病毒巨噬细胞炎症蛋白-Ⅱ对载脂蛋白B mRNA编辑酶催化样蛋白3G和正常T细胞表达分泌的调节活化蛋白表达的影响

目的:明确病毒巨噬细胞炎症蛋白-II(viral macrophage inflammatory protein-II,vMIP-II)对HIV-1抑制因子:载脂蛋白BmRNA编辑酶催化蛋白3G(apolipoproteon B mRNA-editing catalytic polypeptide-3G,APOBEC 3G)和正常T细胞表达分泌的调节活化蛋白(regulated upon activation normal T expressed and secreted,RANTES)表达的影响。方法:构建vMIP-II真核表达载pEGFP-N3-vMIP-II,分别采用电穿孔和脂质体转染的方法将其转染至Jurkat和293T细胞。荧光定量PCR检测vMIP-II基因对Jurkat细胞和293T细胞内的抗艾滋病基因APOBEC3G和RANTES表达水平的影响。结果:测序结果显示成功构建了的vMIP-II真核表达载体,荧光显微镜观察估计转染效率达到50%左右。与空载体组相比较,转染pEGFP-N3-vMIP-II组的Jurkat细胞内的APOBEC3G和RANTES分别上调4.97倍和7.31倍,293T细胞内的APOBEC3G和RANTES分别上调为5.73倍和8.14倍。结论:vMIP-II基因不同程度的上调了Jurkat细胞和293T细胞内的抗艾滋病基因APOBEC3G和RANTES的表达。     

vMIP-Ⅰ激活Jurkat细胞抗-HIV基因表达的研究

本研究通过构建真核表达载体pEGFP-N3-vMIP-Ⅰ,电穿孔法将其转染至Jurkat细胞,荧光定量PCR检测vMIP-Ⅰ基因对Jurkat细胞内CCL5、APOBEC3G、APOBEC3F、等抗-HIV基因表达水平的影响,从而探讨vMIP-Ⅰ抗HIV感染的机制。结果显示:成功构建了pEGFP-N3-vMIP-Ⅰ载体,电穿孔转染效率达到40%左右,与转染空载体组相比,vMIP-Ⅰ转染组的Jurkat细胞内CCL5、A3G、A3F和MX1分别上调7.37倍、1.58倍、2.42倍和2.06倍。研究结果表明:vMIP-Ⅰ基因可激活Jurkat细胞内一些抗HIV相关基因的表达,这可能是vMIP-Ⅰ基因抗HIV感染的机制之一。     

vFLIP激活Jurkat细胞表达HIV抑制因子的研究

目的:检测KSHV编码的vFLIP蛋白对Jurkat细胞HIV抑制因子表达的影响。方法:本研究首先构建真核表达载体pIRES2-EGFP-vFLIP,再将其电穿孔至Jurkat细胞,然后使用实时荧光定量PCR技术检测Jurkat细胞中A3G、A3F、CCL5等HIV抑制因子的表达水平,从而探讨vFLIP抗HIV/AIDS的作用及机制。结果:成功构建了真核表达载体pIRES2-EGFP-vFLIP,电转染效率达到40%左右。与对照载体转染组相比,vFLIP转染组内Jurkat细胞中CCL5、A3G、A3F及Mx2基因的表达分别上调了6.71、2.20、1.52及14.47倍。结论:vFLIP蛋白可以激活Jurkat细胞内某些HIV抑制因子的表达,提示其具有一定的抗HIV/AIDS作用。     

RANTES在新疆卡波氏肉瘤中的表达及其与KSHV感染的相关性探讨

目的:探讨调节活化正常T细胞的表达与分泌因子(RANTES)在新疆卡波氏肉瘤(KS)组织中的表达及其与卡波氏肉瘤相关病毒(KSHV)感染的相关性.方法:利用免疫组化方法对新疆卡波氏肉瘤组织及正常皮肤中趋化因子RANTES进行蛋白表达水平的检测.利用感染KSHV的人脐静脉内皮细胞(HUVECs)作为卡波氏肉瘤研究的细胞模型,分别在含有佛波酯(TPA)和不含佛波酯的ECM培养基中培养.培养1d,3d,5d后各收取一批细胞,通过RT-PCR检测HUVEC细胞中趋化因子RANTESmRNA的表达水平.结果:KS切片中RANTES的阳性表达率明显高于正常皮肤组.感染KSHV的HUVEC,在含有TPA和不含有TPA的培养基中RANTES的表达都呈现出先低后高的变化趋势.5 d后,接受TPA刺激的HUVEC中RANTES表达显著上调.结论:RANTES在新疆卡波氏肉瘤组织中高表达,并且其表达受KSHV调节,感染细胞初期抑制RANTES的表达,之后促进其表达.     

人APOBEC-3F和-3G的克隆表达、亚细胞定位及其抑制HBV的研究

载脂蛋白B mRNA编辑酶催化多肽(apolipoprotein B mRNA-editing enzyme catalytic-polypeptide,APOBEC)家族成员,是近年来发现的具有抗病毒作用的天然免疫分子,对HIV和HBV等多种病毒具有抑制作用,为研究APOBEC的抗病毒作用,对APOBEC-3F和-3G进行克隆、表达及亚细胞定位分析．HBV主要的生物合成发生于细胞核中,利用核定位信号(NLS)及二联核定位信号(B-NLS)与APOBEC-3F和-3G融合表达,以进一步了解APOBEC-3F和-3G的亚细胞定位及功能．利用RT-PCR从植物凝集素(PHA)刺激的人外周血淋巴细胞RNA中,对APOBEC-3F和-3G进行克隆,利用HindⅢ和KpnⅠ双酶切插入到pcFlag载体中,脂质体转染MDCK细胞后利用免疫荧光检测APOBEC重组蛋白的亚细胞定位．电泳结果表明,RT-PCR扩增得到APOBEC-3F和-3G基因,双酶切鉴定结果表明,APOBEC真核表达载体构建成功,基因序列经DNA测序证实．免疫荧光结果表明,转染表达后的APOBEC-3F和-3G均定位于细胞胞浆中．APOBEC-3F和-3G可以在HepG2.2.15细胞中显著抑制HBV的复制．可以有效转运绿色荧光蛋白(GFP)入核的NLS及B-NLS,不能将APOBEC-3F和-3G有效地转运至核中．成功地对APOBEC家族中的两个重要成员APOBEC-3F和-3G进行了克隆、表达及亚细胞定位研究,为进一步利用APOBEC家族蛋白开发抗HIV及HBV药物提供基础．     

HIV-1感染对细胞内宿主因子TSG101及与Alix表达的影响

探讨HIV-1感染宿主细胞后对其宿主蛋白肿瘤易感基因101蛋白(Tumor Susceptibility Gene 101,TSG101)及ALG-2相互作用蛋白X(ALG-2-interacting protein X,Alix)表达的影响。以HIV-1感染性克隆病毒pNL4-3感染TZM-bl PM1、Jurkat细胞株和人外周血单个核细胞(PBMCs),感染24h后收获细胞提取总RNA,逆转录PCR检测在RNA水平各因子的表达差异;感染48h后收获细胞提取总蛋白,Western-blot检测各因子在蛋白水平的表达差异。结果显示:HIV-1感染对原代PBMC与细胞系表达Alix与TSG101影响显著不同,细胞系主要表现为下调,而原代PBMC主要表现为TSG101上调;细胞系中的下调又细分为Jurkat细胞的Alix与TSG101的双下调、TZM-bl细胞的Alix单下调以及PM1细胞无影响三种情况。HIV-1感染对细胞宿主分子TSG101及Alix在RNA和蛋白水平的表达均有影响,这种影响因细胞的不同而有差异。HIV-1感染调节Alix与TSG101的机制生物学意义尚有待于进一步阐明。     

人类免疫缺陷病毒1型与细胞间的相互作用

自从发现人类免疫缺陷病毒1型(HIV-1)是引起获得性免疫缺陷综合征(AIDS)的病原体后,人们对HIV-1与人体相互作用的过程进行了深入研究.通过研究发现了HIV-1与机体相互作用的多种机制,例如HIV-1主要侵犯人体以CD4 T细胞为主的表达其结合表位(如CCR5和CXCR4)的免疫活性细胞[1].目前研究者在正常机体内发现多种物质与HIV-1致病有关.例如APOBEC蛋白(人体内主要为APOBEC3G),当HIV-1侵入机体后,该蛋白表达减少,这一过程在HIV-1的致病过程中发挥重要作用.通过对这些蛋白或分子的研究,进一步揭示了HIV-1的致病机制,为治疗HIV感染/AIDS提供了新思路.同时不同的HIV-1感染细胞模型的构建为AIDS的研究提供了多种工具.     

APOBEC3G与Vif在HIV-1感染中的作用

载脂蛋白B mRNA编辑催化多肽样（apolipoprotein B mRNA-editing catalytic polypeptide-like,APOBEC）蛋白是一组胞嘧啶脱氨基酶,具有天然的抗病毒活性,对多种病毒具有抑制作用,特别是逆转录病毒. APOBEC3蛋白能够抑制人类免疫缺陷病毒(HIV-1)的感染,其中APOBEC3G和APOBEC3F的作用最强. APOBEC3G能够通过胞嘧啶脱氨基作用和非胞嘧啶脱氨基作用抑制病毒感染. HIV-1病毒感染因子(Vif) 蛋白主要经泛素-蛋白酶体途径介导APOBEC3G降解,从而拮抗其抗病毒作用. APOBEC3G和Vif之间相互作用的研究对于寻求新的抗HIV治疗靶点具有重要意义.     

APOBEC3F的克隆、真核表达及其抗HBV效应的初步研究

目的克隆、体外真核表达载脂蛋白BmRNA编辑酶催化多肽样3F（APOBEC3F）基因并研究其抗病毒效应。方法应用RT—PCR的方法从人PBMC细胞中扩增APOBEC3F基因,构建HA—APOBEC3F融合蛋白真核表达载体pXFA3F,pXFA3F和具有复制能力的1．3倍HBV载体pHBVl．3共转染HepG2细胞,Western印迹检测APOBEC3F融合蛋白在HepG2细胞中的表达,ELISA方法检测细胞培养上清液中的HBsAg和HBeAg水平,实时定量PCR检测HBV相关mRNA水平变化。结果克隆了APOBEC3F基因,其经测序与GeneBank公布序列一致;构建的APOBEC3F真核表达载体pXFA3F经转染可在HepG2细胞中瞬时表达;APOBEC3F可抑制乙型肝炎表面抗原和e抗原的分泌,可使转染细胞内HBV相关mRNA水平下降。结论成功克隆并真核表达了APOBEC3F基因,其在体外具有抗HBV生物学效应。     

反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的表达

目的:利用反转录病毒载体构建猪载脂蛋白B mRNA编辑酶催化多肽样蛋白(APOBEC)3F重组质粒,并实现其在猪肾细胞PK15中的表达。方法:用RT-PCR方法扩增五指山猪来源的外周血淋巴细胞APOBEC3F基因,将其定点插入反转录病毒载体pMSCV neo中,同时于插入位点两侧分别添加FLAG和GFP标签,构建重组质粒pMSCV-FLAG-A3F-GFP,并进行酶切、测序鉴定;将鉴定正确的重组质粒与pVSV-G、pGag-Pol共转染包装细胞HEK293T,分别于转染后48~72 h收集细胞的培养上清以获得假型病毒粒子;用该假型病毒感染猪源细胞PK15,通过PCR、Western印迹检测目的基因的整合及表达。结果:PCR扩增到1254 bp的猪APOBEC3F基因,重组质粒pMSCV-FLAG-A3F-GFP经酶切、测序,结果无误;3质粒共转染HEK293T细胞包装出的假型病毒感染PK15细胞后观察到GFP表达;从感染假型病毒的PK15细胞基因组中扩增到1254 bp的猪APOBEC3F基因,Western印迹检测到78.1×103的猪APOBEC3F蛋白的表达。结论:实现了反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的整合与表达,为深入研究该分子对猪内源性反转录病毒(PERV)的抑制作用奠定了基础。     

NPC1L1:固醇脂质吸收的关键蛋白质

NPC1L1是最近发现的一种与NPC1同源的蛋白质。在体内的分布有物种差异性,其亚细胞定位存在很大争议。近些年发现NPC1L1在固醇类脂质代谢途径中起着重要作用,是肠道吸收固醇类脂质尤其是胆固醇的关键蛋白质,这项新发现使得人们对固醇类脂质的吸收机制有了了解。高胆固醇血症是心血管系统疾病的一个高危因子,因此,对NPC1L1的研究具有重大的实际意义,正逐渐成为研究的热点。     

Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels

Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K(+) Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 alpha-subunits. We found that homomeric channels, formed by monomeric or dimeric alpha-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory subunits.     

Variation in salinity tolerance and shoot sodium accumulation in Arabidopsis ecotypes linked to differences in the natural expression levels of transporters involved in sodium transport

Salinity tolerance can be attributed to three different mechanisms: Na⁺ exclusion from the shoot, Na⁺ tissue tolerance and osmotic tolerance. Although several key ion channels and transporters involved in these processes are known, the variation in expression profiles and the effects of these proteins on Na⁺ transport in different accessions of the same species are unknown. Here, expression profiles of the genes AtHKT1;1, AtSOS1, AtNHX1 and AtAVP1 are determined in four ecotypes of Arabidopsis thaliana. Not only are these genes differentially regulated between ecotypes, the expression levels of the genes can be linked to the concentration of Na⁺ in the plant. An inverse relationship was found between AtSOS1 expression in the root and total plant Na⁺ accumulation, supporting a role for AtSOS1 in Na⁺ efflux from the plant. Similarly, ecotypes with high expression levels of AtHKT1;1 in the root had lower shoot Na⁺ concentrations, due to the hypothesized role of AtHKT1;1 in retrieval of Na⁺ from the transpiration stream. The inverse relationship between shoot Na⁺ concentration and salinity tolerance typical of most cereal crop plants was not demonstrated, but a positive relationship was found between salt tolerance and levels of AtAVP1 expression, which may be related to tissue tolerance.     

人骨髓间充质干细胞向神经细胞分化过程中Notch通路信号分子表达的变化

本研究探讨Noah信号通路在人骨髓间充质干细胞（human mesenchymal stem cells,hMSCs）体外增殖及向神经细胞分化过程中的作用。采集健康自愿者骨髓,体外培养获得hMSCs,取第3代hMSCs,在诱导剂（β-ME,DMSO,BHA）作用下向神经细胞分化。诱导后用免疫细胞化学鉴定神经元特异性烯醇化酶（neuron-specific enolase,NSE）和尼氏体的表达以确定诱导效果：用流式细胞术检测细胞生长周期时相的变化。在诱导前后,用免疫荧光和RT-PCR方法检测Notch通路中Notch1受体蛋白、配体Jagged1（JAG1）、调节蛋白活化相关物早老素1（presenilin 1,PS1）、靶基因hairy and enhancer of split1（HES1）信号分子表达的变化。结果显示：诱导前,处于G0/G1期的hMSCs占58．5％,S＋G2/M期的细胞占41．5％;诱导后,G0/G1期细胞比例升高,而S＋G2/M期细胞比例下降,NSE阳性细胞率达（77±0．35）％,细胞质中可见深蓝色的块状或颗粒状尼氏体。免疫荧光显示,诱导前后hMSCs内Notch1和JAG1均呈阳性表达,但RT-PCR检测发现诱导后Notch1、JAG1、PSl和HES1 mRNA表达量较诱导前明显降低（均P〈0．05）。结果表明,诱导hMSCs向神经细胞分化能抑制Notch信号分子表达,低水平的Notch信号激活可能有利于神经细胞的分化。     

Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

SIRT1 is an NAD⁺-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1.     

Enhancement of hepatic microsomal drug oxidation and glucuronidation in rat by 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT)

A single dose of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (160 mg/kg i.p.) enhanced the monooxygenase step of drug biotransformation in rat liver. The O-demethylation of p-nitroanisole was especially increased, a peak in activity approximately 5-fold compared with controls being attained in 7 days. On the other hand, there was only a 2-fold increase in aryl hydrocarbon hydroxylase activity.DDT increased the cytochrome P-450 content of the liver, this increase coincided well with that in p-nitroanisole O-demethylation activity.The UDPglucuronosyltransferase activity of liver microsomes was not enhanced by DDT administration, unless the microsomes were pretreated to reveal latent activity prior to assay. After trypsin digestion of microsomes a maximum increase in activity of approximately 3-fold was observed as a result of DDT dosage. The canonic surfactant cetylpyridinium chloride was less active in revealing the latent UDP-glucuronosyltransferase activity, and two other membrane perturbants, the detergent digitonin and phospholipase A, were unable to show enhancement in UDPglucuronosyltransferase as a result of DDT dosage.     

CYP1A1基因单核苷酸多态性与肺癌的相关性研究

目的:探讨代谢酶CYP1A1基因MspI位点多态性与新疆汉族人群肺癌遗传易感性之间的相关性.方法:应用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)技术检测59例新疆汉族肺癌和84例新疆汉族健康人的CYP1A1基因MspI位点多态性分布频率,并分析了CYP1A1基因MspI位点多态性与新疆汉族人群肺癌遗传易感性和患者性别之间的相关性.结果:(1)CYP1A1基因MspI位点3种多态基因型分布频率在两组间比较差异有统计学意义(χ2=6.682,P=0.035),CC基因型在病例组的分布频率显著高于正常对照组.(2)携带突变CC基因型的个体较携带TT基因型的个体患肺癌的危险性增加(OR=3.759.95%CI=1.228-11.494,P=0.035).(3)男女肺癌患者的CYP1A1基因MspI位点基因型及等位基因频率的差异均无显著性(P>0.05).结论:(1)CC突变基因型可能是新疆汉族人群的肺癌易感因素.(2)CYP1A1基因MspI位点多态性可能与新疆汉族肺癌患者的性别无关.     

粘膜系统鳞状细胞癌(SCC)相关蛋白(DCUN1D1)的表达、纯化和晶体生长

目的:SCCRO/RP42/DCUN1D1是粘膜系统鳞片状细胞癌(SCC)发生时人类基因组3q区域扩增的潜在靶标之一,其蛋白作用机制尚不清楚,本文拟通过表达并大量纯化SCC相关蛋白DCUN1D1用于蛋白结晶以求获得其三维结构。方法:使用人肝脑组织RNA反转录产物为模板扩增出DCUN1D1基因cDNA片断并将其克隆至原核表达载体PGEX-6P-1中,通过IPTG诱导获得大量可溶性表达,再经过GST亲和层析和Sephadex G-200层析柱纯化。结果:获得了纯度95%以上的蛋白,采用悬滴气相扩散法筛选蛋白晶体,获得显微镜下可见的微晶。结论:初步得出DCUN1D1晶体生长条件及范围,为解析DCUN1D1的三维结构并进一步认识其生物功能奠定了基础。