自然流产模型小鼠蜕膜细胞凋亡及相关基因的表达

目的 通过比较正常妊娠模型小鼠及自然流产模型小鼠蜕膜细胞凋亡及Bcl-2、Bax、Fas、FasL蛋白的表达,从细胞及分子水平探讨自然流产的发病机制.方法建立正常妊娠模型CBAXBALB/c和自然流产模型CBAXDBA/2.用免疫组化SABC法测定两组模型孕13 d蜕膜细胞Bcl-2、Bax、Fas、FasL蛋白的表达,并通过MIAS-2000医用彩色病理图像免疫组化测量系统对其表达进行半定量分析,其结果用平均灰度值表示;同时应用DNA缺口原位末端标记技术(TUNEL)测定两组模型孕13天蜕膜细胞凋亡情况.结果与正常妊娠模型相比,自然流产模型蜕膜细胞Bcl-2蛋白的表达降低(P＜0.01);Bax蛋白的表达明显升高(P＜0.01);FasL的表达明显升高(P＜0.01);Fas的表达两组比较无明显差异(P＞0.05).蜕膜细胞凋亡指数(AI),自然流产模型明显高于正常妊娠模型(P＜0.01).结论 早孕期蜕膜组织细胞凋亡异常是自然流产的机制之一,Bcl-2/Bax,Fas/FasL途径可能是诱导早孕期蜕膜细胞凋亡的重要因素.     

HDV/HBV感染树鼠句肝组织中的肝细胞凋亡

目的探讨HDV/HBV感染树鼠句肝组织中Fas/FasL、Bcl2/Bax和ICE表达与HDV感染之间的关系,以及Fas/FasL、Bcl2/Bax和ICE在丁型肝炎肝细胞凋亡中的作用.方法采用免疫组化技术对HDV/HBV感染树鼠句肝组织中Fas/FasL、Bcl2/Bax和ICE的表达进行检测;应用原位末端标记技术对肝细胞凋亡进行检测.结果HDAg表达与Fas/FasL、Bcl2/Bax和ICE表达之间关系密切(P＜0.05),HDAg表达越强,Fas、FasL、Bax和ICE表达也越强,而Bcl2表达则越弱.Fas/FasL、Bcl2/Bax和ICE表达与肝细胞凋亡之间关系密切(P＜0.05),Fas、FasL、Bax和ICE表达越强,凋亡细胞越多;相反,Bcl2表达越强,凋亡细胞越少.结论肝细胞内HDAg表达可诱导Fas、FasL、Bax和ICE表达,但对Bcl2表达无明显诱导作用;Fas/FasL、Bcl2/Bax和ICE在肝细胞凋亡中起重要作用.     

大剂量过敏原诱导哮喘小鼠T细胞不反应性及机制研究

目的 探讨大剂量过敏原诱导哮喘小鼠T细胞不反应性及其机制.方法 采用不同浓度的过敏原在体外处理哮喘小鼠DC及T细胞,ELISA法检测DC分泌IL-10和TGF-β1以及T细胞Th1/Th2细胞因子的分泌,并分析其相关性.结果 ①在0～10 mg/mL范围内,哮喘小鼠DC分泌IL-10、TGF-β1水平随处理浓度增高而增高,正常小鼠DC各浓度处理组之间无显著差异.②在0～1 mg/mL范围内,Th2细胞因子的分泌随处理浓度增高而增高,但在10 mg/mL的OVA作用的下,IL-4、IL-5等细胞因子的分泌水平则较其他组明显降低(P＜0.05).IL-2和IFN-γ的分泌也受到明显抑制(P＜0.05).而对正常小鼠T细胞,OVA质量浓度的变化对其Th1/Th2细胞因子的分泌无影响.③Th1/Th2细胞因子水平与DC分泌的IL-10和TGF-β1水平无明显相关性.结论 大剂量过敏原(10 mg/mL的OVA)可诱导哮喘小鼠T细胞不反应性,但与DC分泌IL-10和TGF-β1水平无明显关系.     

FasL诱导EAE小鼠淋巴细胞凋亡的实验研究

为了解FasL在诱导EAE淋巴细胞凋亡中发挥的作用 ,我们用髓鞘碱性蛋白 (MBP )致敏小鼠 ,建立动物模型 实验性自身免疫性脑脊髓膜炎 (EAE )。分析了EAE动物淋巴细胞膜上Fas及FasL分子的表达水平 ,检测了分泌的细胞因子 ;用FasL分子诱导了EAE淋巴细胞凋亡。结果表明 ,应用MBP致敏KM小鼠 ,成功地建立了EAE模型 ,KM小鼠的发病率为6 8 3% ,发病程度为 1～ 2分 ;小鼠特异性淋转比对照组高 ,并与发病评分成正比 ;细胞因子分泌水平提示以Th2为主 ;EAE淋巴细胞Fas和FasL的表达及凋亡显著高于对照组 ;FasL诱导淋巴细胞凋亡剂量依赖曲线表明 ,在一个较小的浓度范围内呈现正相关 ,若加入单抗可部分阻断FasL诱导的凋亡。结果提示Fas FasL在EAE动物淋巴细胞凋亡中起了重要作用。     

SLE患者外周血T细胞亚群凋亡特点及其相关机制的研究

目的:研究系统性红斑狼疮(SLE)患者外周血CD3+、CD4+、CD8+T细胞亚群的凋亡特点及其相关的凋亡机制。方法:采用三色荧光流式细胞术检测高活动性SLE患者、低/非活动性SLE患者以及正常对照者外周血T细胞亚群的百分比例、T细胞亚群膜表面Fas/FasL的表达率、T细胞亚群早期凋亡(AV+PI-)的情况;采用ABC-ELISA法测定各组SLE患者和正常对照者血清IL-10水平;对10例血清IL-10水平异常升高的高活动性SLE患者进行体外PBMCs培养实验,其中分别加入抗FasL抗体和抗IL-10抗体,48小时常规培养后分别检测PBMCs中T细胞亚群百分比例、T细胞亚群膜表面Fas/FasL的表达率、T细胞亚群早期凋亡的变化。结果:SLE患者外周血T细胞凋亡异常增多,其中以高活动性SLE患者CD4+T细胞亚群的凋亡尤为显著(P0.05),CD4+T细胞的异常凋亡与CD4+和CD8+T细胞膜表面表达增高的Fas/FasL密切相关(P0.05);各组SLE患者外周血血清IL-10水平均明显升高(P0.01),高活动性SLE患者更为明显,血清IL-10水平升高不仅与SLE主要临床检验指标相关,而且与CD4+/CD8+比值减少、CD4+T细胞高表达FasL相关(P0.05);随访研究显示,随着SLE病情好转、稳定,血清IL-10水平下降明显、T细胞亚群Fas/FasL的表达率明显减少、T细胞亚群凋亡逐渐减少,以上变化均在CD4+T细胞亚群中体现得最为明显(P0.05)。结论:SLE患者T细胞活动性异常升高,尤其是CD4+T细胞亚群凋亡增加,是SLE疾病发展的重要病理机制,IL-10作为能诱导T细胞高表达Fas/FasL的重要调节因子,参与了SLE患者CD4+T细胞凋亡的免疫调节,SLE患者异常增高的IL-10很可能通过Fas/FasL途径促进了T细胞尤其是CD4+T细胞亚群的凋亡。     

龟龄集对 AD 小鼠皮层和海马 Fas/ FasL 表达及神经元凋亡的影响

目的 探究龟龄集对阿尔茨海默病 (Alzheimer’s disease, AD) 小鼠皮层和海马 Fas/ FasL 表达及 神经元凋亡的影响。 方法 构建 AD 小鼠模型, 小鼠随机分为对照组、 模型组、 多奈哌齐组及龟龄集低、 中、 高剂量组。 Morris 水迷宫检测干预前后小鼠的学习记忆能力; HE 染色检测小鼠皮层、 海马神经元病理 学改变情况; TUNEL 染色检测神经元凋亡情况; Western 印迹及 Real time PCR 分别检测 Fas、 FasL 的蛋白 表达水平及 mRNA 表达水平。 结果 与模型组比较, 龟龄集各组和多奈哌齐组小鼠学习记忆能力明显提高 (P< 0. 05), 皮层和海马神经元病理学损害及神经元凋亡改善 (P< 0. 05), Fas、 FasL 蛋白和 mRNA 水平下 降 (P< 0. 05)。 结论 龟龄集可能通过抑制 Fas/ FasL 表达抑制 AD 模型小鼠皮层、 海马神经元凋亡, 并改 善其学习记忆能力。     

血必净对活化诱导T细胞凋亡的调节

目的 观察活化诱导对脾脏T淋巴细胞凋亡、凋亡相关基因mRNA表达及caspase3活性的影响,以及活血化瘀中药的调节作用.方法 提取BALB/c小鼠脾脏T淋巴细胞并培养,以Con A+IL-2诱导T细胞活化凋亡,MTT法检测细胞增殖活性,流式细胞仪检测细胞凋亡率,RT-PCR检测Fas、FasL、Bcl-2、Bax、IL-2 mRNA表达水平,分光光度法测定caspase3酶活性,并观察活血化瘀中药对上述各项指标的影响.结果 活化T淋巴细胞于诱导18h后凋亡率明显增加,于诱导6h时未见FasL、Bax mRNA表达,Fas、Bcl-2 mRNA表达无明显变化;于诱导18 h后Fas、FasL、Bax mRNA表达升高,Bel-2 mRNA表达下降,caspase3活性增高.活血化瘀中药可降低T细胞凋亡,并可分别降低Fas、FasL、Bax mRNA表达,提高Bcl-2 mRNA表达,减轻easpase3酶活性.在活化诱导早期(6 h)促进T淋巴细胞内IL-2 mRNA表达,在晚期(18 h)减少IL-2 mRNA表达.结论 过度活化是脾脏T淋巴细胞异常凋亡的诱发因素,而凋亡的发生与Fas、FasL、Bax、Bcl-2 mRNA表达的改变有关.活血化瘀中药可通过调节IL-2及凋亡相关基因mRNA表达而减轻脾脏T淋巴细胞凋亡,同时可以促进T淋巴细胞的增殖活性.     

杂色曲霉素对小鼠脾细胞IL-2及IFN-γ分泌和表达的影

目的探讨杂色曲霉素(ST)对体外培养的小鼠脾细胞IL-2及IFN-γ mRNA表达及其蛋白分泌的影响.方法分别采用半定量RT-PCR及ELISA方法,研究5种不同剂量ST(0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L,2 mg/L)预处理对小鼠脾细胞IL-2及IFN-γ mRNA表达及其蛋白分泌的影响.结果不同剂量ST预处理2 h均可引起小鼠脾细胞IL-2及IFN-γ表达的改变,但不同剂量影响不同,小剂量ST处理组(0.125、0.25 mg/L)在ST处理后2 h,可诱导脾细胞IL-2及IFN-γ mRNA的表达;而大剂量ST处理组(1 mg/L、2 mg/L)可明显抑制脾细胞IL-2及IFN-γ mRNA表达及其相应蛋白的分泌,尤以ST 1 mg/L的抑制作用最明显.结论ST可影响小鼠脾细胞IL-2及IFN-γ表达和分泌的改变,较小剂量组(0.125、0.25 mg/L)表现为诱导作用,而较大剂量组(1 mg/L、2 mg/L)则表现为明显抑制作用.     

顺铂联合exosomes抗小鼠肝癌效应的实验研究

目的:评价顺铂(DDP)与exosomes联用的抗肿瘤效果,研究其可能机制.方法:采用MTT法检测顺铂对小鼠肝癌H22细胞增殖的影响,用H21源exosomes瘤苗免疫小鼠,3 H-TdR释放法检测exosomes诱导产生的CTL活性及顺铂对CTL杀伤的增敏作用,RT-PCR检测顺铂作用后H22细胞中Fas及exosomes免疫后脾淋巴细胞FasL的mRNA表达水平,Western blot检测顺铂对H22细胞Fas的蛋白表达水平的影响.以小鼠肝癌H22细胞接种BALB/c小鼠建立动物模型,观察顺铂联合exosomes治疗对小鼠生存期的影响.结果:顺铂抑制H22细胞生长呈量效关系;exosomes免疫小鼠可诱导产生针对H22细胞的CTL反应,经2.5 mg/L顺铂预处理24 h的H22细胞对CTL杀伤的敏感性增强(P＜0.05).顺铂在mRNA和蛋白水平,显著增强H22Fas的表达;exosomes免疫小鼠后,脾淋巴细胞FasL表达增加.顺铂联合exosomes组小鼠生存期较单独治疗组及对照组明显延长(P＜0.05).结论:顺铂与exosomes联合治疗有协同抑制肿瘤的作用,产生协同作用的机制与增强CTL活性有关.     

超抗原SEB体外诱导胸腺细胞凋亡的研究

目的：观察超抗原SEB能否在APC的辅助下体外诱导胸腺细胞凋亡，并研究其机制。方法：SEB作用于与APC混合培养的胸腺细胞，用DNA凝胶电泳、DNA片段测定等判定胸腺细胞凋亡并作定量分析；用间接免疫荧光法标记并以流式细胞仪检测Fas、FasL的表达。结果：SEB处理组胸腺细胞有凋亡表现且凋亡率明显高于对照组，Fas、FasL的表达明显高于对照组。结论：SEB可以于体外在APC辅助下诱导胸腺细胞凋亡，Fas、FasL的高表达可能在凋亡中起重要作用，这可能为体外研究胸腺细胞阴性选择过程提供一种方法。     

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.     

Uteroglobin in the developing rabbit conceptus in vivo and in vitro

Summary Uteroglobin (UGL) was measured in day- 4 to day-10 rabbit conceptuses by a competitive ELISA. Levels in blastocyst fluid, tissues, coverings and in the early fetus were determined separately. The total amount of UGL increased from 18.4 ng to 6.8 g per conceptus. The UGL content of individual day-6 blastocysts was studied in vitro. Culturing was carried out up to 60 h in Ham's F10 medium with polyvinylpyrrolidone as macromolecular component, with and without progesterone, and with progesterone plus estradiol. UGL was determined in the blastocyst fluids, tissues with coverings and in the culture media. After labelling with [³⁵S]-methionine, protein patterns of total blastocysts and of culture media were analysed by two-dimensional gel electrophoresis and fluorography. The morphology of cultured blastocysts was examined by electron microscopy. During 60 h of culture, the blastocysts expanded in diameter by 84%, and released 19% of their initial UGL content into the medium, independent of the hormonal substitution. Neither de novo synthesis, nor degradation of UGL was found: the protein remained unlabelled in fluorography, and its total quantity was not significantly different from that of non-cultured controls. Trophoblast, endoderm and embryoblast cells showed well preserved cell organelles and intercellular junctions, while the morphological differentiation of the germ layer was inhibited.