Impact Analysis and Mechanism Investigation to Rat’s Adjuvant Arthritis Model Using Anti-DR5 mAb

类风湿性关节炎（RheumatoidArthritis，RA）是以关节慢性炎症为特征的自身免疫性疾病。虽然RA的确切发病机制还不清楚，但是T细胞、B细胞、巨噬细胞、嗜中性粒细胞和滑膜成纤维细胞在关节发炎及疾病发展的过程中处于重要地位。细胞因子、Th1/Th2细胞平衡、细胞凋亡等在RA的发病过程中也起着重要作用。RA与滑膜组织中多种淋巴细胞及滑膜成纤维细胞凋亡不足有关，因此诱导这些细胞凋亡防止其过度增殖成为治疗RA的有效途径。 TRAIL（TNFrelatedapoptosisinducingligand）为Ⅱ型跨膜蛋白，属于TNF超家族。TRAIL能诱导表达TRAIL特异性受体的细胞发生凋亡...Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation of the joints. Although the precise pathogenesis of RA remains unclear, T cells, B cells,macrophages, neutrophils and synovial fibroblasts are central to the mechanisms of joint inflammation and disease progression.RA is connected with the apoptosis inhibition of synovioblasts and lymphocytes in synovial tissu...学位：理学硕士院系专业：生命科学学院生物学系_动物学学号：2172006115212

The preparation and tumor cell apoptosis-inducing activity assay of anti-human DR5 single-chain antibody

作者简介: 张佳锴,男,硕士研究生,主要从事肿瘤免疫学研究, Tel: 0592-2180587，E-mail: baroce@ 163.com; 庄国洪，通信作者，E-mail: [email protected]。[中文摘要]目的构建、表达、纯化抗人死亡受体5(DR5)单链抗体,并检测其诱导肿瘤细胞凋亡的活性。方法采用RT-PCR获取鼠源性抗人DR5单克隆抗体重链和轻链可变区基因序列,以一柔性连接肽连接二者,转入表达载体,以大肠杆菌表达融合蛋白,亲和色谱纯化后用MTT实验和凋亡检测试剂盒检测其凋亡诱导活性。结果获得的序列经比对为抗体重链和轻链可变区基因,表达纯化后的重组蛋白具有接近完整抗体的肿瘤细胞凋亡诱导活性。结论抗人DR5 scFv可作为诱导肿瘤细胞凋亡的候选药物,为肿瘤免疫学研究提供材料。[英文摘要]Purpose To construct，express，purify anti-human DR5 scFv，and to assay its activity of apoptosis induction for tumor cell lines． Methods Variable region sequences of heavy chain and light chain to murine anti-human DR5 monoclonal antibody were acquired by RT-PCR， then they were linked through a flexible linker peptide and was cloned into the expression vector． After being expressed by E．coli and purified using affinity chromatography， the recombinant protein was employed to MTT assay as well as apoptosis assay kit in order to detect its apoptosis-inducing activity． Results The sequences we've got were established as the variable region genes of antibody's heavy and light chain，while the recombinant proteins expressed and purified possessing the apoptosis-inducing activity come near to complete antibody．Conclusion Anti-human DR5 scFv can supply material for tumor immunology research as drug candidate inducing tumor cell apoptosis．福建省自然科学基金资助项目(C0710046

抗人死亡受体5单链抗体ZF1对鼠H22肝癌细胞的作用分析

目的:研究抗人死亡受体5单链抗体ZF1对H22肝癌细胞体内外抑制增殖作用。方法:MTT法检测ZF1对H22肝癌细胞体外杀伤作用,流式细胞术检测ZF1诱导H22的凋亡率,建立H22移植瘤模型,随机分为PBS组、ZF1组、EPI组和ZF1/EPI联合组四组。观察肿瘤生长和小鼠体重变化情况。治疗13天后分离肿瘤组织,进行HE染色检查、TUNNEL法检测细胞凋亡。结果:体外实验显示:ZF1可抑制H22细胞的增殖,呈剂量依赖性,抑制率最高为84.5%。体内实验结果显示单独应用ZF1或联合应用ZF1/EPI时,肿瘤增长受到明显抑制。HE染色和TUNNEL分析结果表明ZF1可有效诱导肝癌肿瘤凋亡,ZF1/EPI联合组效果更明显,而对正常肝细胞无毒性。结论:单链抗体ZF1具有良好抑制H22细胞增殖的作用。ZF1和EPI联合应用效果更明显

抗人DR5单链抗体诱导HepG2细胞凋亡的实验研究

目的探究抗人DR5单链抗体(scFv)ZF1对肝癌细胞株HepG-2的凋亡作用及机制。方法 MTT法检测ZF1对HepG-2的细胞毒效应;流式细胞术检测ZF1诱导HepG2的凋亡率;荧光显微镜观察经ANNEXIN-V/PI双染的HepG-2细胞;DNA Ladder检测HepG-2细胞的DNA特点;Western blotting检测Bcl-2、PARP蛋白的表达。结果 MTT结果显示ZF1抑制HepG-2细胞生长呈剂量依赖性,ZF1终浓度分别为0.225、0.45、0.9mg/ml、1.2mg/ml200μl时,HepG-2细胞的生长抑制率分别为28.8%、52.3%、65.3%、89.8%;流式细胞仪检测结果显示,终浓度分别为0.225、0.45、1.2mg/ml2ml作用HepG-2细胞4h,凋亡率分别为32.9%、56%、83.2%;荧光显微镜下可见早期凋亡细胞,细胞膜呈绿色荧光,细胞内有凋亡小体的形成,伴有核结构的变化;DNALadder出现明显的彗星尾状条带;Western blotting检测到ZF1诱导凋亡的细胞内Bcl-2、PARP蛋白表达上调。结论 ZF1可诱导HepG2细胞株凋亡,这种作用与HepG2细胞株表达Bcl-2、PARP蛋白蛋白表达上调相关

Fas胞外区基因的构建、表达、纯化及多克隆抗体的制备

目的构建Fas胞外区(eFas)基因的表达载体,表达纯化重组蛋白,进行多克隆抗体的制备,为进一步功能研究奠定基础。方法通过重叠PCR获得eFas基因的编码序列,构建pET-22b(+)/eFas表达载体,转化大肠杆菌Rosetta-gami,IPTG诱导表达,Ni-NTA柱亲和纯化,SDS-PAGE鉴定重组蛋白的纯度。将纯化的eFas融合蛋白免疫新西兰白兔制备多克隆抗体,通过ELISA方法检测多克隆抗体的效价。结果获得了eFas的编码序列与表达载体,目的蛋白主要在包涵体中表达,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。结论eFas融合蛋白基因的构建、表达、纯化以及多克隆抗体的制备,为进一步研究Fas提供了材料

Characterization of single chain antibody against DR5

目的探究抗人死亡受体5(dr5)单链抗体(Adr5SCfV)的特异性。方法 ElISA检测Adr5SCfV的效价、亲和力和表位分析。WESTErn blOTTIng检测单链抗体的特异性。MTT检测Adr5SCfV对结肠癌细胞SW480的生长抑制。结果 ElISA显示抗人死亡受体5单链抗体抗体效价为1.2x104。通过间接ElISA证实Adr5SCfV与Adr5MAb识别dr5胞外段(Edr5,系本实验室自己构建)的同一个位点。Adr5SCfV的亲和力常数为2.12x10-2。Edr5与Adr5SCfV能够特异结合。MTT检测结果显示,Adr5SCfV抑制SW480细胞生长呈剂量依赖性,Adr5SCfV终质量浓度分别为0.225 Mg/Ml、0.45 Mg/Ml、0.9 Mg/Ml、1.2 Mg/Ml。200μl时,SW480细胞的存活率分别为97.7%,70.9%、70.1%、23.6%。结论 Adr5SCfV能与Edr5特异性结合,并有较强的活性。The aim of our study is to characterize the property of aDR5ScFv.We employed ELISA to determine the titer,relative affinity and epitope recognition of aDR5ScFv,while Western blotting to analyze the specificity of aDR5ScFv.We also measured the inhibitory effects of aDR5ScFv on colon cancer cell SW480 growth by MTT.We found that the titer of aDR5ScFv was 1.2×104;aDR5ScFv and aDR5mAb recognized the same epitope on DR5 molecule;and the affinity constant of aDR5ScFv was 2.12×10-2.Furthermore,aDR5ScFv could recognize eDR5 specifically and inhibit the growth of SW480 cells in a dose-dependent manner(aDR5ScFv final concentrations of 0.225,0.45,0.9,1.2 mg/ml correspond to SW480 viabilities of 97.7%,70.9%,70.1%,23.6%).Western blotting showed the binding of aDR5ScFv to DR5 was specific.Thus,we concluded that binding of aDR5ScFv to DR5 is specific and aDR5ScFv had strong activity.福建省自然科学基金(C0710046

Induction of apoptosis by Anti-DR5 mAb in synovial cells of adjuvant arthritis rats and its possible mechanisms

目的:探讨AnTI-dr5 MAb诱导佐剂型关节炎(AdJuVAnT ArTHrITIS,AA)大鼠滑膜细胞凋亡作用及其可能机理。方法:注射弗氏完全佐剂建立AA大鼠模型,分离并体外培养大鼠滑膜细胞。MTT检测、dnA倍体分析及流式细胞术分析AnTI-dr5 MAb对大鼠滑膜细胞的凋亡作用,WESTErn blOT检测大鼠滑膜细胞CASPASE-3、bCl-2蛋白的表达,CASPASE抑制剂实验分析AnTI-dr5 MAb对大鼠滑膜细胞凋亡作用的机制。结果:MTT实验表明AnTI-dr5 MAb能抑制大鼠滑膜细胞的增殖,流式细胞术分析这种作用模式为细胞凋亡。AnTI-dr5 MAb处理后的大鼠滑膜细胞中CASPASE-3蛋白水平上升,bCl-2蛋白水平下调。CAS-PASE抑制剂实验证明AnTI-dr5 MAb诱导大鼠滑膜细胞的凋亡是通过CASPASE途径实现的。结论:AnTI-dr5 MAb诱导大鼠滑膜细胞的凋亡,这种作用与滑膜细胞表面CASPASE-3、bCl-2蛋白表达相关。Objective:To study the effects of Anti-DR5 mAb on inducing synovial cells of adjuvant arthritis(AA) rats and its mechanisms.Methods:AA was induced by CFA in rats.The synovial cells of rats were separated and cultured in vitro system.The apoptosis induced by anti-DR5 mAb on synovial cells was measured by MTT analysis,DNA fragmentation and flow cytometry.Caspase-3 and Bcl-2 expression in synovial cells were detected by Western blot.The involvement of the apoptotic pathway was further proved by a caspase inhibition assay.Results:MTT result showed that Anti-DR5 mAb could inhibit synovial cells growth.And flow cytometry suggested that the cell death mode was apoptosis.The protein level of caspase-3 in synovial cells treated with anti-DR5 mAb was raised,while Bcl-2 level declined.When the caspase inhibitor was added to the synovial cells treated with anti-DR5 mAb,it was showed in a dose-dependence inhibition effect,indicating that anti-DR5 mAb inducing apoptosis might be through the caspase pathway.Conclusion:Anti-DR5 mAb could induce synovial cell apoptosis through caspase pathway.And this effect may be related to the protein level of Caspase-3 and Bcl-2.福建省自然科学基金项目(C0710046

人死亡受体5全长基因的构建及转染胶质瘤细胞

目的构建人死亡受体5(death receptor5,DR5)全长基因真核细胞表达载体pcDNA3.1/DR5,pcDNA3.1/GFP/DR5,并观察其转染胶质瘤细胞U138的效果。方法通过重叠PCR获得DR5全长编码序列,构建pcDNA3.1/GFP/DR5,pcDNA3.1/DR5表达载体,利用脂质体转染试剂盒,分别将2种质粒pcDNA3.1/GFP/DR5、pcDNA3.1/GFP共转染胶质瘤细胞U138,转染后72h,半定量逆转录聚合酶链反应(RT-PCR)检测DR5mRNA的表达,流式细胞术检测DR5的表达强度、检测Anti-DR5对胶质瘤细胞U138生长的影响。结果获得了DR5全长编码序列,成功瞬时转染U138,RT-PCR、流式细胞术检测结果表明,转染后U138细胞DR5mRNA、蛋白水平的表达明显增加,Anti-DR5可以明显抑制U138细胞的生长。结论获得了DR5全长编码序列,探索到成功转染DR5的最佳方法,为稳定筛选高表达DR5的U138细胞提供依据

A Fault Detection Method for Control Valve Based on Noise Analysis

针对调节阀的在线故障检测问题,文中提出了一种基于噪声分析的故障检测方法。该方法首先利用小波分解技术提取过程信号的噪声;然后,通过数字滤波减少干扰信号;最后,从统计角度出发,计算过程噪声的统计数字特征(自相关函数)来检测故障是否发生。仿真结果表明:该方法可以检测出气动调节阀的阀门堵塞故障,同时验证了该方法的可行性与有效性

Anti-DR5 mAb诱导佐剂型关节炎大鼠滑膜细胞凋亡及机理初探

目的:探讨Anti-DR5 mAb诱导佐剂型关节炎(Adjuvant Arthritis,AA)大鼠滑膜细胞凋亡作用及其可能机理。方法:注射弗氏完全佐剂建立AA大鼠模型,分离并体外培养大鼠滑膜细胞。MTT检测、DNA倍体分析及流式细胞术分析Anti-DR5 mAb对大鼠滑膜细胞的凋亡作用,Western blot检测大鼠滑膜细胞Caspase-3、Bcl-2蛋白的表达,Caspase抑制剂实验分析Anti-DR5 mAb对大鼠滑膜细胞凋亡作用的机制。结果:MTT实验表明Anti-DR5 mAb能抑制大鼠滑膜细胞的增殖,流式细胞术分析这种作用模式为细胞凋亡。Anti-DR5 mAb处理后的大鼠滑膜细胞中Caspase-3蛋白水平上升,Bcl-2蛋白水平下调。Cas-pase抑制剂实验证明Anti-DR5 mAb诱导大鼠滑膜细胞的凋亡是通过Caspase途径实现的。结论:Anti-DR5 mAb诱导大鼠滑膜细胞的凋亡,这种作用与滑膜细胞表面Caspase-3、Bcl-2蛋白表达相关