Treatment on Adjuvant Arthritis Rat Model by DcR3 and Its Mechanisms

类风湿性关节炎(RheumatoidArthritis，RA)是一种慢性、全身性的炎性自身免疫病。细胞因子、Th1/Th2细胞平衡、细胞凋亡等在RA的发病过程中起着重要作用。人诱骗受体(DcR)3是新近发现的TNFR超家族成员之一。DcR3能促进T细胞向Th2细胞转化及促进经TRAIL途径的细胞凋亡，对一些自身免疫性疾病如系统性红斑狼疮(SLE)有一定的治疗作用。到目前为止，DcR3对RA作用的研究尚未报道。本文探讨了DcR3对佐剂型关节炎（AA）大鼠模型的治疗作用及其相关机理。 采用重叠PCR技术获得了DcR3基因，构建原核表达载体pET-22b(＋)/DcR3，通过优化其表达条件，实现目...Rheumatoid arthritis (RA) is a chronic inflammatory disorder. Cell cytokine, Th1/ Th2 cell balance and cell apoptosis have an important effect on RA, but the complete etiology of RA has not yet been clarified. Decoy receptor 3 (DcR3) is a novel member of the TNF receptor superfamily. DcR3 can stimulate T cells to transform into Th2 cells, and some kinds of cells to generate apoptosis induced by TR...学位：理学硕士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：20042609

Functional Analyses of Dnt1 as an Inhibitor of Dma1 in Early Mitosis in Fission Yeast

人类肿瘤抑制因子Chfr是一种E3泛素连接酶，当细胞受到不良条件刺激(如微管解聚药物等)时，Chfr可以阻止细胞进入有丝分裂。然而，人们目前还不清楚Chfr的功能是否也受到调控。Dma1是Chfr在裂殖酵母中的同源蛋白，两者在序列上存在一定的相似性。已有的研究表明，Dma1在有丝分裂晚后期负调控有丝分裂的退出(mitoticexit)和胞质分裂(cytokinesis)。最近，为了进一步研究Dma1的功能，我们实验室通过串联亲和纯化(tandemaffinitypurification，TAP)的方法在裂殖酵母细胞中纯化了Dma1蛋白复合物，并配合质谱分析的方法鉴定出与Dma1一起被纯化的蛋白...The human tumor suppressor Chfr is an E3 ubiquitin ligase required to block mitotic entry in response to mitotic stress. However nothing is known about how the activity of Chfr is regulated. Previous studies showed that Dma1, the fission yeast potential functional relative of Chfr, acts in late mitosis as a negative regulator of mitotic exit and cytokinesis. Recently, our laboratory has identified...学位：理学博士院系专业：生命科学学院生物医学科学系_细胞生物学专业学号：2162007015381

ニチジョウゴ トシテノ ヒニク ハツワ ニツイテノ ジッショウテキ ケンキュウ : シロウト リロン ニ モトズイタ アタラシイ ブンルイホウ ノ テイアン シャカイ ガクブ カイコウ 30シュウネン キネン ロンブンシュウ

皮肉は伝統的には言語学者などの専門家の定義に基づいて議論されることが多いが，専門家ではない一般の人々の思う皮肉はそれとは必ずしも同一のものであるとは限らない。しろうと理論（Furnham, 1998）に基づき，大学生の考える皮肉発話を質的研究により分類し，ボトムアップ方式での皮肉の分類法を提案し，先行研究と比較する。調査参加者は80名の大学生で，皮肉が発話されたエピソードを自由に記述してもらった。その文字データを研究者がKJ法により分類し，合計で9つのカテゴリが抽出された。また，先行研究と同様に，皮肉発話者は多くの場合，話し手や状況に対してネガティブな感情を抱いていると判断された

Preparation and identification of polyclonal antibody raised against heat shock protein 90 of fission yeast

作者简介:李文珠(1983－),女,博士研究生,主要研究方向:细胞生物学 通信联系人：靳全文，教授，主要研究方向：细胞周期调控及表观遗传学， [email protected][中文文摘]为制备兔抗裂殖酵母Hsp90多克隆抗体,在通过PCR获得了裂殖酵母Hsp90(Swo1)基因后,构建了pMALc2x-Swo1表达载体,可用于表达编码正确氨基酸序列的目的基因。转化大肠杆菌BL21(DE3),IPTG诱导表达,Amylose Resin柱纯化。目的蛋白表达量占菌体总蛋白的30%以上。纯化后,蛋白纯度达95%以上。纯化后的MBP-Swo1融合蛋白抗原加福氏完全佐剂背部皮内注射首次免疫新西兰大白兔,第28天用MBP-Swo1融合抗原加福氏不完全佐剂同样剂量加强免疫,第35天时再次免疫。第49天心脏采血。收集血清后,用免疫印迹(westernblot)检测Swo1多克隆抗体的特异性。免疫印迹检测结果显示该抗体能够特异性识别内源性的裂殖酵母Hsp90/Swo1蛋白,但并不识别人类细胞中的Hsp90α/β。[英文文摘]To prepare the polyclonal antibody of Hsp90(Swo1) of fission yeast Schizosaccharomyces pombe,the encoding gene swo1+was amplified from the yeast genome and then inserted into expression vector pMALc2x.The resulted plasmid pMALc2x-Swo1 was transformed and expressed in E.coli BL21(DE3).The expressed fusion protein was purified through Amylose Resin column.The proteins were expressed mainly as secretion with the yield of more than 30% of total bacterial proteins.After purification,the purity of the proteins was about 95%. The New Zealand white rabbits were immunized with Freund’s complete adjuvant plus purified MBP-Swo1 fusion antigen through the back skin intradermal injection for the first time. The same dose of Freund’s incomplete adjuvant plus MBP-Swo1 was injected to strengthen the immunity after 28 and the 35 days respectively. After 49 days, blood sample was collected from heart, and antisera were extracted. The specificity of Swo1 polyclonal antibody was examined by Western blot. It showed that the antibody obtained had a high specificity to detect endogeneous Swo1 from fission yeast, but it could not recognize Hsp90α/β in human cells. Key words: fission yeast；Hsp90/Swo1；polyclonal antibody.高等学校博士学科点专项科研基金资助项目(20100121110003);国家自然科学基金资助项目(30871376);教育部科学技术研究重点项目(108076

Function of heat shock protein 90 (Hsp90) in heterochromatic gene silencing in fission yeast

鉴于高等生物中HSP90与ArgOnAuTE蛋白的功能相关性,研究了裂殖酵母中HSP90/SWO1与AgO1蛋白之间的相互关系以及对异染色质区基因沉默的影响。结果表明,裂殖酵母中SWO1蛋白通过与AgO1蛋白的相互作用,可以稳定AgO1蛋白,并且这种相互作用依赖于SWO1的n端和中央结构域以及AgO1的n端和PAz结构域。在着丝粒的OTr区和IMr区,SWO1+基因的突变会引起区域内基因沉默的解除,并且与rnAI组分双突变后(AgO1Δ或dCr1Δ),基因沉默解除的效果加强。在交配型区,SWO1+基因突变后也会引起显著的基因沉默解除现象。当SWO1+基因突变后,依赖于TAS3的人工异染色质区基因沉默解除。研究发现了裂殖酵母中热激蛋白HSP90的新功能,即参与异染色质区的基因沉默调控。Given that Hsp90 functionally correlates with Argonaute in higher eukaryotes,we examined the interaction between Hsp90(Swo1) and Ago1and the function of Hsp90 on heterochromatic gene silencing in fission yeast Schizosaccharomyces pombe.The results showed that Swo1 could stabilize Ago1 through interaction with Ago1 in fission yeast.The interaction between Swo1 and Ago1 depends on N domain and the central region of Swo1,as well as the N and PAZ domains of Ago1.The centromeric silencing was alleviated in the swo1-26 mutant,but the silencing of reporter gene at telomere and rDNA regions remained unchanged.Double mutants of swo1-26 dcr1Δ and swo1-26 ago1Δ showed enhanced silencing defects at centromeric regions.In the mating type region,we observed a significant derepression of gene silencing in the swo1-26 mutant.The artificial RITS complex-dependent silencing system was also defective in the swo1-26 mutant.Our results found a new role of Hsp90 in gene silencing at heterochromatin regions in S.pombe.高等学校博士学科点专项科研基金资助项目(20100121110003);国家自然科学基金面上项目(30871376);教育部科学技术研究重点项目(108076

Fas ligand and Anti-human DR5 monoclonal antibody induce tumor cell lines apoptosis

目的探讨FasL、Anti-DR5mAb对肿瘤细胞Hela、BGC823、MCF-7、L342、H9101、D6的杀伤作用及机制。方法采用RT-PCR、MTT比色法、电泳、DNA倍体分析、Westernblot等方法。结果H9101、Hela细胞株DR5mRNA水平有表达,D6细胞无表达;H9101、L342细胞株FasmRNA水平有表达,D6细胞无表达。H9101、L342细胞株对FasL、Anti-DR5mAb敏感并呈剂量依赖性;MCF-7、BGC823细胞株对FasL敏感,对Anti-DR5mAb相对敏感。Hela对FasL相对敏感,对Anti-DR5mAb敏感;D6对两种凋亡诱导剂耐受。结论FasL、Anti-DR5mAb能不同程度地诱导肿瘤细胞凋亡,其机制与Fas、DR5、Caspase-8、Bcl-2的表达有关。 【英文摘要】 Objective:To study the cytotoxic effects on tumor cell lines Hela,BGC823,MCF-7,L342,H9101,D6 induced by Fas ligand and anti-human DR5 monoclonal antibodies(Anti-DR5 mAb) and the underlying mechanism.Methods:Fas/DR5 mRNA were detected by RT-PCR. Cytotoxicity exerted by FasL/Anti-DR5 mAb on tumor cell lines was measured by MTT colorimetry and the induced apoptosis was determined by agarose gel electrophoresis.Results:The expression of DR5 on BGC823 and Hela cells were higher and DR5 didn’t express in D6. The ...厦门大学科研启动基金(No.Z03103)资

重组人RGD-FasL对胶质瘤细胞U138/U343/U373的体外抗肿瘤的活性分析

目的研究重组人RGD-FasL对3株胶质瘤细胞U138/U343/U373的杀伤作用及机制。方法采用RT-PCR方法、MTT比色法、DNA倍体分析、流式细胞术检测融合蛋白对3株胶质瘤细胞的杀伤作用;利用Western blot法探讨其作用机制。结果FasmRNA在U138和U343细胞中有表达,在U373细胞中未有可见表达;DcR3mRNA在3株细胞中均有表达,在U373细胞中强表达。采用流式细胞术检测肿瘤细胞表面两种受体的表达情况,结果与RT-PCR相符。U138细胞株对RGD-FasL敏感并呈剂量依赖性;U343细胞株对RGD-FasL相对敏感;U373细胞对RGD-FasL不敏感。用PI检测细胞周期与凋亡表明RGD-FasL能使U138/U343细胞停留在G1期,推迟进入S期,抑制细胞生长并诱导细胞发生凋亡。Western blot实验表明RGD-FasL作用细胞后caspase-8/3/9的表达升高,Bcl-2的表达降低。结论重组人RGD-FasL可以不同程度的诱导胶质瘤细胞凋亡,其机制与其受体的表达和caspase-8/3/9、Bcl-2的表达有关

Studies of preparation and properties of RGD-tTF water-based ferrofluids

目的探讨RGD-tTF水基磁流体通过磁场和RGD多肽在体外对内皮细胞双靶向的功能。方法通过化学沉淀法以柠檬酸钠为表面活性剂制备水基磁流体(MnFe2O4),弱酸改性后与重组的RGD-tTF融合蛋白结合,利用H-600透射电镜观测纳米粒径,以SUQID鉴定磁性,用MTT法、因子X活化检测和流式细胞仪检测RGD-tTF磁流体生物活性。结果成功制备出的水基磁流体能在磷酸盐缓冲液中稳定分布且具有生物兼容性,实验表明RGD-tTF与水基磁流体结合后对RGD和tTF生物活性均无显著影响,并证实在磁场的作用下能实现了水基磁流体对RGD-tTF的定位作用。结论一种具有内皮细胞双靶向功能的RGD-tTF水基磁流体已制备成功。 【英文摘要】 Purpose To study the property of RGD-tTF water-based ferrofluids double targeting EC304 cells in vitro by magnet and RGD peptide.Methods Water-based ferrofluids(MnFe2O4) were prepared by chemical precipitation method using citrate as surfactant, dispersed in weak acid to create surface charges,and coated with recombinant RGD-tTF protein.Proteins coated ferrofluids were characterized by H-600 transmission electron microscopy(TEM)and Superconducting Quantum Interference Device(SQUID),and its biological activi...教育部和厦门大学出国留学人员启动基金资

抗人DR5单克隆抗体的制备、鉴定及活性分析

目的制备抗人DR5单克隆抗体(mAb),鉴定其特性,并进行生物学活性分析。方法以纯化的可溶性DR5(sDR5)免疫Balb/c小鼠,杂交瘤技术制备抗人DR5mAb;运用ELISA、SDS-PAGE电泳方法测定抗DR5mAb与sDR5结合的特性;Ig亚类ELISA试剂盒鉴定抗DR5mAb亚类;间接ELISA法检测腹水mAb效价;流式细胞仪检测肿瘤细胞表面DR5的表达水平;流式细胞仪检测抗DR5单克隆抗体(mAb)诱导肿瘤细胞凋亡的功能。结果获得1株可分泌抗DR5mAb的杂交瘤细胞系R150。SDS-PAGE电泳检测证实,获得的R150可特异性地识别DR5;R150的Ig亚类为IgGI(λ型);腹水效价为1×106;通过流式细胞仪可敏感地检测到肿瘤细胞表面DR5的表达水平及R150诱导肿瘤细胞凋亡情况。结论获得1株可分泌抗DR5mAb的细胞系R150,抗体具有效价高、特异性强等特点并能有效诱导肿瘤细胞凋亡,具有较好的应用价值

死亡受体5胞外区域的重组、表达及活性鉴定

目的构建死亡受体5(deathreceptor5,DR5)胞外区域(eDR5)的表达载体,表达纯化重组蛋白并鉴定其生物特性。方法通过重叠PCR获得DR5胞外段编码序列,构建pET-22b(+)/DR5表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达,Ni2+柱亲和纯化,SDS-PAGE、直接ELISA鉴定纯化产物的纯度和特异性,用MTT法检测eDR5蛋白阻断DR5单克隆抗体FMU1.5和TRAIL诱导人胶质瘤细胞株U343(高表达DR5)、U373(低表达DR5)细胞凋亡的作用。结果获得了DR5胞外段编码序列,目的蛋白在上清及包涵体中都有表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度达95%以上,蛋白产量达9mg/ml。ELISA结果表明所纯化蛋白为eDR5。eDR5蛋白可部分阻断FMU1.5和TRAIL诱导人胶质瘤细胞株U343细胞凋亡的作用,其阻断率与DR5表达相关。结论死亡受体5胞外段基因的成功重组、表达及纯化,为进一步的功能研究奠定了基础