胰高血糖素样肽-1改善过氧化氢诱导的血管内皮氧化损伤

目的:探讨胰高血糖素样肽-1(GLP-1)对过氧化氢(H2O2)诱导的内皮细胞氧化损伤的保护作用及机制.方法:取人脐静脉内皮细胞(HUVECs),分为正常对照组、H2O2组、GLP-1组、H2O2+浓度10 nmol/L GLP-1组、H2O2+浓度100 nmol/L GLP-1组、H2O2+浓度1 000 nmol/L GLP-1组.采用荧光显微镜检测细胞内活性氧自由基(ROS)水平、观察细胞骨架F-actin变化;采用流式细胞仪检测细胞凋亡比率;Western Blotting检测磷酸化的雷帕霉素靶蛋白(p-m TOR)蛋白水平;实时荧光定量聚合酶链反应(QPCR)检测抗氧化酶:过氧化氢酶(CAT)、超氧化物歧化酶2(SOD2)和谷胱甘肽过氧化物酶1(GPX1)基因的表达变化.结果:H2O2组细胞骨架破坏明显,细胞内ROS含量增加,细胞凋亡率增加;加入GLP-1后,细胞骨架破坏受到显著抑制,ROS含量减少,凋亡率降低.Western Blotting结果显示,H2O2组p-m TOR表达降低,GLP-1可以提高p-m TOR表达,使蛋白水平趋于正常;加入GLP-1受体拮抗剂艾塞纳肽(9~36)后,GLP-1不能上调p-m TOR表达.H2O2作用后,CAT、SOD2和GPX1基因表达降低,而GLP-1可以逆转这些基因表达的降低.结论:GLP-1通过上调抗氧化应激酶对抗H2O2诱导的内皮细胞损伤,且可能是通过活化m TOR通路起保护作用.     

环境污染物PFOS可引起大鼠海马干细胞的增殖

目的:观察全氟辛烷磺酰基化合物(PFOS)大鼠海马干细胞增殖情况.方法:利用大鼠腹腔内连续注射不同剂量的PFOS 14天,建立动物模型.以Brdu作标记物,利用免疫组织化学染色的方法检测不同剂量PFOS作用下大鼠海马干细胞增殖情况.结果:以Brdu作标记物,高剂量PFOS作用下大鼠海马内可检测到大量阳性细胞存在,对照组和低剂量PFOS作用下无此阳性结果产生.结论:推测PFOS可引起大鼠海马干细胞的增殖.     

ANXA2对肝癌细胞F-actin表达及细胞微结构的调节

为了研究ANXA2对人肝癌细胞F-actin表达的调节,通过siRNA技术抑制肝癌细胞SMMC-7721的ANXA2表达,以免疫荧光法观察了F-actin表达及细胞微结构的变化.结果显示:抑制ANXA2基因表达后,细胞聚合成束的F-actin明显减弱,并由聚集分布变成弥散分布,且细胞的伪足、微绒毛等微结构生长受到抑制.结果提示,ANXA2表达水平的变化可能改变了SMMC-7721细胞F-actin的表达、聚合、重排与分布,从而导致细胞的细胞骨架重建,进而引起细胞的运动力与形态发生一系列变化.上述结果对进一步阐明ANXA2如何影响F-actin的动态组装以及ANXA2在细胞骨架系统重塑过程中的作用机制等提供了证据.     

La3+通过重组细胞骨架影响大鼠成骨细胞增殖、分化

研究了稀土离子La3 对体外培养的成骨细胞增殖、分化及细胞骨架的影响,并初步探讨了相关机理.用细胞计数法检测了成骨细胞的增殖.用RT-PCR技术测定了碱性磷酸酶(ALP)、骨钙素(OC)、骨桥蛋白(OPN),骨涎蛋白(BSP)以及cbfa-1 mRNA水平.采用激光扫描共聚焦显微镜观察了细胞中F肌动蛋白(F-actin)的变化.结果显示:La3 在48h促进了成骨细胞增殖;在4 d促进了早期分化指标ALP,BSP和cbfa-1 mRNA的表达;在21d促进了晚期分化指标OC和OPN mRNA的表达.与此同时,La3 使成骨细胞骨架发生重组.另外,Western Blot分析证实La3 作用于成骨细胞短时间即可激活粘着斑激酶(FAK)酪氨酸磷酸化.结果提示,La3 通过提高FAK酪氨酸的磷酸化水平,改变细胞骨架的分布和聚合,从而促进成骨细胞的增殖和分化.     

以鼠腹腔微环境研究三氧化二砷对食管癌EC109细胞骨架的影响

目的:细胞骨架微丝是与肿瘤细胞生长密切相关的因素之一,本文以生物机体腹腔为免疫的微环境,研究化疗药物三氧化二砷(As2O3)对人食管癌细胞株微丝骨架的影响.方法:利用鬼笔环肽(Phalloidin)及碘化丙碇(Propidium Iodide,PI)标记,以流式细胞仪技术分析小鼠腹腔液中食管癌EC109细胞周期的各期细胞内F-actin的变化.结果:诱导肥大细胞(mast cell,MC)迁移入腹腔的同时,迅速升高G0/G1期细胞及降低S期细胞内的F-actin含量,DNA检测结果显示G0/G1期的肿瘤细胞数量迅速增加(p<0.05),S期细胞含量降低;三氧化二砷作用后,食管癌细胞各期的F-actin含量均降低,尤其S期为甚.MC和As2O3共同作用后,食管癌细胞各期的F-actin含量均也减少,但G0/G1期细胞数量却显著增加.结论:在小鼠腹腔微环境中,免疫功能改变(免疫细胞MC的聚集)引起G0/G1期细胞数量迅速升高、S期细胞的数量降低,可能促使EC109细胞G0/G1期向S期跨越的延迟;在此环境中,As2O3也可能通过抑制S期EC109细胞内F-actin的重组来延迟细胞从G0/G1期进入S期;诱导肥大细胞迁入腹腔的同时加入药物As2O3,其作用主要表现为短期效应,促进了肿瘤细胞内F-actin含量的降低,G0/G1期细胞数量较高,出现短暂的延迟G0/G1期向S期跨越,增强了对肿瘤细胞生长的抑制作用.因此,以生物机体为研究环境,可能更真实地呈现化疗药物对肿瘤细胞的治疗效果.     

紫外光助零价铁活化过硫酸盐降解全氟辛烷磺酸盐

针对全氟辛烷磺酸盐(PFOS)去除,构建紫外光助零价铁活化过硫酸盐降解体系。考察了体系中零价铁粉浓度、过硫酸盐浓度和pH值对降解效果的影响。结果表明:紫外光和零价铁共同激活过硫酸盐产生硫酸根自由基(SO_4·~-)攻击水中PFOS,至反应24 h时,体系对PFOS的降解率达到91%;适度增加体系零价铁粉浓度、过硫酸钠浓度会提高体系对PFOS的降解率,但持续增加零价铁粉浓度、过硫酸钠浓度,不会持续增加体系对PFOS的降解率;酸性适度(pH=3,pH=4)条件下体系对PFOS的降解效果优于强酸(pH=2)和弱酸偏中性(pH=5,pH=6)条件下的降解效果。     

水杨酸、水杨酸甲酯对亚硝胺合成阻断作用研究

用紫外光解法研究水杨酸(SA)、水杨酸甲酯(MS)对亚硝胺合成的阻断效果,并与相同条件下抗坏血酸(Vc)进行对照,探讨浓度、pH值、反应温度、反应时间对SA、MS阻断亚硝胺效果的影响.研究结果表明:反应浓度为1.28 mmol/L、pH3.00、反应温度37℃、反应时间1 h时,SA、MS对亚硝胺的阻断率最高,阻断能力依次是:VcMSSA.     

Rho激酶对大鼠海马神经元骨架相关蛋白mRNA表达的影响

目的:探讨Rho激酶对大鼠海马神经元突起生长和骨架相关蛋白mRNA表达的影响.方法:用Rho激酶的抑制剂Y-27632和激动剂溶血磷脂酸(LPA)干预海马神经元,观察突起的生长并用RT-PCR检测大鼠海马神经元神经丝结合蛋白(Dbn1)、微丝肌动蛋白(F-actin)、微管相关蛋白(Tau)、α-微管蛋白(α-tubulin) mRNA的表达.结果:用LPA干预2 h后突起从远端逐渐退缩,长度缩短,细小突起退缩明显;Y-27632干预2 h后细胞胞体和突起的远侧端新生出细小突起.RT-PCR实际扩增长度与设计长度相吻合.内参照β-actin电泳条带在不同分组灰度较均一,呈高表达.Dbn1呈较高水平表达,激动剂LPA组表达下降(P<0.05),抑制剂Y-27632组表达量增多(P<0.05);F-actin和Tau表达呈低水平,激动剂作用后均使表达量相应的减少(P<0.05),抑制剂组表达增多(P<0.05);α-tubulin表达量最高,激动剂组表达量出现明显下降(P<0.05),抑制剂组表达增加(P<0.05).结论:激活Rho激酶下调Dbn、F-actin、Tau、α-tubulin mRNA的表达并诱导突起缩短,抑制则增加其表达并促进突起生长.     

稀土Gd~(3 )的掺杂对Sr_2CeO_4∶Eu中Eu~(3 )发光的影响

采用共沉淀方法,合成了Gd3 和Eu3 共掺杂的Sr2CeO4荧光体.当Eu3 浓度较小(掺杂浓度为2%)时,Gd3 离子对Sr2CeO4的蓝带发射及附着在其上面的Eu3 的特征跃迁起猝灭作用;而当Eu3 的浓度较高(掺杂浓度为8%)时,Gd3 离子对Sr2CeO4的蓝带发射及附着在其上面的Eu3 的特征跃迁起敏化作用,尤其是当Gd3 离子的掺杂浓度为3%时,Eu3 的5D0-7F2跃迁发射(615nm)增强为原来的132%.     

全氟辛烷磺酸对大型蚤的慢性毒性研究

采用大型蚤21天暴露和子代21天恢复实验法,研究了PFOS对大型蚤的慢性毒性效应及其子代F1(1st)和F1(3rd)的恢复情况。结果显示:PFOS质量浓度高于20mg/L时,F0代总产卵量、终点体长和内禀增长率都受到显著抑制。3个指标在最高浓度组与空白组相比,依次降低了62.2%,40.6%,34.0%(p<0.01)。随PFOS暴露浓度增加,其对大型蚤毒性逐渐加重,PFOS暴露浓度与对大型蚤毒性之间具有明显的剂量-效应关系。当PFOS质量浓度高于30mg/L时,F1(lst)代总产卵量和终点体长受到显著抑制。F1(3rd)代比F1(1st)代大型蚤恢复程度更好,除暴露于50mg/L的总产卵量、终点体长和内禀增长率仍受显著抑制,其他指标都恢复到接近空白组水平。大型蚤总产卵量、终点体长和内禀增长率作为最敏感的指标,建议将其用于评价PFOS的慢性毒性。     

全氟辛磺酸对于爪蟾的甲状腺激素干扰效应

运用两栖类变态实验(AMA)研究了全氟辛磺酸(PFOS)对于非洲爪蟾(Xenopus laevis)蝌蚪的生长、变态和甲状腺组织结构的影响.结果显示,与对照组相比,7d后,10,50和250μg.L-1PFOS暴露组蝌蚪的后肢长分别减小了17%,11%和18%,蝌蚪的发育延缓了1~2个阶段;19d后,10,50和250μg.L-1PFOS组蝌蚪的体重分别增加了17%,10%和20%,体长分别增加了5%,4%和6%,而10μg.L-1和250μg.L-1PFOS组蝌蚪的后肢长分别减小了12%和13%;暴露组甲状腺组织结构出现滤泡数目减少、上皮细胞增生、胶质减少甚至滤泡溶通等现象.结论是,在本实验浓度下,PFOS对于非洲爪蟾的蝌蚪生长有促进作用,对于变态发育过程却有抑制作用.参照AMA的方法,可以判断PFOS为抗甲状腺激素干扰物.     

In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte.

The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.     

河蚬(Corbicula fluminea)对氧化石墨烯和全氟辛烷磺酸类物质(PFOS)联合胁迫的生理生化响应

为了探索碳纳米材料氧化石墨烯(GO)和全氟辛烷磺酸类物质(PFOS)在淡水底栖贝类体内的联合毒性效应, 以河蚬为研究对象, 考察 1 mg/L GO和500 ng/L PFOS单独及联合暴露28天后对河蚬体长、体重、滤食率、活性氧水平、抗氧化系统酶活性和丙二醛含量的影响, 并采用优化的综合生物标志物响应指数(EIBR)进行整体评估。结果表明, 暴露结束后, 河蚬的体长和体重没有明显的变化。与空白对照组和溶剂对照组相比, GO和PFOS的单独暴露组及联合暴露组的滤食率均显著下降。在河蚬的鳃和内脏团中, GO和PFOS的胁迫都会引起抗氧化系统酶活性响应的显著变化, 且两器官中变化趋势一致。EIBR结果表明, 鳃和内脏团中联合暴露组的毒性比 PFOS或GO单独暴露组的毒性更强。     

Characterization of Perfluorooctane Sulfonate by Gas Chromatography/ Mass Spectrometry

With quantitative conversion to volatile derivatives,substance such as perfluorooctane sulfonate (PFOS) that is difficult to analyze can be separated by conventional techniques and then be characterized. In this paper,the methods and procedures in which perfluorooctane sulfonic acids were converted to corresponding sulfonic ester by reaction with triethylorthoacetate(TEOA) in the presence of solvent were described,and then PFOS was analyzed as ethyl ester derivatives. Mass spectra (MS) were generated using electron ionization (EI) mode. And the EI spectra of the volatile derivatives show ions characteristic of both the fluorinated hydrocarbon and the sulfonic ester portion of the molecules.     

微塑料和全氟辛烷磺酸类物质共暴露对翡翠贻贝滤食率和抗氧化系统的影响

为了探索微塑料和全氟辛烷磺酸类物质(PFOS)在海洋生物体内的复合污染毒性机制, 以翡翠贻贝为研究对象, 在聚苯乙烯微塑料(0.2 μm) 4.55×10⁸个/L以及PFOS浓度为10, 100和1000 μg/L的条件下, 研究PFOS或微塑料单独暴露以及二者复合暴露对翡翠贻贝滤食率、活性氧水平(ROS)、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、谷胱甘肽巯基转移酶(GST)活性和谷胱甘肽还原酶(GR)活性的影响。结果表明, 与溶剂对照组相比, 暴露于微塑料和PFOS后翡翠贻贝的滤食率均没有显著变化。在翡翠贻贝的鳃、内脏团和性腺中, 微塑料或PFOS的胁迫都会引起酶响应的显著变化。与PFOS单独暴露组相比, 微塑料和 1000 μg/L PFOS共同暴露时, ROS水平在鳃和性腺中显著提升, 在内脏团中显著下降; MDA含量在鳃和内脏团中显著提升, 在性腺中显著下降; GST和GR活性在鳃中显著下降, 在性腺中显著提高。研究结果说明微塑料会改变翡翠贻贝对PFOS的氧化应激响应。     

PFOS暴露对秀丽线虫寿长的影响及其与IIS相关基因的关联

以秀丽隐杆线虫(C.elegans)为模型,本文研究了全氟辛烷磺酸(PFOS)对机体寿长的影响及初步机理.结果显示0.2~200μmol/L的PFOS暴露50 h导致野生型秀丽线虫寿长呈剂量依赖性缩短.在4类转基因线虫上,观察到Insulin/IGF-l.1信号通路(IIS)相关的daf-16、daf-2和age-1基因突变或敲除能影响线虫的寿长.进一步观察PFOS暴露导致4类转基因线虫的寿长变化率,并与野生型线虫比较.在CF1139和CF1580突变种上daf-16或daf-2的突变均未改变PFOS的缩短寿长效应.而在CF1295和TJ1052转基因型上发现daf-16b的基因敲除或age-1基因突变阻断PFOS的减寿效应.结果表明PFOS慢性暴露能加速动物衰老,缩短寿长.PFOS作用与IIS信号通路关系密切,daf-16b和age-1基因在其中起重要作用.     

Characteristics of the binding features of Nelin with F-actin and screening Nelin interactive proteins

The interaction of nelin, a cardiac-specific expressed protein of human novel gene nelin, with F-actin was studied by both F-actin cosedimentation in vitro and colocalization assays. The results showed that nelin is a new F-actin binding protein and is colocolized with F-actin in cytoplasm of cells. Three new nelin binding proteins, filamin C subtype, titin N2B subtype and inter-alpha trypsin inhibitor heavy chain precursor (ITIH), were identified from human heart cDNA library using yeast two-hybrid screening. The binding activity of filamin C with nelin was confirmed by coimmunoprecipitation. Filamin C binds to nelin through its C-terminal region. It is indicated that nelin is a cytoskeleton associated protein and acts as a membrane-cytoskeleton associated protein involved in the formation of focal adhesions.     

Correlation between platelet gelsolin levels and different types of coronary heart disease

Gelsolin is an important cytoskeletal protein of platelets and studies have shown a close relationship between gelsolin and cardiovascular disease.However,the role of gelsolin in the development of coronary heart disease(CHD) is unclear.In this study,we record the distribution of gelsolin in human platelets and plasma and its association with different types of CHD.This study included 114 cases,with 33 stable angina pectoris(SAP) cases,81 acute coronary syndrome(ACS) cases—composed of 39 unstable angina pectoris(UAP) and 42 acute myocardial infarction(AMI) cases,and 31 healthy control participants.Gelsolin concentration in platelet rich plasma(PRP) and platelet poor plasma(PPP),actin filament(F-actin) and Gc-globulin of PPP were determined by enzyme-linked immunoadsorbent assay(ELISA).The fluorescence intensity of CD62p and cytoplasmic calcium([Ca2+] i) in human platelets measured by flow cytometry.We also used turbidimetry to detect the platelet aggregation rate(PAR).We analyzed the correlation between platelet gelsolin concentration and CD62p or plasma F-actin levels among each different patient group.Compared with the control group,the gelsolin level in PRP of UAP and AMI groups increased significantly(P<0.01),while the gelsolin level in PPP of all the three patient groups decreased markedly(P<0.01),and the CD62p,PAR,[Ca2+] i of platelets,F-actin and Gc-globulin of the UAP and AMI groups increased significantly(P<0.01).Compared with the SAP group,the gelsolin level in PRP,the PAR,[Ca2+] i of platelets and CD62p of other two groups increased significantly(P<0.01),F-actin of the AMI group increased markedly(P<0.01).Platelet cytoskeleton protein dynamics vary among the different types of CHD.Platelet gelsolin levels are markedly increased and accompanied by increased platelet activity,F-actin and [Ca2+] i of ACS patients,while gelsolin levels in PPP are markedly lower.Abnormally increased platelet gelsolin levels show high positive correlation with the level of platelet activity.Therefore,platelet gelsolin might be a novel molecular marker and/or a new potential therapeutic target of anti-platelet therapy of ACS.     

Perfluorooctane sulfonate （PFOS） and related fluorochemicals in chicken egg in China

The ubiquitous occurrence of perfluorinated compounds （PFCs） in environmental samples has drawn much attention. Recent human exposure studies found relatively high perfluorooctane sulfonate （PFOS） concentrations in blood samples from several cities in China when compared with other countries. The objectives of the present study were： （1） to measure PFC concentrations and compositions in chicken egg samples from local markets in China; and （2） to conduct a preliminary human health risk assessment of egg consumption. Eight pooled egg samples from eight locations were analyzed for 11 PFCs. The results showed that close to 100% of the PFOS in the egg was distributed in egg yolk and PFOS was not detected in egg white （〈0.08 ng/g wet weight, w/w）. Of the perfluoroalkylsulfonates, only PFOS was detected in all egg samples, while of the perfluoroalkylcarboxylates, perfluoroundecanoic acid （PFUnDA） was detected in all samples, followed by perfluorooctanoate （PFOA） （75% occurrence） and perfluorodecanoic acid （PFDA） （50% occurrence）. PFOS concentrations in egg ranged from 45.0 to 86.9 ng/g w/w. The results suggested that current concentrations of PFOS in domestic chicken eggs are unlikely to cause immediate harm to Chinese populations.     

人肽基脯氨酰顺反异构酶基因的克隆、表达、纯化及热变性交联

蛋白质分子间交联是普遍存在的现象．然而，蛋白质交联的分子机理还不太清楚．为了进一步探测蛋白质交联的分子机理，以及交联能否在异源肽链间发生，本实验室克隆了人肽基脯氨酰顺反异构酶(human Peptidylproly-Cis-trans-isomerase，hPPI)cyclophilin cDNA基因，并纯化出了PPI蛋白．最后，将PPI和．lysozyme蛋白进行热变性交联实验，结果显示在同源和异源肽链间都有二聚体和多聚体形成．并证实蛋白质交联可经三步完成：1)蛋白质构象包括二级结构改变；2)形成分子间二硫键；3)形成分子间异肽键．