EF延缓HPAT轴衰老的基因表达谱研究

目的：淫羊藿总黄酮(EF)延缓下丘脑-垂体-肾上腺-胸腺(HPAT)轴衰老的分子机制。方法：利用基因芯片技术，分别检测淫羊藿总黄酮组、右归饮组、桃红四物汤组、老年大鼠对照组及青年大鼠对照组下丘脑、垂体、肾上腺、脾脏淋巴细胞的基因表达谱。结果：老年大鼠和青年大鼠相比，HPAT轴多种神经递质、激素、细胞因子或其受体表达下调；EF组HPAT轴多种神经递质、激素、细胞因子或其受体表达上调；右归饮组及桃红四物汤组未见广泛的调节作用。结论：老年大鼠HPAT轴与生长、发育、衰老相关的基因表达以衰退的表现为主；EF能上调神经递质受体的表达并通过NEI网络的下行通路激活神经内分泌和免疫系统；通过下调促凋亡、抗增殖基因，上调抗凋亡、促增殖基因的表达，重塑淋巴细胞基因表达的平衡，延缓免疫衰老。     

枸杞多糖调控老年大鼠T细胞凋亡及相关基因表达的研究

目的 :探讨枸杞多糖对老年大鼠T细胞过度凋亡及相关基因表达的调节作用。方法 :利用TUNEL标记的流式细胞术和荧光实时定量PCR技术 ,研究了老年大鼠和年轻大鼠T细胞凋亡百分率及抗凋亡和促凋亡基因 (Fas,FasL ,TNFR1,Bax ,Bcl 2 ,TNFR2 )mRNA表达情况 ,并研究了枸杞多糖对老年大鼠T细胞凋亡百分率及促凋亡和抗凋亡基因mRNA表达的影响。结果 :枸杞多糖能够有效地降低老年大鼠T细胞的过度凋亡 ,而且可以下调促凋亡的TNFR1基因mRNA表达并上调抗凋亡的Bcl 2基因mRNA表达。结论 :枸杞多糖下调促凋亡基因表达的同时上调抗凋亡基因的表达 ,从而改善老年大鼠T细胞过度凋亡的状态     

早期凋亡的T淋巴细胞诱导生成耐受性树突状细胞的实验研究

目的： 探讨早期凋亡T淋巴细胞的抑制性免疫调节性能。方法： 采用定时紫外线照射诱导T淋巴细胞早期凋亡，深低温反复冻融获得坏死T淋巴细胞。体外诱导、纯化并培养骨髓源性不成熟树突状细胞（imDCs）,imDCs分别和早期凋亡或坏死T淋巴细胞共培养。用流式细胞仪、双夹心ELISA、［³H］掺入混合淋巴细胞反应等方法分析imDCs吞噬早期凋亡或坏死T淋巴细胞后，在不同处理条件下，MHC-Ⅱ、CD40、CD80、CD86的表达水平、分泌IL-12 p70以及刺激T淋巴细胞增殖能力的差异。结果： imDCs和坏死细胞碎片共培养后明显趋于成熟，其MHCⅡ和CD40、CD80、CD86的表达水平显著上调；分泌较高水平的IL-12 p70；和同种异体处女T淋巴细胞混合培养后显著刺激处女T淋巴细胞增殖。 imDCs和早期凋亡的T淋巴细胞共培养后,其MHC-Ⅱ、CD40、CD80和CD86的表达维持较低水平；仅分泌较低水平IL-12 p70；和同种异体处女T淋巴细胞混合培养后不能刺激淋巴细胞增殖。此外，早期凋亡的T淋巴细胞孵育上清显著抑制了吞噬坏死细胞碎片后的imDCs表达共刺激分子CD40、CD80、CD86。当TGFβ₁中和抗体和早期凋亡T淋巴细胞同加入imDCs，在表达MHC-Ⅱ、CD40、CD80、CD86，分泌IL-12 p70，刺激处女T淋巴细胞增殖等方面和吞噬坏死T淋巴细的DCs相比无显著差异。结论： 早期凋亡T淋巴细胞通过释放免疫抑制性细胞因子TGFβ₁，诱导imDCs呈现出耐受性DCs（TolDCs）的免疫表型及生物学特征，从而发挥抑制性免疫调节作用。     

芍药苷抑制佐剂性关节炎大鼠FLS 异常增殖

目的:研究芍药苷(PF)对佐剂性关节炎(AA)大鼠关节滑膜成纤维样滑膜细胞(FLS)异常增殖的影响。方法:采用完全弗氏佐剂足跖注射法制备AA大鼠,造模后第28天股动脉放血处死大鼠,分离培养FLS,大鼠足爪肿胀评分法检测PF灌胃治疗对AA大鼠的治疗作用,ELISA检测PF对AA大鼠FLS细胞因子IL-8表达的影响,MTT检测PF加入FLS培养液对AA大鼠FLS增殖的影响,Real time q PCR检测PF加药对AA大鼠FLS促凋亡基因Bax、凋亡因子Caspase 3、抗凋亡基因Bcl-2、RA病理基因Fibronectin表达的影响。结果:PF显著抑制AA大鼠足爪肿胀程度,PF加入FLS培养液后对FLS增殖具有一定的抑制作用,对AA大鼠FLS中Bax、Caspase 3表达具有显著上调作用,对Bcl-2、Fibronectin、IL-8表达具有显著抑制作用。结论:PF可能通过抑制AA大鼠FLS异常增殖抑制AA大鼠病理发展。     

胰岛淀粉样多肽对高糖刺激下INS-1细胞凋亡及其相关基因表达的影响

目的探讨高糖刺激下转染过表达人胰岛淀粉样多肽基因的大鼠胰岛素瘤细胞(INS-1)细胞凋亡的影响及其相关基因表达。方法应用酶联免疫吸附法(ELISA)检测高糖刺激后INS-1及转染人胰岛淀粉样多肽的INS-1细胞(h IAPP/INS-1)上清液的胰岛素水平;利用Hoechst33258染色检测细胞凋亡百分率;采用RTPCR方法检测高糖刺激培养后的Bcl2、Bax m RNA的水平。结果高糖刺激下,与未转染人胰岛淀粉样多肽INS-1细胞相比较,h IAPP/INS-1细胞胰岛素分泌功能显著下降,凋亡细胞比例明显增加,抗凋亡基因Bcl2 m RNA表达水平下调,促凋亡基因Bax m RNA表达水平上调,Bcl2/Bax比例明显降低。结论人胰岛淀粉样多肽上调促凋亡基因表达和抑制抗凋亡基因表达可能与糖尿病的发生发展相关。     

腺病毒介导的IL-24表达对PC-3人前列腺癌细胞体内外抑癌效应

目的:研究腺病毒介导的IL-24基因表达对前列腺癌细胞体内外的抑癌效应及分子机制。方法:将扩增的Ad-IL-24感染PC-3细胞,用RT-PCR和Western blot法检测IL-24基因在PC-3细胞中的表达;MTT法检测IL-24基因表达对PC-3细胞的生长影响;流式细胞术(FCM)检测细胞凋亡变化;RT-PCR法检测IL-24基因的表达对PC-3细胞中的Bcl-2、Bax、p53和Caspase3凋亡相关基因表达的影响。用Ad-IL-24在裸鼠PC-3移植瘤的瘤体内注射治疗,通过免疫组化法检测Bcl-2、Bax、Caspase-3、CD34等与细胞凋亡和血管形成相关因子的表达。结果:IL-24基因在PC-3细胞中成功表达,对PC-3细胞增殖有明显抑制作用,可上凋p53、Bax、Caspase-3基因和下凋Bcl-2基因表达,进而诱导细胞凋亡。Ad-IL-24能显著抑制裸鼠前列腺癌移植瘤生长,瘤重的抑制率可达54%;免疫组化结果显示Ad-IL-24不仅能明显上调Bax和Caspase-3基因和下调Bcl-2基因表达,而且能引起与血管形成标记基因CD34表达水平下降。结论:腺病毒介导的IL-24基因表达在体内外可明显抑制PC-3细胞的生长,诱导其凋亡。其机制可能与上凋P53、Bax、Caspase-3基因和下凋Bcl-2和CD34基因表达水平有关。     

具核梭杆菌诱导的肠上皮细胞Caco-2炎症反应及凋亡

目的研究具核梭杆菌诱导人肠上皮细胞(Caco-2)的炎症反应与凋亡,并初步探讨其机制。方法具核梭杆菌感染Caco-2细胞后,采用RT-PCR及ELISA检测促炎细胞因子IL-1β、IL-8和TNF-α的转录水平和蛋白水平的表达,利用透射电镜、流式细胞术、Western blot和RT-PCR等技术检测具核梭杆菌诱导的肠上皮细胞凋亡情况及促凋亡相关蛋白PARP剪切、BECN1、Caspase3剪切和AKT磷酸化的情况。结果具核梭杆菌感染人肠上皮细胞系Caco-2后,无论在转录水平还是蛋白水平,促炎细胞因子IL-1β、IL-8和TNF-α的表达均比对照组显著升高,其中感染6 h后促炎细胞因子表达最高。其次,具核梭杆菌能够诱导Caco-2细胞凋亡,其中感染6 h凋亡率最高;具核梭杆菌感染6 h后,凋亡通路相关蛋白PARP剪切、Caspase3剪切显著增加,BECN1显著上调,而抗凋亡蛋白AKT磷酸化则显著下调。结论具核梭杆菌能够诱导人肠上皮细胞的炎性反应和凋亡。     

脂肪源间充质干细胞治疗aGVHD分子机制的初步研究

目的初步探讨成人脂肪源间充质干细胞（adult adipose tissue-derived mesenchymal stem cells．AMSC）治疗急性移植物抗宿主病（acute graft-versus-host disease，aGVHD）的分子机制。方法3例行异基因造血干细胞移植术后发生aGVHD患者，以每公斤体质量2×10^6个细胞剂量静脉输注AMSC；应用RT-PCR扩增患者AMSC使用前后外周血单个核细胞的TCR Vβ 24个亚家族的CDR3，了解患者TCR Vβ T细胞的分布情况；应用尼龙毛柱分离外周血T淋巴细胞，再经CD8磁珠分选出CD8^＋ T淋巴细胞．应用流式细胞术检测发生aGVHD患者使用AMSC前后外周血CD8^＋T细胞亚群的变化。结果患者发生aGVHD时，有TCR Vβ3及其他亚家族基因表达，输注AMSC后，GVHD得以有效控制。Vβ3不表达；当患者GVHD复发时，Vβ3基因又表达，治疗后不表达；与输注AMSC前相比，输注AMSC后，CD8^＋T细胞中的CD8^＋CD28^-亚群显著上调（P〈0．05），同时．患者的aGVHD得以有效控制。结论AMSC治疗aGVHD的分子机制可能与其抑制TCR Vβ亚家族基因表达有关，TCR Vβ3可能是AMSC作用的靶基因；同时，AMSC治疗aGVHD的作用机制可能与其上调CD8^＋CD28^-T细胞亚群有关．CD8^＋T细胞可能是AMSC作用的靶细胞。     

GZMA、GZMB、TSP-1、TLR和IL-15条细胞凋亡通路基因在肝再生中的表达变化

目的探讨颗粒酶A(GZMA)、颗粒酶B(GZMB)、血小板反应蛋白-1(TSP-1)、Toll样受体(TLR)和白细胞介素-1(IL-1)5条细胞凋亡通路基因在肝再生(LR)过程中的表达变化。方法大鼠随机分为33组,每组6只,用Rat Genome 230 2.0芯片检测大鼠部分肝切除(PH)后不同恢复时间点5条细胞凋亡通路基因的表达情况,采用Real-time PCR和Western blotting技术对芯片结果进行验证,并用生物信息学方法对凋亡通路基因在肝再生中的表达变化进行分析。结果 Real-time PCR和Western blotting均与Rat Genome 230 2.0芯片的检测结果趋势一致;GZMA、GZMB、TSP-1、TLR和IL-1 5条细胞凋亡通路中9、8、24、31和34个基因与肝再生相关。它们主要在肝再生启动阶段起始表达,在细胞增殖阶段表达的基因数最多。表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调相近等5类,大多数基因表达加强,少数基因表达降低。它们表达的时间相关性分为13组。基因协同作用模型(Et)分析表明,GZMA介导的细胞凋亡通路在肝再生后期促进细胞凋亡;TLR介导的细胞凋亡通路几乎均在整个肝再生中促进细胞凋亡;GZMB、TSP-1和IL-1介导的细胞凋亡通路可能在肝再生中不发挥细胞凋亡作用。结论 GZMA和TLR两条通路调控肝再生中的细胞凋亡。     

SARI基因促进急性髓性白血病细胞凋亡机制的探讨

目的：通过慢病毒载体过表达SARI（Suppressor of AP-1,regulated by IFN）基因,探讨SARI基因促进急性髓性白血病（Acute myeloid leukemia,AML）细胞凋亡的分子机制。方法：构建SARI基因过表达的慢病毒载体,感染AML细胞HL-60和NB4,应用实时荧光定量PCR和Western blot对感染后细胞进行过表达的鉴定。两株AML细胞均设空白对照组（CON组）、空载体对照组（NC组）及SARI组,应用Western blot检测SARI过表达后两株AML细胞组凋亡相关蛋白Bcl-2、Bcl-xl、Bax和凋亡途径相关蛋白CytoC、Caspase8、Caspase9、Caspase3、Actived-Caspase3、PARP的表达变化。结果：SARI组细胞的SARI mRNA和蛋白表达水平均显著高于CON组和NC组（P＜0.05）,表明SARI过表达成功。SARI过表达的HL-60和NB4细胞中抗凋亡蛋白Bcl-2、Bcl-xl表达均下调,促凋亡蛋白Bax表达上调。CytoC表达增加,Caspase8、Caspase9、Caspase3剪切激活,Actived-Caspase3上调,PARP下调,PARP剪切体上调。结论：SARI基因促进AML细胞凋亡可能是通过激活外源性凋亡途径及与外源性凋亡途径交叉的内源性凋亡途径。     

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.