共表达不同水平的IL-12对于HBV DNA疫苗质粒免疫原性的影响

目的 在HBsAg DNA疫苗质粒中共表达IL-12佐剂分子,研究IL-12的表达水平对于HBV DNA疫苗质粒免疫原性的影响.方法 构建携带来源于中国地区C型HBV参照序列CHN-HBV07-C经密码子优化的preS2-S基因的DNA疫苗质粒pHBV,并将3个不同IL-12表达水平的佐剂分子表达盒序列分别克隆入该疫苗质粒中,通过瞬时转染293T细胞以检测重组质粒IL-12分子及乙肝表面抗原的表达情况.将不同IL-12表达水平的疫苗质粒免疫BALB/c雌性小鼠,IFN-γ的ELISPOT方法检测HBsAg抗原特异性的细胞免疫应答,化学发光定量ELISA法检测HBsAg特异的抗体水平.结果 成功构建3个不同IL-12表达水平的HBV DNA疫苗质粒,293T细胞转染结果显示:不携带IL-12分子表达盒的对照疫苗质粒pHBV的HBsAg表达水平可达70 ng/ml;低表达IL-12的疫苗质粒pHBV-12l的HBsAg表达水平为18 ng/ml;而高表达IL-12的重组疫苗质粒pHBV-12h的HBsAg表达水平仅为6 ng/ml.BALB/c小鼠的免疫结果表明:高表达IL-12分子的疫苗质粒pHBV-12h所诱导的细胞免疫及体液免疫水平相对于对照疫苗质粒pHBV均显著降低了.低表达IL-12分子的疫苗质粒pHBV-12l所诱导的抗体水平也显著降低了,但其细胞免疫应答水平却显著提高了.结论 在同一疫苗质粒中,高表达IL-12分子的质粒可能会影响到抗原蛋白的表达,而低表达IL-12分子的疫苗质粒却能增强细胞免疫应答.因此,平衡好佐剂分子及抗原蛋白的表达水平对于诱导高水平的免疫应答是个重要的因素.     

pVR-IL15基因佐剂对表达HBsAg重组病毒免疫效果的影响CSCD

目的 探索pVR-IL15基因佐剂在表达HBsAg病毒治疗型疫苗的不同免疫策略中对免疫应答的影响.方法 采用了携带乙肝病毒S基因片段的DNA、重组Ankara痘病毒和重组腺病毒序贯免疫策略免疫BALB/c小鼠,在序贯免疫策略的基础上联合pVR-IL15免疫.用ELISA和IFN-γELISPOT方法检测不同免疫组别中小鼠的细胞免疫和体液免疫反应强度.结果 联合pVR-IL15免疫组的体液免疫应答水平和细胞免疫应答水平均显著高于无联合pVR-IL15免疫组,差异具有统计学意义.结论 pVR-IL15基因佐剂能够增强表达HBsAg重组病毒诱导的小鼠免疫应答.     

结核分枝杆菌MPT64重组母牛分枝杆菌免疫学特性

目的:研究结核分枝杆菌MPT64重组母牛分枝杆菌疫苗免疫学特性.方法:用分泌表达MPT64的重组母牛分枝杆菌疫苗免疫BALB/c小鼠,ELISA法检测免疫小鼠的特异性抗体滴度和抗体亚类.分离免疫小鼠脾淋巴细胞,检测淋巴细胞增殖、IFN-γ和IL-12产生水平、CD4+细胞和CD8+细胞数、脾淋巴细胞特异性CTL杀伤效应.毒株攻击后对脾脏细菌负荷计数.结果:MPT64重组母牛分枝杆菌疫苗免疫可诱导小鼠高水平的体液免疫应答,免疫小鼠脾淋巴增殖明显,IFN-γ和IL-12含量增加,CD4+和CD8+细胞百分比明显增加,CTL杀伤效应明显,对MTB H37Rv攻击后有一定的保护作用.结论:MPT64重组母牛分枝杆菌疫苗可诱导小鼠有效的体液和细胞免疫应答,有可能作为新型TB疫苗候选.     

重组弓形虫棒状体蛋白5诱导小鼠的免疫应答及其抗弓形虫感染的保护作用

为探讨重组弓形虫棒状体蛋白5诱导的免疫应答及其免疫保护效应,本研究克隆表达了弓形虫棒状体蛋白5,并分析其诱导的细胞免疫、体液免疫应答和抗弓形虫感染的保护作用。采用PCR方法从刚地弓形虫cDNA中扩增出ROP5基因片段,将该基因片段克隆至pET-28a原核表达载体表达,用重组ROP5蛋白免疫BALB／c小鼠,ELISA检测免疫后小鼠抗体和细胞因子的变化。以强毒型RH株弓形虫攻击感染免疫小鼠,统计小鼠不同时间存活率,评价重组蛋白产生的免疫保护性。结果表明ROP5重组蛋白疫苗免疫BALB／c小鼠诱导机体产生高水平的IgG、IgG1、IgG2a抗体和IFN—y、IL-2及IL-10细胞因子。与PBS及佐剂对照组相比,重组蛋白免疫组小鼠的存活时间明显延长。本实验证实了ROP5重组蛋白免疫BALB／c小鼠能够诱导其产生高水平的体液免疫和细胞免疫应答,以及一定的抗弓形虫感染保护作用。     

IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠(H-2d)特异性免疫应答的影响

目的：观察IL-12和IL-18基因免疫对HBcAg核酸疫苗诱导小鼠（H-2d）特异性体液免疫和细胞免疫应答的影响.方法：用肌肉注射法将HBV核心区DNA疫苗、IL-12质粒和IL-18 质粒接种BALB/c小鼠;ELISA法检测小鼠血清抗-HBc（IgG）及IgG亚类（IgG1、IgG2a）;LDH释放法检测小鼠脾细胞HBcAg特异性CTL活性.结果：免疫6周后,HBcAg DNA疫苗联合IL-12质粒、IL-18质粒和IL-12＋IL-18质粒组小鼠的血清抗HBc终点滴度均明显高于单纯注射HBcAg DNA疫苗组小鼠（P＜0.05）,抗HBc IgG亚类以IgG2a占优.DNA疫苗免疫的各组小鼠,HBcAg特异性细胞毒性T淋巴细胞杀伤率均高于对照组（P组）,其中C＋IL-18组和C＋IL-12＋IL-18组中CTL值明显高于C组,尤以C＋IL-12＋IL-18组中的CTL杀伤率最高.结论：IL-12和IL-18基因与HBcAg DNA疫苗联合免疫,不仅能增强HBcAg特异性体液免疫应答,而且能增强HBcAg特异性CTL的杀伤活性.     

结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察

目的:构建编码结核分枝杆菌Ag85A分泌蛋白重组真核表达质粒,研究其与hIL-12联合免疫小鼠后的细胞免疫应答。方法:(1)构建质粒:采用PCR法从H37Rv菌株中扩增Ag85A编码基因,用限制性内切酶消化后,插入克隆载体PMD20-T中,经酶切鉴定与序列测定证实后,以亚克隆法构建于真核表达载体PCDNA3.1的相应酶切位点。(2)动物实验:50只C57BL/6N小鼠随机分为:①Ag85A基因疫苗+hIL-12质粒组(联合免疫组);②重组Ag85A基因疫苗组;③卡介苗BCG组(阳性对照);④空载体组(阴性对照);⑤PBS组(空白对照)。基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106CFU BCG免疫1次,约0.3 ml/只。第三次免疫小鼠后28天,处死各组小鼠,分离脾细胞,ELISA法检测脾细胞培养上清液中IFNγ-、IL-2、IL-4水平;乳酸脱氢酶释放法检测脾细胞杀伤活性;分离的脾细胞经TB-PPD刺激后,XTT比色法检测脾淋巴细胞增殖活性。结果:(1)成功构建结核分枝杆菌Ag85A基因DNA疫苗。(2)联合免疫组能诱导较强烈的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-r和IL-2水平显著高于Ag85A基因疫苗组,与BCG组相当,IL-4分泌减少;特异性CTL杀伤活性明显增强;淋巴细胞增殖活性也明显高于其他组别。结论:hlL-12表达质粒能够增强结核分枝杆菌Ag85A基因DNA疫苗所诱导的小鼠免疫应答。     

铝佐剂吸附CpG ODN增强重组乙肝疫苗免疫效应

目的探索新佐剂CpG ODN在重组乙肝疫苗中的免疫效应及吸附状态。方法 CpG ODN佐剂与重组乙肝疫苗混合免疫小鼠,收集小鼠血清检测。通过免疫小鼠后的体液和细胞免疫应答水平来评价双佐剂乙肝疫苗对重组乙肝疫苗的增强作用。CpG ODN佐剂与乙肝疫苗在不同温度条件下混合吸附,计算在不同吸附时间下CpG ODN佐剂在乙肝疫苗中的吸附率。结果CpG ODN佐剂可以提高重组乙肝疫苗在体液及细胞中的免疫效果。CpG ODN佐剂在室温条件下1 h即可在乙肝疫苗中达到吸附平衡,于2~8℃放置5 d的条件下其吸附率约在50%左右。结论 CpG ODN能够增加乙肝疫苗的免疫应答,其免疫效果可提高5倍以上。     

IL-12对结构优化的HBV核心抗原DNA疫苗诱导免疫应答的影响

目的 探讨IL-12对一种结构优化的HBV核心抗原(HBcAg)DNA疫苗免疫效果的影响。方法 将小鼠IL-12基因插入结构优化的DKA疫苗pST-HBc，构成pST-HBc/IL12，将pST-HBc／IL12转染COS7细胞，ELISA检测培养上清中的HBcAg和小鼠IL-12。分别将pST-HBc／IL12和pST-HBc肌肉注射接种BALB／c小鼠，检测小鼠的体液和细胞免疫应答。结果 ELISA检测到培养上清液中的HBcAg和小鼠IL-12，pST-HBc／IL12诱导的抗-HBc总IgG阳转率和抗体水平不及pST-HBc，但其诱导的脾细胞增殖反应和细胞毒性T淋巴细胞反应均强于pST-HBc。结论 IL-12可进一步增强这种HBcAgDNA疫苗诱导的细胞免疫应答。     

甲乙肝联合疫苗中不同配比对小鼠Th1/Th2型细胞因子变化的影响

目的 通过注射甲乙肝联合疫苗和单价甲肝、乙肝疫苗来观察小鼠在免疫过程中细胞因子的变化情况,衡量应答的性质,探讨两种抗原对小鼠细胞因子变化的不同作用以及乙肝抗原对甲肝抗原的作用.方法 选用150只 Balb/c 小鼠随机分成注射联苗 640 Eu/mL:10 μg/mL组、640 Eu/mL:20μg/mL组、单价甲肝抗原640 Eu/mL组、单价乙肝抗原10μg/mL组、单价乙肝抗原20μg/mL组和佐剂AL(OH)3,对照组,每组25只.于第4周加强免疫1次,分别在初次免疫注射后第1、2、3、4周及加强免疫后第2周,眼眶采血,分离血清,每组各5只小鼠,上流式细胞仪检测小鼠血清5种细胞因子(TNF、IFN-γ、IL-5、IL-4、IL-2)含量.结果 疫苗诱导产生的Th1/Th2型细胞因子均随时间的变化而变化,5种细胞因子均约在第3周达最大值,其中小鼠产生最多的是TNF;加强免疫后,640 Eu/mL:20 μg/mL联苗组刺激小鼠产生细胞因子水平高于640 Eu/mL:10μg/mL联苗组.结论 甲乙肝联合疫苗能刺激机体产生特异性细胞免疫应答,甲乙肝联合疫苗能诱导机体产生与单价相同或略强的Th1/Th2型细胞因子.     

双歧杆菌完整肽聚糖对重组乙肝表面抗原细胞免疫应答的影响

目的:探讨双歧杆菌完整肽聚糖(BWPG)对重组乙肝表面抗原(rHBsAg)诱导细胞免疫应答的影响。方法:以BWPG作为rHBsAg的佐剂免疫BALB/c小鼠,观察免疫小鼠的生长状态及中枢淋巴器官的发育情况,检测NK细胞和CTL杀伤活性的变化,脾淋巴细胞的增殖和细胞因子的产生。结果:免疫小鼠未出现可见的毒副反应,胸腺和脾脏组织呈增生状态,脾脏NK细胞和CTL的杀伤活性增强,脾淋巴细胞体外增殖活跃,细胞因子产生增多。结论:BWPG能促进中枢淋巴器官增生,增强机体对rHBsAg的细胞免疫应答。     

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.