H1N1的体外抑制作用及其时效关系.结果:知母宁对甲型人流感病毒具明显的综合抑制作用及抑制病毒吸附后的复制增殖作用,这两种作用的半数有效浓度(EC50)分别为0.70及0.76 mg·ml-1.知母宁对病毒吸附的预防作用弱,无直接杀伤病毒的作用.随药物作用时间延长,知母宁在低浓度抗病毒有效率呈减小趋势,在高浓度时(≥1.4 mg·ml-1)其抗病毒能力基本不变.同时知母宁可明显降低病毒的感染性.结论:知母宁具有明显的体外抗甲型人流感病毒作用.     

基于生物信息学分析方法设计流感病毒A的通用寡核苷酸探针

目的:设计流感病毒A诊断芯片的寡核苷酸(Oligo)探针.方法:利用BLAST软件对流感病毒A的基因序列进行比对,得到有意义的特异序列;用生物学软件Array Designer4.2设计特异性高、Tm值相近、长度均一的Oligo探针.结果:获得10条30 mer的Oligo探针.结论:利用BLAST软件和生物学软件Array Designer4.2设计流感病毒A诊断芯片的探针,可为后期打印成基因芯片,用于流感病毒A的检测打下基础.     

The Protective Effect of Eupatilin against Hydrogen Peroxide-Induced Injury Involving 5-Lipoxygenase in Feline Esophageal Epithelial Cells

In this study, we focused to identify whether eupatilin (5,7-dihydroxy-3'',4'',6-trimethoxyflavone), an extract from Artemisia argyi folium, prevents H₂O₂-induced injury of cultured feline esophageal epithelial cells. Cell viability was measured by the conventional MTT reduction assay. Western blot analysis was performed to investigate the expression of 5-lipoxygenase by H₂O₂ treatment in the absence and presence of inhibitors. When cells were exposed to 600 µM H₂O₂ for 24 hours, cell viability was decreased to 40%. However, when cells were pretreated with 25~150 µM eupatilin for 12 hours, viability was significantly restored in a concentration-dependent manner. H₂O₂-treated cells were shown to express 5-lipoxygenase, whereas the cells pretreated with eupatilin exhibited reduction in the expression of 5-lipoxygenase. The H₂O₂-induced increase of 5-lipoxygenase expression was prevented by SB202190, SP600125, or NAC. We further demonstrated that the level of leukotriene B₄ (LTB₄) was also reduced by eupatilin, SB202190, SP600125, NAC, or nordihydroguaiaretic acid (a lipoxygenase inhibitor) pretreatment. H₂O₂ induced the activation of p38MAPK and JNK, this activation was inhibited by eupatilin. These results indicate that eupatilin may reduce H₂O₂-induced cytotoxicity, and 5-lipoxygenase expression and LTB₄ production by controlling the p38 MAPK and JNK signaling pathways through antioxidative action in feline esophageal epithelial cells.     

某区甲型H1N1流感病毒分离鉴定及同源性的分析

目的检测并分离甲型H1N1流感病毒,对开封地区首次分离到的病毒株进行全基因组序列测定及同源性分析,为研究流感病毒的流行及变异规律提供科学依据。方法采用Real-time RT-PCR方法检测,筛选确定出甲型H1N1流感病毒阳性标本;利用狗肾传代细胞分离得到甲型H1N1流感病毒株A/Kaifeng/01/2009(H1N1);测定并分析其全基因组序列;利用序列比对进行了同源性分析。结果从1828份流感样病例中检出甲型H1N1流感病毒阳性标本286份,阳性率15.6%。在开封地区首次获得甲型H1N1流感病毒株及全基因组序列。基因组序列分析表明:该毒株与2009年大流行株高度同源,为同一进化分支。与以往流行的猪流感病毒株对比发现,HA基因有12个碱基发生了点突变。结论 MDCK细胞对甲型H1N1流感病毒具有较高敏感性;开封地区首例甲型H1N1流感病例分离病毒株与北美流行株高度同源;相对于以往古典型猪流感代表株出现了HA蛋白抗原性漂移;为今后进一步开展甲型H1N1流感病毒分子生物学研究奠定基础。     

人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性研究

 目的   比较人甲型H7N9禽流感全病毒灭活疫苗和裂解疫苗在小鼠中的免疫原性,为该疫苗的类型选择提供初步依据。 方法   采用相同血凝素含量（5 μg）的H7N9全病毒灭活和裂解疫苗（含或不含氢氧化铝佐剂共4种类型）,分别对BALB/c小鼠进行1针或2针免疫。免疫后,用血凝抑制（hemagglutination inhibition,HI）试验检测血清抗体滴度,比较不同类型疫苗的免疫效果。 结果   小鼠免疫1针全病毒灭活疫苗后,全部血清阳转,HI抗体几何平均滴度（geometric mean titer,GMT）为149;免疫2针后,抗体GMT为243。小鼠免疫1针裂解疫苗后无抗体阳转;免疫2针后全部抗体阳转,GMT为139。两种疫苗添加铝佐剂后,诱导的HI抗体GMT仅略有增加。 结论   H7N9全病毒灭活疫苗在小鼠中的免疫原性较强。在同样类型和剂量的情况下,裂解疫苗需要免疫两次才能达到与全病毒疫苗相同的效果。铝佐剂对免疫原性提升不明显。     

红霉素对白介素-4诱导人支气管上皮细胞表达白介素-6及还原型谷胱甘肽的影响研究

目的:探讨红霉素对白介素(IL)-4诱导人支气管上皮细胞(HBE)表达IL-6及还原型谷胱甘肽(GSH)的影响。方法:培养HBE,取对数生长期细胞以每孔104个接种于96孔培养板中,分组为正常对照组(生理盐水20μL)、IL-4组(IL-410μL,生理盐水10μL)、红霉素低、高剂量组(IL-410μL,红霉素5、50μg.mL-110μL),加入相应药物,每组8个复孔,孵育24、48h后检测各组细胞存活情况及细胞中IL-6、GSH水平。结果:与正常对照组比较,IL-4组细胞孵育24、48h后活力明显降低(P<0.01),细胞中IL-6、GSH水平明显升高(P<0.05或P<0.01);与IL-4组比较,红霉素低、高剂量组细胞孵育48h后活力明显升高(P<0.05或P<0.01),细胞中IL-6、GSH水平明显降低(P<0.05或P<0.01),但孵育24h几乎无明显变化。结论:红霉素可抑制IL-4诱导HBE炎症介质IL-6的释放,下调GSH的合成。     

2003年～2005年北京地区甲3型流感病毒流行株金刚烷胺耐药性调查

目的:为我国制订流感大流行防备计划提供参考。方法:对2003年～2005年北京地区流感样患儿分离的流感病毒流行株30株,用RT-PCR一步法扩增流感病毒M2基因片段、测定核酸序列和进行生物信息软件分析,确定耐药性氨基酸位点。结果:M2基因31位氨基酸被天冬酰胺置换的有20株,显示对金刚烷胺类药耐药率为66.7%。结论:应在我国进一步开展抗流感病毒耐药性监测。     

Recombinant chimeric lectins consisting of mannose-binding lectin and L-ficolin are potent inhibitors of influenza A virus compared with mannose-binding lectin

MBL structurally contains a type II-like collagenous domain and a carbohydrate recognition domain (CRD). We have recently generated three novel recombinant chimeric lectins (RCL), in which varying length of collagenous domain of mannose-binding lectin (MBL) is replaced with that of L-ficolin (L-FCN). CRD of MBL is used for target recognition because it has a broad spectrum in pathogen recognition compared with L-FCN. Results of our study demonstrate that these RCLs are potent inhibitors of influenza A virus (IAV). RCLs, against IAV, show dose-dependent activation of the lectin complement pathway, which is significantly higher than that of recombinant human MBL (rMBL). This activity is observed even without MBL-associated serine proteases (MASPs, provided by MBL deficient mouse sera), which have been thought to mediate complement activation. These observations suggest that RCLs are more efficient in associating with MASP-2, which predominantly mediates the activity. Yet, additional serum further increases the activity while RCL-mediated coagulation-like enzyme activities are diminished compared with rMBL, suggesting reduced association with MASP-1, which has been shown to mediate coagulation-like activity. These data suggest that RCLs may interfere less with host coagulation, which is advantageous to be a therapeutic drug. Importantly, these RCLs have surpassed rMBL for anti-viral activities, such as viral aggregation, reduction of viral hemagglutination (HA) and inhibition of virus-mediated HA and neuraminidase (NA) activities. These results are encouraging that novel RCLs could be used as anti-IAV agents with less side effect and that RCLs would be suitable candidates in developing a new anti-IAV therapy.     

Exploring the Requirements for the Hydrophobic Scaffold and Polar Amine in inhibitors of M2 from Influenza A Virus

Inhibitors targeting the influenza A virus M2 (A/M2) proton channel, have lost their effectiveness due to widespread resistance. As a first step in the development of new inhibitors that address this problem, we have screened several focused collections of small molecules using two electrode voltage patch clamp assays (TEVC) on Xenopus laevis Oocyte. Diverse head groups and scaffolds of A/M2 inhibitors have been explored. It has been found that not only amine, but also hydroxyl, aminooxyl, guanidine and amidine compounds are active against the A/M2 proton channel. Moreover, the channel is able to accommodate a wide range of structural variation in the apolar scaffold. This study offers information to guide the next generation of A/M2 proton channel inhibitor design.     

人源性及牛源性乳铁蛋白抑制丙型肝炎病毒复制作用的对比研究

目的：探讨人乳铁蛋白(HLF)及牛乳铁蛋白(BLF)在体外HepG2细胞中对丙型肝炎病毒(HCV)感染增殖的抑制作用，为乳铁蛋白用于临床治疗HCV感染提供理论和实验依据。方法：以HepG2细胞作为HCV复制体系，以RT nPCR方法，测定培养细胞提取物中病毒正、负链，并用活性细胞定量鉴定法(MTT)对两种乳铁蛋白的细胞毒性作用及药物剂量进行研究。结果：细胞毒实验显示，实验中两种乳铁蛋白的浓度小于1.5 mg·mL 1时对HepG2细胞生存率无显著影响。HCV阳性血清接种HepG2细胞后，HCV正、负链可于第8，10，12天被检测到。当两种乳铁蛋白(浓度分别为1.5，1.0，0.5，0.1 mg·mL 1)先与HCV阳性血清作用后再接种细胞时，不同时期提取物中均未测定到HCV正、负链。而当乳铁蛋白(浓度同上)先与细胞作用后洗去再与HCV阳性血清作用时，不同时期细胞提取物中可测定到HCV正、负链。结论：乳铁蛋白能在非细胞毒性剂量时能防止HCV感染HepG2细胞；乳铁蛋白可能主要是通过与病毒颗粒本身相互作用发挥上述功能；两种来源的乳铁蛋白在体外抑制HCV复制的作用差异无显著性。     

抗H7N9流感病毒中和抗体快速检测方法的建立及应用

目的：对建立的抗H7N9流感病毒中和抗体快速检测方法进行方法学验证及初步应用。方法：分别采用不同代次细胞对高、中、低不同滴度的阳性血清进行多次平行检测,考察细胞代次对检验结果的影响;采用NIBSC提供的参考品对方法学的特异性进行验证;同时应用抗H7N9的阳性血清检测进一步评估方法的准确性和精密性;采用ELISA-MNT法和血凝抑制（hemagglutination inhibition,HI）试验分别接种H7N9灭活流感疫苗免疫的小鼠血清样本20份,分析两种方法检测结果的相关性。结果：采用ELISA-MNT中和法,使用不同代次MDCK细胞（25、30和35代）检测相同的血清样本的中和抗体滴度结果相同;该方法只对羊抗H7N9的血清具有较高保护力,对其他血清基本没有交叉反应;该方法准确性良好;该方法组间、组内精密性的平均变异系数分别为4%和11%。该方法测定H7N9型流感疫苗免疫后的小鼠血清抗体效价,其结果与HI抗体的相关系数为0.61,表明两种方法的检测结果之间呈良好的正相关性。结论：建立的微量病毒中和法能够满足H7N9流感病毒中和抗体效价检测的要求,可用于H7N9新型大流行流感疫苗的免疫评价。     

呼吸道合胞病毒与甲型流感病毒混合感染患儿血清炎症因子表达水平及临床意义

目的：探讨呼吸道合胞病毒（RSV）、季节性流感病毒A型（Inf-A）混合感染时患儿体内常见的炎症因子表达水平及其临床意义。方法：选取Inf-A和RSV检测阳性的患儿及部分健康儿童共115例纳入研究,将所有研究对象分为4组：Inf-A感染组、RSV感染组、混合感染组（MIX组）及健康对照组（HC组）。采用悬液芯片技术和酶联免疫吸附试验（enzyme-linked immunosorbent assay,ELISA）法分别检测4组患儿体内白介素IL-1β、IL-2、IL-8、肿瘤坏死因子TNF-α、干扰素IFN-γ等致炎性因子及IL-4、转化生长因子TGF-β等抗炎性细胞因子的表达水平,比较4组患儿的炎症因子水平与病程严重程度的相关性。结果：急性期Inf-A感染组、RSV感染组、MIX组患儿IL-1β、IL-8、TNF-α、IFN-γ水平均不同程度升高。RSV组IL-2较治疗前无变化,MIX组升高不明显。MIX组IL-1β、TNF-α、IL-8水平较治疗前均明显升高。在疾病恢复期,IL-1β、TNF-α、IL-8、IFN-γ水平较治疗前均明显下降,但与HC组相比TNF-α、IFN-γ水平仍明显偏高。RSV组、Inf-A组及MIX组IL-4与TGF-β均无明显升高,与HC组比较差异无统计学意义。结论：Inf-A、RSV混合感染患儿体内炎症因子占主导地位,以IL-1β、IL-8、TNF-α水平升高为主,而IL-1β、IL-8水平的变化可能是反应病情严重程度较敏感的指标。     

基于Calpain-ERK信号通路研究Calpeptin对雌激素诱导人乳腺上皮细胞MCF-10A转化及干性标志物表达的影响

目的:研究钙激活中性蛋白酶(Calpain)抑制剂Calpeptin对雌二醇(E2)诱导人乳腺上皮细胞MCF-10A转化及干性标志物表达的影响,并探讨其作用机制。方法:以人乳腺上皮细胞MCF-10A为研究对象,采用E2诱导制备转化细胞模型。将细胞分为对照组[0.1%二甲基亚砜(DMSO)]、E2转化组(50 nmol/L)、E2转化+Calpeptin组(50 nmol/L E2+1μmol/L Calpeptin),以相应含药培养基连续培养15代。然后采用MTT法检测细胞的增殖率(24、48 h),采用平板克隆试验检测细胞的克隆形成率,采用悬浮成球试验检测细胞的成球数;采用实时荧光定量-聚合酶链式反应法检测细胞中干性标志物(CD44、Nanog、OCT4)和细胞外信号调节激酶(ERK)m RNA表达水平,并采用Western blotting法检测细胞中CD44、Nanog、OCT4、ERK和磷酸化ERK(p-ERK)蛋白表达水平。另取E2转化细胞分为对照组(0.1%DMSO)和U0126(ERK抑制剂)组(10μmol/L),按上述方法测定细胞的克隆形成率、成球数以及细胞中CD44...     

Human breast milk oligosaccharides attenuate necrotizing enterocolitis in rats by suppressing mast cell accumulation,DPPI activity and TLR4 expression in ileum tissue,and regulating mitochondrial damage of Caco-2 cells

Necrotizing enterocolitis (NEC), a devastating infant disease characterized by severe intestinal necrosis, its pathogenesis is poorly understood, but appears to be multifactorial and highly associated with immaturity of gastrointestinal tract and immature innate-immune system. Breast-milk is effective strategy to protect infants against NEC. This study is using a NEC rat model to investigate the pathological mechanism of NEC involved intestinal-damages, and the therapeutic mechanism of sialylated human milk oligosaccharides (SHMOs) on NEC rats; also using cell model to investigate the effects of SHMOs on colon-epithelial cells (Caco-2) in-vitro.Extraction and characterization of SHMOs from breast milk, establishment of a NEC rat model, histopathological analysis and mast cell accounting of the terminal ileum were taken; The levels of DPPI, TLR4, IL-6, TNF-α, MMP-2/9 and glutathione were measured using various methods. Caco-2 cells were pre-treated with SHMOs and cultured with LPS, histamine, chymase or DPPI, cell viabilities and mitochondrial membrane potential were examined; flow cytometry was used to detect cell cycle.The accumulation of mast cells was found in the ileum of NEC rats, but prohibited by SHMOs treatment; the increased levels of TLR4, DPPI, IL-6, TNF-α, MMP-2/9 in NEC ileum were suppressed by SHMOs in-vivo. SHMOs prevented Caco-2 cells from LPS, histamine, chymase induced damages by surviving cell viability, regulating G0/G1 and S phase in cell cycles, and increasing mitochondrial membrane potential. These findings provide a new insight into the pharmacological mechanism of SHMOs treatment for NEC and suggest that SHMOs needs well attention for therapeutic aims.