大蒜新素对MCMV感染小鼠转录因子T-bet和GATA-3基因表达的影响

目的探讨大蒜新素(即大蒜素注射液)对鼠巨细胞病毒(murinecytomegalovirus,MCMV)感染小鼠脾转录因子Tbet和GATA3基因表达的影响。方法20只BALBc小鼠被随机分成大蒜新素治疗组(10只)和安慰剂组(10只)。均在MCMVSmith株接种24h后分别用大蒜新素一般剂量(每天25mgkg)和等量生理盐水腹腔注射,每天1次,连续2周。另设正常对照组(10只)不接种病毒,仅用等量生理盐水治疗。用PFUml测定小鼠脾组织病毒滴度;用RTPCR方法检测小鼠脾转录因子TbetGATA3mRNA表达强度;用双抗体夹心ELISA法检测小鼠脾细胞培养上清细胞因子IFNγ和IL10表达水平。结果MCMV感染小鼠14d,TbetmRNA和IFNγ的表达基本下降至基线水平,而GATA3mRNA和IL10的表达显著增强;大蒜新素能诱导MCMV感染模型鼠转录因子TbetmRNA和IFNγ的表达显著增加(P<0.01),而转录因子GATA3mRNA和IL10的表达显著下降(P<0.01);同时显著降低小鼠脾组织匀浆中病毒滴度(P<0.01)。结论MCMV感染可导致TH1TH2类细胞因子表达失衡,主要表现为IFNγ表达明显降低和IL10表达明显增高;大蒜新素通过上调转录因子TbetmRNA的表达促进IFNγ的分泌,并下调转录因子GATA3mRNA的表达抑制IL10的分泌。     

巨细胞病毒感染对小鼠心肌组织TH1/TH2类细胞因子及其特异性转录因子T-bet/GATA-3表达的影响

目的动态观测MCMV感染对小鼠心肌组织TH1/TH2类细胞因子及其特异性转录因子T-bet／GATA-3表达的影响。方法建立小鼠巨细胞病毒感染心肌炎模型。观察小鼠血清删浓度变化，心肌组织病理损害和心肌组织细胞因子IFN-γ、IL-4表达水平以及脾组织中转录因子T-bet、GATA-3表达强度。结果心肌组织病理积分和血清cTnI浓度在MCMV感染后第3天增高，第7天达高峰，第14天仍维持较高水平；IFN-γ在MCMV感染第3天时表达迅速增高达到峰值，随后下降，第14天基本下落至正常水平；IL-4在MCMV感染第5天表达迅速增高达到峰值，随后下降，在第14天又升高；MCMV感染第3天转录因子T-bet蛋白表达达峰值，随后逐渐降低，到第14天时降低更为显著。转录因子GATA-3在MCMV感染第7天时表达明显增强，第14天时增强更为显著。结论MCMV感染通过上调GATA-3转录因子和叫表达，下调T-bet转录因子和IFN-γ表达，导致TH1/TH2平衡失调，表现为TH2细胞优势应答状态，使心肌损害加重。这可能是CMV感染致宿主特异性细胞免疫功能降低并造成组织损伤的机制之一。     

BALB/c小鼠哮喘进展过程中T-bet和GATA3的表达变化

目的:初步探讨转录因子T-bet和GATA3在小鼠哮喘模型哮喘进展过程中的改变及其意义.方法:18只BALB/c小鼠随机分为正常对照组、哮喘组和药物组(地塞米松组),每组各6只.采用Western blot法检测各组小鼠肺组织T-bet和GATA3的表达,同时运用流式细胞仪检测小鼠脾细胞胞内细胞因子白介素4(IL-4)和干扰素γ(IFN-γ)的表达.结果:哮喘组与正常对照组相比,药物组与哮喘组相比,肺组织T-bet和GATA3均有明显改变(P＜0.01).哮喘组T-bet明显减低,而GATA3明显升高;地塞米松治疗后,T-bet和GATA3均明显减低,后者减低更为明显.流式细胞仪分析显示:哮喘组CD4 T细胞细胞内IL-4/IFN-γ的比值显著高于对照组(P＜0.01);经地塞米松处理后的哮喘小鼠CD4 T细胞细胞内IL-4/IFN-γ的比值显著降低(P＜0.01).结论:转录因子T-bet和GATA3与哮喘进展密切相关,调控它们的表达,可以改变IL-4/IFN-γ比值,纠正哮喘的Th2漂移,可能成为哮喘治疗的新靶点.     

黄芪在树突状细胞水平对哮喘TH 1/TH 2平衡的调节作用

目的 探讨黄芪在树突状细胞(DC)水平对过敏性哮喘TH/TH2平衡的调节作用.方法 用rhGM-CSF和rhIL-4诱导培养外周血来源的DC并予鉴定,ELISA法检测其分泌的细胞因子IL-12、IL-10以及与自身T细胞反应后,RT-PCR检测T-bet和GATA-3 mRNA含量,流式细胞术检测T细胞分泌的胞内细胞因子IL-4和IFN-γ水平.结果 哮喘患儿外周血DC分泌IL-10高于对照组(P＜0.05);黄芪干预后DC分泌IL-10降低,与哮喘组比较差异有统计学意义(P＜0.05).哮喘患儿外周血DC分泌IL-12低于对照组(P＜0.05);黄芪干预后DC分泌IL-12增加,但与哮喘组比较差异无统计学意义.混合培养第7天哮喘组T细胞内IL-4水平显著高于正常对照组(P＜0.01);而IFN-γ水平则显著低于正常对照组(P＜0.05);哮喘组IL-4/IFN-γ比值高于正常对照组(P＜0.01).黄芪干预后T细胞内IL-4水平与哮喘组比较差异无统计学意义,而IFN-γ水平增加,与哮喘组比较差异有统计学意义(P＜0.05),IL-4/IFN-γ比值降低,与哮喘组比较差异有统计学意义(P＜0.01).哮喘组T-bet mRNA的表达强度明显低于正常对照组(P＜0.01);而哮喘组GATA-3 mRNA的表达强度则明显高于正常对照组(P＜0.05);哮喘组GATA-3/T-bet比值高于正常对照组(P＜0.05).黄芪干预后T细胞GATA-3 mRNA的表达强度与哮喘组比较差异无统计学意义,而T-bet mRNA水平增加,与哮喘组比较差异有统计学意义(P＜0.05),GATA-3/T-bet比值降低,与哮喘组比较差异有统计学意义(P＜0.01).结论 哮喘患儿DC功能缺陷,产生IL-12减少、IL-10增加导致TH2优势分化,从而使TH1/TH2平衡向TH2倾斜,合成IFN-γ减少,进而造成气道慢性炎症、气道高反应性而致哮喘发作.黄芪对DC的调节主要通过降低IL-10的分泌水平,从而降低其抑制TH0细胞向TH 1分化的功能,即间接抑制了TH0细胞向TH2的分化.     

CD4+CD25+调节性T细胞抑制效应性T细胞在MCMV感染中的免疫作用

目的 通过细胞水平探讨CD4+CD25+调节性T细胞(Treg)在T淋巴细胞与感染小鼠巨细胞病毒(MCMV)的小鼠胚胎成纤维细胞(MEF)共培养体系中发挥的免疫作用.方法 建立小鼠T细胞与感染MCMV的同系MEF(MEFMCMV)体外共培养细胞模型.通过噬斑法检测共培养上清中感染性病毒量;Western blot方法检测辅助性T细胞亚群TH1/TH2特异性转录因子T-bet/GATA-3蛋白表达水平;ELISA法检测共培养上清中细胞因子IL-4、IL-10和IFN-γ表达水平.结果 去除Treg的T细胞(TdepTreg)与MEFMCMV共培养3 d后可显著减少上清中的感染性病毒量;同时THl/TH2上游特异性转录因子T-bet/GATA.3和下游细胞因子IL-4、IL-10和IFN-γ的蛋白质表达水平显著升高.向共培养体系中添加Treg后病毒负荷量显著增加;T-bet/GATA-3和IFN-γ蛋白表达水平下降;IL-10蛋白表达水平在Treg比率为1%～2%时与TdepTreg组比较无显著差异,在Treg比率增至5%～20%时较TdepTreg组显著增加;而IL-4表达水平与TdepTreg组比较无显著差异,上述效应均与Treg添加比率成剂量相关性.结论 巨细胞病毒感染小鼠成纤维细胞后能激活效应性T细胞增殖活化,而Treg可抑制效应性T细胞在MCMV感染中的免疫保护作用.     

吡格列酮对哮喘模型TH1/TH2细胞因子表达的影响

目的 初步探讨过氧化物酶增殖活化受体-γ（PPAR-γ）激动剂吡格列酮对哮喘模型TH1/TH2细胞因子表达的影响及其机制。方法 18只BALB／c小鼠随机分为正常对照组、哮喘组和药物组，每组各6只。采用Western blot方法检测各组小鼠肺组织T-bet和GATA3的表达，同时运用流式细胞仪检测小鼠脾细胞胞内细胞因子白细胞介素4（IL-4）和干扰素-γ（IFN-γ）的表达。结果 哮喘组肺组织T-bet的表达与正常对照相比显著升高（P〈0．01），GATA3无显著变化（P〉0．05）；经吡格列酮治疗的小鼠肺组织T-bet的表达与哮喘鼠比显著增高（P〈0．01），而GATA3无显著变化（P〈0．05）。哮喘鼠T细胞内IL-4／WN-γ比值与正常组相比明显升高（P〈0．01）；经吡格列酮治疗后细胞内IL-4，IFN-γ比值明显降低（P〈0．01）。结论 PPAR-γ激动剂吡格列酮可能通过参与调控TH1细胞转化过程中重要转录因子T-bet的表达，改变IL-4／IFN-γ比值，从而改善相应的炎性症状，PPAR-γ可能成为哮喘治疗的新靶点。     

PPAR-γ激动剂吡格列酮对Jurkat T细胞分化的调节作用

目的 探讨吡格列酮对Jurkat T细胞T-bet/GATA-3表达的影响及其与调节TH1/TH2细胞分化作用机制之间的关系.方法 不同浓度的吡格列酮刺激Jurkat T细胞,在不同时间点分别用ELISA法检测TH1/TH2细胞因子表达谱及用RT-PCR检测T-bet和GATA-3 mRNA表达的变化.为探讨实验结果是否为过氧化物酶体增殖激活受体γ(PPAR-γ)依赖性,同时设立加有PPAR-γ特异性拮抗剂GW9662(终浓度为10 mol/L)的对照组.结果 吡格列酮对Jurkat T细胞分泌细胞因子IFN-γ和IL-10的表达均起抑制作用,抑制T-bet和GATA-3 mRNA的表达,并具有浓度和时间依赖性.GW9662可缓解吡格列酮抑制IFN-γ分泌及T-bet mRNA表达,但对于IL-10和GATA-3 mRNA的受抑程度则无明显影响.结论 吡格列酮抑制TH0向TH1细胞分化是PPAR-γ依赖性地通过转录因子Tbet进行调节,对TH2细胞的抑制作用则非PPAR-γ依赖性的转录因子GATA-3途径的负性调节.     

脑脊髓损伤大鼠局部Th1/Th2细胞相关因子的表达及意义

目的 分析大鼠急性脑脊髓性损伤后脑脊髓中Th1和Th2细胞相关因子的表达水平,探讨其在持续性二次损伤中可能的作用.方法 制备SD大鼠急性脑脊髓性损伤模型,随机分为损伤组和脂多糖(LPS)处理组,各组又分别对脑和脊髓进行实验处理.用荧光定量PCR分别检测不同组别大鼠脑脊髓中Th1和Th2细胞相关因子,并进行相关性分析.结果 与对照组相比,损伤和LPS处理的脑组织Th1细胞相关的细胞因子IFN-γ表达明显增高(P＜0.05),而T-bet的表达并无明显改变;在损伤和LPS处理的脊髓中,IFN-γ和转录因子T-bet、HLX的表达均显著升高(P＜0.05).无论是脊髓损伤组还是LPS处理组,细胞因子IL-4和转录因子GATA3均与对照组无异,呈低表达状态.结论 脑脊髓损伤后呈现不同程度的Th1细胞相关因子上调,尤以可溶性的细胞因子IFN-γ为显著,脊髓损伤时出现T-bet与HLX表达上调.     

T-bet、GATA3及相关因子的表达与胃癌及转移的相关性研究

目的：分析胃癌患者Th1和Th2两类细胞因子的基因表达与转录因子T-bet和GATA3表达的相关性,从细胞因子与转录因子角度考证胃癌患者Th1/Th2细胞分化趋势,探讨p53与T-bet的相关性,了解T-bet与肿瘤转移之间的关系。方法：利用荧光定量PCR技术检测55例胃癌患者及45例正常人外周血单个核细胞（PBMC）中T-bet、GATA3转录因子和IFN-γ、IL-4等细胞因子的水平,应用免疫组化法检测胃癌组织中p53的表达状况。结果：55例胃癌患者T-bet、GATA3、IFN-γ、IL-4的表达率依次为51%（28/55）、78%（43/55）、27%（15/55）、69%（38/55）。定量PCR结果显示,病人组的T-bet和IFN-γ明显低于正常对照组（P〈0.01）,而GATA3和IL-4则明显高于正常对照组（P〈0.01）。T-bet与IFN-γ在病人体内的mRNA表达存在正相关性（P〈0.01）,正常人则没有。GATA3与IL-4的表达在两组中均呈明显正相关（P〈0.01和P〈0.05）,且在p53阳性的患者中,T-bet的表达率较低,而GATA3的表达率较高。结论：胃癌患者有明显Th2漂移现象,且p53的表达与T-bet的表达呈负相关。     

地塞米松对实验性哮喘小鼠TH1/TH2细胞因子的影响及其机制的探讨

目的：初步探讨地塞米松对小鼠哮喘模型哮喘进展过程中T_H细胞因子的影响及其机制。方法： 18只BALB/c小鼠随机分为正常对照组、哮喘组和地塞米松处理组，每组各6只。运用流式细胞仪检测小鼠脾细胞胞内细胞因子白细胞介素-4和干扰素-γ的表达,用Western blotting方法检测各组小鼠肺组织转录因子 T-bet 和GATA-3的表达，用组织学观察肺组织炎症程度。结果：哮喘鼠T细胞内IL-4/IFN-γ比值明显高于正常组（P﹤0.01）；地塞米松组细胞内IL-4/IFN-γ比值明显低于哮喘组（P﹤0.01）。哮喘组肺组织T-bet的表达明显低于正常对照组（P﹤0.01），GATA-3明显高于正常对照组（P﹤0.01）;地塞米松组的小鼠肺组织T-bet和GATA-3的表达均明显低于哮喘组（P﹤0.01）， GATA-3降低更为明显。结论：调控T_H1和T_H2细胞转化过程中重要转录因子T-bet和GATA-3的表达，可以改变IL-4/IFN-γ比值，纠正哮喘的T_H2漂移，从而改善相应的炎性症状，这可能是地塞米松抑制哮喘的重要机制之一。     

Development in in vitro toxicology

There are three principal pressures driving the development of in vitro toxicology: (1) the need for more efficient testing systems to cope with the large number of xenobiotics currently being developed; (2) public pressure to reduce animal experimentation; and (3) a need for a better understanding of the mechanisms of toxicity. Within this, in vitro toxicology is focused on local, systemic, and target-organ toxicity. It is becoming increasingly apparent that a step or decision-tree approach using input of a variety of experimental data (physicochemical properties, biokinetics, cytotoxicity) provides the most efficient system for predicting toxicity. Examples of the use of in vitro toxicity systems for prediction of systemic toxicity and target-organ (liver) toxicity are presented.Originally presented at ECCP 93.     

Seasonal variations in acute toxoplasmosis in pregnant women in Slovenia

Between December 1999 and December 2004, 40 081 pregnant women were examined for toxoplasmosis with Toxo-IgG, Toxo-IgM enzyme immunoassay. Women with positive results were then retested with the Toxo-IgG avidity assay for recent toxoplasmosis. Recent acute toxoplasmosis in pregnant women was found to be significantly more frequent (p < 0.01) during winter than summer. The incidence of acute toxoplasmosis during winter-spring was also significantly more frequent (p < 0.025) than summer-autumn. This phenomenon should be taken into account when formulating preventive measures for toxoplasmosis, especially for pregnant women.     

Age-related changes in polyamines in memory-associated brain structures in rats

Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines and have important roles in maintaining normal cellular function, regulating neurotransmitter receptors and modulating learning and memory. Recent evidence suggests a role of putrescine in hippocampal neurogenesis, that is significantly impaired during aging. The present study measured the polyamine levels in memory-related brain structures in 24- (aged), 12- (middle-aged) and 4- (young) month-old rats using liquid chromatography/mass spectrometry and high performance liquid chromatography. In the hippocampus, the putrescine levels were significantly decreased in the CA1 and dentate gyrus, and increased in the CA2/3 with age. Significant age-related increases in the spermidine levels were found in the CA1 and CA2/3. There was no difference between groups in spermine in any sub-regions examined. In the parahippocampal region, increased putrescine level with age was observed in the entorhinal cortex, and age did not alter the spermidine levels. The spermine level was significantly decreased in the perirhinal cortex and increased in the postrhinal cortex with age. In the prefrontal cortex, there was age-related decrease in putrescine, and the spermidine and spermine levels were significantly increased with age. This study, for the first time, demonstrates age-related region-specific changes in polyamines in memory-associated structures, suggesting that polyamine system dysfunction may potentially contribute to aged-related impairments in hippocampal neurogenesis and learning and memory.     

休克过程中肾上腺髓质素变化的研究进展

Adrenomedullin (AM) is a new peptidergic regulator of vascular function. AM serves as a hormone, which has many biological properties, plays an important role in the many pathophysiological processes, especially shock. This review will highlight the structure, biological properties of AM and the relationship between AM and shock.     

Secular change in menarche in women in Madrid

The age at menarche was estimated by recollection in 1617 women between the ages of 18 and 60 in Madrid and a nearby suburb, Pinto. The population of Pinto is working-class and the Madrid group, taken from residential neighbourhoods , belongs to the upper middle class. In both groups we found a diminution in average age at menarche, from 14.04 to 13.02 years in Madrid and from 14.55 to 13.16 years from about 1935 to about 1965 in Pinto. These changes have been more intense in the group which is less well-off economically, where living conditions have varied much more drastically.     

Pitfalls in TRAP assay in routine detection of malignancy in effusions

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.