aDR5scFv specific identification and functional analysis in vitro and vivo

背景及目的 肺癌是目前世界上最常见的恶性肿瘤，从大体上可分为小细胞肺癌（SCLC）和非小细胞肺癌(NSCLC)。其中大部分为非小细胞肺癌，约占80%，小细胞肺癌约占20%。约65%~70%的非小细胞肺癌在就诊时就已处于晚期。早期小细胞肺癌可以取得较好的治疗效果。但晚期的非小细胞肺癌无论手术或者放射治疗均不能取得满意的治疗效果。单克隆抗体作为一种新的治疗方法，虽然在临床上还不够成熟，但是它是一种具有很大潜力的免疫学治疗方法。 目前现有的研究证实，抗DR5单克隆抗体(aDR5mAb)可通过与DR5的特异性结合，诱导表达DR5的肿瘤细胞凋亡，但同时也有报道证明抗DR5单克隆抗体亦存在肝毒性。目前...ABSTRACT Background & objective Lung cancer is one of common malignant tumors in the world。There are two kinds of Lung cancer.One is SCLC.The other is NSCLC. The number of people who suffer from NSCLC accounted for most of the number of people who have Lung cancar It's about 80 pecent.About 65%~70% of NSCLC are found to be in the advanced stage.Althought the expectation of patients who have SCLC...学位：医学硕士院系专业：医学院临床医学系_肿瘤学学号：2452008115347

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

Inhibitory effect of anti-human NRP-1 monoclonal antibody on hepatocellular carcinoma cell line HepG2 and its mechanism in vitro

目的研究抗人神经纤毛蛋白-1(Neuropilin1,NRP-1)单克隆抗体对肝癌Hep G2细胞的生长抑制作用及其机制。方法小鼠腹水法制备抗NRP-1单克隆抗体(NRP-1 m Ab)并用r Protein A亲和柱纯化抗体,间接ELISA检测抗体的滴度水平。Western blot检测NRP-1 m Ab对Hep G2细胞的特异性,细胞免疫荧光和流式细胞术检测NRP-1蛋白在肝癌细胞株Hep G2上的表达,MTT法检测NRP-1 m Ab对Hep G2的生长抑制作用,Western blot检测ERK1/2、P-ERK1/2、Akt、P-Akt蛋白的表达水平。结果 SDS-PAGE和间接ELISA检测纯化的NRP-1 m Ab纯度为95%以上,效价为1×10~(-6);Western blot检测结果显示NRP-1 m Ab可与Hep G2细胞膜上的NRP-1蛋白特异性结合。细胞免疫荧光染色结果显示NRP-1定位于Hep G2细胞膜,流式细胞术结果显示NRP-1蛋白在Hep G2细胞上表达水平较高;MTT法检测结果显示NRP-1 m Ab对Hep G2细胞有生长抑制作用。Western blot检测到在不同浓度NRP-1 m Ab作用下,Hep G2细胞裂解液P-ERK1/2、P-Akt蛋白的条带信号逐渐减弱。结论纯化的NRP-1m Ab能抑制Hep G2细胞的生长,其抑制作用是通过EGF和HGF信号通路实现的。The aim of the experimental is to investigate the inhibitory effect of anti-human nerve cilia protein1(Neuropilin-1,NRP-1) monoclonal antibody(NRP-1 mAb) on hepatocellular carcinoma cell line HepG2 and its mechanism in vitro.Anti-human NRP-1 monoclonal antibody(NRP-1 mAb) was prepared from mouse ascites and purified by rProteinA affinity column assay.The titer of antibody was determined using indirect ELISA assay;the characteristic of NRP-1 mAb binding to NRP-1 was determined using Western blotting;the expression of NRP-1protein in hepatocellular carcinoma cell line HepG2 was determined using immunofluorescence assay and flow cytometry assay.Growth inhibition of HepG2 cells treated with different concentrations of NRP-1 mAb was determined using MTT assay,while Western blotting was used to detect the expression levels of ERK1/2,P-ERK1/2,Akt and P-Akt proteins.The results of SDS-PAGE and indirect ELISA showed that the purity of purified NRP-1mAb was more than 95%and the titer was 1×10~(-6).Western blotting analysis suggested that NRP-1 mAb could bind specifically to NRP-1 on HepG2 cell;immunofluorescence staining showed that NRP-1 was located in the membrane of HepG2 cells.Flow cytometry analysis showed that the expression level of NRP-1 on HepG2 cell was relatively high.Western blotting analysis suggested that P-ERK1/2 and P-Akt expression levels were down-regulated after having incubated HepG2 cells with different concentrations of NRP-1 mAb.In conclusion,NRP-1 mAb could inhibit the growth of HepG2 cells(P<0.05),and its inhibitory effect is achieved by reducing the P-ERK1/2 and P-Akt expression.南京军区医学科技创新项目(12MA061,15MS104

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

重组人血管内皮生长抑制因子对人肺腺癌裸鼠移植瘤的放射效应影响

目的:探讨重组人血管内皮抑制因子(recombinant human vascular endothelial growth inhiloitor-192,rhVEGI-192)对人肺腺癌裸鼠移植瘤的放射增敏作用。方法:采用原核表达rhVEGI-192,获得目的蛋白。通过肿瘤倍增时间,计算药物的增敏系数。通过建立人肺腺癌裸鼠移植瘤模型,荷瘤裸鼠随机分为4组:对照组、10Gy、rhVEGI-192、rhVEGI-192+10Gy。采用6MV-X线进行照射,照射剂量为10Gy。获得移植瘤标本,利用免疫印迹法检测移植瘤中VEGF(vascular endothelial growth factor)的表达变化。结果:SDS电泳结果显示,目的蛋白位于22k D左右。10Gy照射时,重组人血管内皮抑制因子的EF(enhancement factor)值为1.5。和空白对照组相比,rhVEGI-192组和10Gy组移植瘤的生长受到抑制(P<0.001),rhVEGI-192+10Gy组移植瘤生长显著抑制(P<0.001),rhVEGI-192+10Gy组移植瘤较10Gy组有明显生长抑制。和空白组相比,rhVEGI-192组VEGF表达减少,而10Gy组VEGF表达变化不明显,rhVEGI-192+10Gy组VEGF表达明显减少。rhVEGI-192+10Gy和rhVEGI-192组相比,VEGF表达减少。结论:rhVEGI-192联合照射能够减少VEGF的表达。这可能是rhVEGI-192的增敏机制之一。中国人民解放军南京军区医学科技创新项目(编号:No.12MA061

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

ERK干扰质粒的构建及对胃癌细胞株BGC823 DcR3表达的影响

目的探讨胃癌发展的分子机制,通过构建ERK1/2shRNA真核表达载体,体外研究其对人胃癌细胞株BGC823ERK1/2蛋白及DcR3表达水平的影响。方法应用PRNAT-U6.1/Neo载体构建ERK1/2基因shRNA重组质粒,经脂质体法导入BGC823细胞中,分别设置对照组、干扰组和U0126抑制剂组。Westernblot法检测转染后BGC823细胞及抑制剂使用后ERK1/2蛋白的表达变化,荧光显微镜检测质粒自带GFP基因的表达情况确认转染效率,ELISA法检测各组细胞上清中DcR3分泌蛋白的表达特点。结果成功构建ERK1/2基因shRNA重组质粒。证明了ERK1/2蛋白的表达与DcR3的分泌水平在BGC823细胞株中呈正相关。结论 ERK1/2干扰质粒明显降低BGC823细胞的ERK1/2蛋白表达水平,ERK信号通路对DcR3的分泌具有重要调控作用,为其下游调控机制的研究奠定了基础

都市住民と農村との交流・協働事業に係る事例報告

本稿では，近年注目されている都市住民と農村との交流・協働事業に着目し，その実践事例を報告し，それぞれ考察するものである。一つ目の事例は，葛飾区郷土と天文の博物館で実践している田んぼの学校による都市住民と農村交流のひろがりについてである。この事例では，博物館という場を通じて都市住民と農村が今後いかにして交流事業を行い，活動を続けていくべきかを考察する。二つ目の事例は，都心から90 分という身近な田舎である南房総での10 年間に渡る実践活動についてである。この事例では，農家と都市住民との“協働”が，未来の食の安全，里山の環境保全へ果たす役割について考察する

肝癌发展中FasL及其受体Fas/DcR3的表达分析

目的研究小鼠肿瘤发生早期肿瘤坏死因子超家族成员FasL及其受体的表达特点,及其与免疫调节相关分子表达的时空关系,探讨其在诱导肿瘤免疫耐受中的作用。方法建立小鼠实体瘤模型,采用RT-PCR方法检测肿瘤组织中可溶型FasL、Fas及DcR3、TGF-β、IL-10在肿瘤发生不同时相的表达,同时,应用ELISA方法定量检测血清中TGF-β、IL-10的表达。结果在早期的肿瘤组织中,Fas的表达先于DcR3,其后DcR3大量表达,而Fas的表达则下调直至丢失,DcR3、FasL同时表达并上调,TGF-β和IL-10也随肿瘤的表达而上调。TGF-β同DcR3的表达具有空间位置的一致性。FasL、DcR3、TGF-β和IL-10的表达水平同肿瘤的生长呈正相关。结论随着肿瘤不断的生长,肿瘤局部的免疫应答逐渐趋向于负调节,FasL、DcR3在诱导肿瘤免疫耐受的过程中可能发挥着重要作用