DC-SIGN在树突状细胞传播HIV-1中作用的实验研究

目的探讨DC—SIGN在树突状细胞（DC）传播HIV-1中的作用。方法用M嗜性或T嗜性HIV-1原代分离株分别刺激未成熟DC（immature DC，iDC））和成熟DC（mature DC，mDC），数量与DC相同的CD4^T细胞作为对照组，与活化的CD4^＋T细胞共培养，用ELISA方法定量检测第4、7、10、14天共培养上清中p24抗原，观察Dc在传播HIV-1的作用。预先加入抗DC—SIGNMcAb和，或抗ICAM-3McAb，观察抗DC-SIGNMcAb和抗ICAM-3McAb对DC传播HIV-1作用的影响。结果用M嗜性HIV-1刺激的iDC以及用M和T嗜性HIV-1刺激的mDC的共培养上清中p24含量均随培养时间延长逐渐增加，显著高于对照组（P=0．001）。加入抗DC．SIGNMcAb后，共培养上清p24抗原明显降低；加入抗ICAM-3McAb后上清中p24抗原并不减少。用T嗜性HIV-1刺激的iDC共培养上清中p24含量不随培养时间延长逐渐增加，与对照组相比差异无统计学意义。结论iDC具有传播M嗜性HIV-1的作用，但不具有传播T嗜性HIV-1的作用；mDC既能将M嗜性也能将T嗜性HIV-1传播给CD4^T细胞。抗DC-SIGN McAb能够抑制DC传播HIV-1的作用，提示DC-SIGN在DC向T细胞播散HIV-1过程中可能发挥重要作用。     

4-1BB信号拮抗肿瘤细胞培养上清诱导的树突状细胞凋亡

为研究4-1BB信号拮抗肿瘤细胞培养上清诱导的树突状细胞(DC)凋亡的作用,采用rmGM-CSF联合rmIL-4诱导小鼠骨髓细胞分化为未成熟树突状细胞(imDC),分别经rmTNF-α、4-1BB激发型单克隆抗体(2A)或TNF-α联合2A激发DC成熟。未成熟或成熟DC分别与不同浓度的肿瘤培养上清混合培养。流式细胞仪测定DC表面CD80、CD86的表达,3H-TdR掺入法测定DC激发T细胞活化增殖能力;JAM法测定未成熟或成熟DC与肿瘤上清混合培养后DNA片断形成率。结果显示:经2A或TNF-α刺激后,DC激发T细胞活化的能力显著提高;5%、10%、20%、50%小鼠肿瘤细胞株A20或B16培养上清与未成熟DC共育24h后,DNA片断形成率分别为16%、29%、41%、51%和20%、27%、37%、58%,且该效应具有时间依赖性;DC经2A或TNF-α激发48h后,不同浓度肿瘤培养上清诱导DNA片断形成率显著降低。因此,肿瘤细胞可以通过分泌可溶性因子诱导未成熟DC凋亡;4-1BBmAb能促进DC的发育成熟,并能有效地抑制肿瘤细胞培养上清诱导的DC凋亡。     

PPD对人PBMC的HIV复制和细胞因子产生的影响

目的：明确结核菌素纯蛋白衍化物(PPD)对PBMC内HIV-1复制的作用及其对细胞因子分泌的影响。方法：HIV-1 SF-33毒株分别感染结核菌素试验阳性(PPD( ))和阴性(PPD(-))健康人的PBMC后，与PPD共孵育72小时，用ELISA方法检测上清中HIV-1 p24抗原、TNF-α和IL-10的含量。结果：在PPD( )的健康人，PPD能显著诱导PBMC内HIV-1的复制，促进HIV-1感染的PBMC分泌TNF-α，同时抑制IL-10的分泌。在PPD(-)的健康人，未见到PPD显著诱导PBMC内HIV-1的复制。结论：在我国PPD( )的健康人，PPD促进HIV-1感染的PBMC分泌TNF-α，并抑制IL-10的分泌，导致HIV-1复制增加。     

妊娠水平孕酮体外对HIV-1感染和复制的影响

目的：研究相当于妊娠后外周血和胎盘局部浓度的孕酮（P4）体外对HIV-1感染及复制的影响。方法：检测终浓度为10^-6mol／L（相当于妊娠妇女外周血水平）和10^-5mol／L（相当于母胎界面局部浓度）的P4对HIV-1介导细胞融合的影响；ELISA法检测感染HIV的MT-2细胞培养上清p24抗原含量来探讨对HIV-1复制的影响；流式细胞术检测对淋巴细胞活化后CD69表达的影响和^1H-TdR掺入法检测对淋巴细胞增殖的影响。结果：10^-5mol／L的P4可以明显抑制HIV-1介导的细胞融合，并且降低感染细胞培养上清中p24抗原含量，两种浓度对淋巴细胞的活化和增殖都有抑制作用。结论：相当于妊娠后母胎界面局部浓度的P4体外可以抑制HIV-1感染和复制，其机制与抑制淋巴细胞活化和增殖有关。     

胆红素衍生物体外抗HIV-1的初步研究

目的 初步研究胆红素衍生物（DTB）体外抑制HIV-1病毒复制的作用。为进一步研究其作用机理奠定基础。方法 将DTB加入到感染HIV-1的细胞中，在适当条件下培养，最终以其培养上清液中的HIV-1p24抗原滴度（ELISA法）及MTT染色细胞毒法（MTT法）判断DTB抑制病毒的效果。结果 DTB在对细胞无毒浓度作用下能够有效抑制HIV-1的复制，当DTB浓度为160，80和40mg/ml时，抑制率分别为93.0%,56.2%和18.1%。结论 DTB可以有效抑制体外培养HIV-1病毒的复制。     

HIV-1 B''亚型R5或R5/X4嗜性毒株在GHOST细胞的感染性分析

目的分析我国HIV-1B′亚型R5或R5／X4嗜性毒株在GHOST细胞的感染性。方法采用传统的共培养方法从HIV-1感染者PBMC中分离并培养病毒，用表达CD4和趋化因子受体CCR5或CXCR4的GHOST细胞系，测定毒株的辅助受体利用情况，使用相同病毒量即2mg的HIV-1 p24分别感染表达不同受体的GHOST细胞系，通过流式细胞仪检测分析绿色荧光蛋白（GFP）的表达反映病毒感染细胞的能力。结果35例B′亚型毒株利用CCR5受体，占22例（62．85％），双嗜性即CCR5／CXCR4均阳性占13例（37．15％）。GHOST-R5／X4细胞的感染性分析结果显示，R5／X4双嗜性毒株的感染性明显高于CD4〉200／μl的R5毒株的感染性（P〈0．05）；R5／X4毒株与CD4≤200／μl的R5毒株感染性比较差异无统计学意义（P〉0．05）；CD4〉200／μl的R5毒株与CD4≤200／μl的R5毒株感染性比较差异有统计学意义（P〈0．05）；GHOST-CCR5细胞感染性分析结果显示：R5／X4双嗜性毒株的感染性明显下降，与CD4〉200／μl的R5毒株的感染性比较差异无统计学意义（P〉0．05）。利用相同剂量的双嗜性毒株分别感染R5、X4或R5／X4的GHOST细胞系，显示双嗜性毒株可同时利用CCR5和CXCR4辅助受体，但69．23％的R5／X4毒株以CCR5受体为主，30．77％的R5／X4毒株以CXCR4受体为主。结论HIV-1B′亚型R5／X4病毒不仅有更广泛的宿主细胞嗜性，而且在GHOST-R5／X4细胞中感染性明显提高。持续使用CCR5受体的毒株在疾病进展的过程中虽然辅助受体的利用是一样的，但病毒感染细胞的能力增加。     

体外无药物选择压力条件下HIV-1耐药基因突变的演变

目的 分离培养体外稳定传代的原代HIV-1耐药毒株,观察失去药物压力下,耐药毒株的体外生长以及主要耐药突变的演化趋势.方法 采集15例服用拉米夫定+司他夫定+萘韦拉平(3TC+D4T+NVP)的HIV-1感染者的外周血单个核细胞(PBMC),用体外共培养的方法从中分离原代HIV-1毒株;RT-PCR扩增耐药毒株历代培养上清的HIV-1 pol区基因并测序,在Stanford HIV Drug Resistance Database数据库进行耐药性分析.结果 15例患者中病毒载量>1000拷贝/ml的有8例,均成功分离出稳定传代的原代毒株,其中2株为耐药毒株,所携带的主要耐药突变分别是K103N/K238T和M184V/K103N/Y181C/H221Y,分别对NVP和3TC/NVP高度耐药;无药物压力的体外培养过程中,M184V、K103N、Y181C和H221Y等耐药突变可以稳定传代,但是K238T发生了回复突变.结论 分离出2株稳定传代的HIV-1耐药毒株,无药物压力情况下,携带K103N突变的毒株具有较好的复制适应性,可稳定传代;携带M184V和K103N/Y181C/H221Y的毒株也能够稳定复制;本研究中发现K238T耐药突变在失去药物的条件下稳定性差,提示该位点易发生回复突变.     

中国HIV-1病毒分离株的生物学特性与疾病进展关系的研究

目的　从HIV AIDS患者应用微量全血法分离中国HIV 1毒株 ,研究HIV 1的生物学特性与HIV AIDS疾病进展相关性。方法　建立微量全血法 ,从HIV AIDS全血标本中分离 17株HIV 1病毒分离株 ;检测这 17株病毒分离株嗜性和复制动力。结果　从 2 6例HIV AIDS病例中分离出HIV 1病毒 ,分离率为 6 5 .4 % (17 2 6 ) ,其中 17例HIV 1感染者的病毒分离率为 5 2 .9% (9 17) ,均为巨噬细胞嗜性 (M嗜性 ,NSI) ;9例AIDS患者的HIV 1病毒分离率为 88.9% (8 9) ,其中 7株为T细胞嗜性 (T嗜性 ,SI) ,1株为巨噬细胞嗜性。通过检测P2 4抗原确定 17株HIV 1病毒分离株的复制动力。在分离到的 17株HIV 1中 ,SI型病毒分离株与AIDS组显著相关 (P <0 .0 5 ) ;AIDS期的病毒分离株的复制动力明显高于HIV感染期 (P <0 .0 5 )。结论　微量全血法可用于病毒分离。 17株分离株的HIV 1复制动力与CD4 + T淋巴细胞计数呈线性负相关 ,与病毒载量呈正相关。     

罗勒多糖对人树突细胞抗原摄取功能及共刺激分子表达的影响

目的探讨罗勒多糖对人外周血单核细胞来源的树突细胞（dendritic cells，DC）抗原摄取功能以及共刺激分子表达的影响。方法从正常人外周血单核细胞诱导未成熟和成熟的DC，实验组加入罗勒多糖（150μg/ml），对照组加入PBS，分别培养24h，收获未成熟的DC，与OVA—FTTC（100μg/ml）共同孵育，流式细胞仪检测DC的抗原摄取能力。同时收获成熟DC，流式细胞仪检测DC表面共刺激分子CD80、CD86的表达情况。结果与对照组比较，罗勒多糖作用组DC的抗原摄取能力显著提高，同时成熟DC表面共刺激分子CD80、CD86的表达也明显升高。结论罗勒多糖可显著增强DC的抗原摄取能力，并且能够提高细胞表面共刺激分子的表达，这可能是罗勒多糖发挥抗肿瘤免疫的机制之一。     

早、中、晚孕期胎盘因子体外抗HIV-1的研究

目的 探讨早、中、晚孕期胎盘因子(PF)体外抗人免疫缺陷病毒-1(HIV-1)及在HIV-1垂直传播中的作用.方法 荧光染料Calcien-AM标记的H9/HIV-1ⅢB分别与早、中、晚孕期不同稀释浓度的PF作用后,与MT2细胞培养,荧光显微镜下观察合胞休的形成;用HIV-1ⅢB感染MT2细胞,并分别与早、中、晚孕期不同稀释浓度的PF作用后,用MTT法检测HIV-1感染细胞的存活率,用ELISA方法检测细胞培养上清中p24抗原水平.结果 各孕期PF并不能抑制MT2和H9/HIV-1ⅢB细胞的融合,但可以增加HIV-1感染细胞的存活率及减少HIV-1 p24抗原的表达,且效应以早孕期PF最大,中孕期PF其次晚孕期PF最小,并与剂量呈正相关.结论 PF在体外具有抗HIV-I的作用,并呈现孕期和剂量相关性,可能在阻断HIV-1垂直传播中具有一定作用.     

Deformation of the dog aortic valve ring during the cardiac cycle

Changes in strain in the line of aortic valve leaflet attachment (aortic ring) were measured during the cardiac cycle by means of an inductive technique. To that purpose coils were sutured to each commissure and base point of the aortic ring, when the animals were on a cardiopulmonary bypass. After bypass and stabilization of the hemodynamic variables changes in the aortic strain were measured at aortic pressures ranging from 4 to 20 kPa. Aortic pressure at the level of the commissure points and left ventricular pressure were measured to assess transvalvular pressure. Commissure strain appeared to depend on aortic and transvalvular pressure throughout the cardiac cycle. At an aortic pressure of 10 kPa (75 mm Hg), the derivative of commissure strain to aortic pressure was found to be 1.9×10^–5±1.2×10^–5 Pa^–1 (mean ±SD). During the ventricular ejection phase commissure strain was 0.04±0.03 higher than during ventricular filling. Maximum variations in basal strain during the cardiac cycle ranged from 0.03 to 0.15. During the ejection phase the basal segments adjoining the myocardium shortened whereas the segment close to the non-contracting anterior mitral valve leaflet lengthened. Strain between a base and a commissure point of the aortic ring were synchronous with the cardiac cycle, but no specific pattern could be found.     

超声弹性成像中应变滤波器的理论及其发展

弹性成像能够提供组织弹性这一基本的力学属性,具有很广阔的应用前景。应变滤波器是超声弹性成像的重要理论框架,可广泛应用于超声弹性成像效果的评估、分析和预测。本文详细综述了应变滤波器的理论和发展,并对文献中的一些笔误作了修正,对实现的一些细节作了说明。通过对应变滤波器的理解,可以加深对超声弹性成像效果的理解,并对采用合适的方法和参数以获得最佳的成像效果以指导。     

Expression patterns of the aquaporin gene family during renal development: influence of genetic variability

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of hypohydration severity on the physiological,psychological and renal hormonal responses to hypoxic exercise

Evidence of the effect of dehydration on physiological responses to hypoxia is limited. The purpose of this study was to determine the effect of hypohydration severity on physiological, renal hormonal and psychological responses to acute hypoxia. Eight males completed intermittent walking tests under normobaric hypoxic conditions (FI O₂ = 0.13) after completing four separate hypohydration protocols, causing change in body mass of approximately 0% (EU), −1% (H1), −2% (H2) and −3% (H3). Physiological and psychological markers were monitored throughout the 125 min test. Fluid controlling hormones were measured pre and post exposure. Heart rate, core temperature, peripheral arterial oxygen saturation (SpO₂), minute ventilation and urine osmolality were found to be significantly different between hydration conditions and correlated with Lake Louise Questionnaire score (LLQ) (P < 0.05). LLQ score increased with hypohydration severity above H2 (EU 1.3 ± 1; H1 1.2 ± 1; H2 2.7 ± 2; H3 3.9 ± 2) (P < 0.001). Antidiuretic hormone and aldosterone increased over the test, but were not different between hydration conditions (P < 0.05). Atrial natriuretic peptide showed no change over time, or with conditions. Therefore, renal hormones are not influenced by hypohydration severity during moderate intensity hypoxic exercise. Hypohydration less than −2% induces greater physiological strain during hypoxic exercise and may cause rise in symptoms such as, fatigue, headache, nausea and lightheadedness.     

二维数字化超声软组织应变成像系统的研制

利用超声散射回波测量生物组织在静态压缩情况下弹性系数的超声弹性成像方法得到了很大的发展.利用国产高速数据采集卡以及商业线阵B型超声诊断仪,建立了一套二维数字化超声软组织应变成像系统.应用这一系统获得了数字化的射频组织超声散射回波信号,并对组织内的应变分布进行成像.实验结果表明,该应变成像系统在获得传统B型超声图像的同时,可以获得组织内部的应变分布图像,从而获取在B超图像上无法得到的组织弹性变化.这一系统的研制成功,不仅为超声弹性成像技术在医学临床上的应用研究打下了基础,同时也为扩宽普通商业B超的应用范围提供了途径.     

不同毒株制备的人用狂犬病疫苗免疫原性比较

目的:比较3种毒株(aG、CTN、CaG)制备疫苗的免疫原性及抗狂犬病毒攻击的能力。方法:应用ELISA法测定3种毒株制备的疫苗免疫家兔、豚鼠、地鼠所获的免疫血清效价;并通过用上述的地鼠免疫血清中和3种病毒后进行小鼠的中和试验。结果:在家兔、豚鼠两组内,细胞适应株毒种制备的疫苗(CaG和CTN株)血清免疫效价明显高于豚鼠脑毒种(aG株)。细胞适应株毒种制备的疫苗免疫地鼠所获得的免疫血清中和3株病毒的能力高于豚鼠脑毒种。结论:使用细胞适应株毒种生产狂犬病疫苗较豚鼠脑毒种具有更好的免疫原性。     

人胚肺成纤维细胞突变株Z-HL16 C转化性质的初步鉴定

目的初步鉴定人胚肺成纤维细胞(HL)突变株Z-HL16C的转化性质。方法采用细胞形态学观察、细胞遗传学染色体分析、细胞在裸鼠体内恶性生长情况的组织病理学检测以及细胞对病毒的敏感性等方法。结果HL细胞转化的Z-HL16C细胞在形态上由梭形的纤维细胞变成多边形的上皮样细胞,且具有稳定无限传代等特点;染色体数目由正常人的46条二倍体,转化成多数为亚三倍体或超三倍体;染色体结构核型中约有30%~40%的染色体为结构异常染色体;裸鼠移植肿瘤发病率为100%,其瘤组织病理检测结果显示,该细胞株为癌变细胞株。病毒实验表明,Z-HL16C细胞对单纯疱疹等多种常见病毒高度敏感。结论人胚肺成纤维细胞的突变株Z-HL16C是对病毒高度敏感的癌细胞株。     

Structural Organization of the Genome of SARS-Associated Coronavirus (Strain SoD) Isolated on the Territory of the Russian Federation

Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined. Phylogenetic analysis revealed maximum similarity between strain SoD genome and Frankfurt 1 strain genome. Three nucleotide substitutions determining two amino acid substitutions were detected.     

Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus

The molecular mechanism involved in cytopathogenicity of rabies virus has not been fully elucidated yet. A fixed rabies virus Nishigahara strain does not induce clear cytopathic effect (CPE) in mouse neuroblastoma (NA) cells, whereas Ni-CE strain, which was established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, induces CPE that is characterized by rounding, shrinkage and detachment of the cells. In this study, to identify which viral gene is associated with the CPE of Ni-CE strain, we analyzed chimeric viruses between Nishigahara and Ni-CE strains generated by reverse genetics systems of both strains. We showed that the matrix gene of Ni-CE strain is responsible for the CPE in NA cells. It was also demonstrated by infection of Nishigahara and Ni-CE mutants with a single amino acid substitution in the matrix protein (M) that an amino acid at position 95 of M is a cytopathic determinant of the virus. We also demonstrated that the CPE is, at least partly, due to apoptosis. This is the first report of identification of an amino acid residue in a rabies virus protein that is important for the cytopathogenicity of the virus.