携带flk1的重组减毒沙门氏菌的构建及其抗肿瘤血管生成的研究

目的:观察携带鼠血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,flk1)的重组减毒鼠伤寒沙门氏菌诱导的flk1特异性免疫应答及抗肿瘤血管生成作用.方法:构建真核表达载体pcDNA3.1-flk1,并将其转化到减毒鼠伤寒沙门氏菌SL7207中,体外感染小鼠巨噬细胞,用Western印迹对表达产物进行鉴定,将重组菌口服免疫小鼠,分析免疫后小鼠体内的特异性CTL应答.采用藻酸盐微囊实验观察其对体内肿瘤血管生成的影响.结果:免疫印迹试验表明携带pcD-NA3.1-flk1表达载体的重组菌感染的小鼠巨噬细胞能表达flk1蛋白,免疫小鼠脾淋巴细胞产生针对flk1的特异性CTL活性.重组疫苗菌的免疫能够明显抑制小鼠体内肿瘤血管的形成.结论:携带flk1的重组减毒沙门氏菌经口服免疫,诱导小鼠产生抗flk1的特异性免疫反应,且能明显产生抗肿瘤血管生成作用.     

口服型VEGFR-2 DNA疫苗的研制及其对小鼠的免疫效应

目的:血管内皮生长因子受体-2(vascular endothelial factor recepto-2,VEGFR-2)在肿瘤生长和转移过程中起着重要作用,制备口服型VEGFR-2 DNA疫苗并研究其生物免疫效应.方法:采用基因重组技术构建VEGFR-2胞外区基因表达载体pcDNA3.1-VR-2, DNA序列分析证实后,脂质体法转染COS-7细胞, Western blotting检测其VEGFR蛋白表达;以氯化钙法将pcDNA3.1-VR-2转化减毒沙门菌SL3261,制备口服型VEGFR-2 DNA疫苗.口服型疫苗免疫C57BL/6小鼠,ELISA法检测免疫后小鼠外周血VEGFR-2抗体水平,MTT法检测小鼠脾淋巴细胞对小鼠血管内皮细胞的体外杀伤活性.结果:成功制备口服型VEGFR-2 DNA疫苗.ELISA法检测显示,疫苗组小鼠抗体滴度随着免疫时间和免疫次数的增加而增高,在免疫6周后产生了高水平抗VEGFR-2 IgG类抗体(1.07±0.018),明显高于生理盐水组(0.14±0.033)和质粒对照组(0.14±0.038),差异均有统计学意义(F=853.17,P=0.000).MTT法检测显示,疫苗组小鼠脾淋巴细胞对靶细胞MS1的杀伤率随着效靶比的增加而增加,在效靶比8∶1时CTL活性(89.38±1.51)明显高于生理盐水组(15.17±2.54)和空质粒对照组(12.99±4.31),差异有统计学意义(F=315.42,P=0.000).结论:成功制备了口服型VEGFR-2 DNA疫苗,该疫苗可激活小鼠特异性的细胞免疫和体液免疫,为进一步抗肿瘤研究奠定了基础.     

口服型VEGFR 2 DNA疫苗的研制及其对小鼠的免疫效应

摘 要 目的: 血管内皮生长因子受体-2(vascular endothelial factor recepto-2,VEGFR-2)在肿瘤生长和转移过程中起着重要作用，制备口服型VEGFR-2 DNA疫苗并研究其生物免疫效应。方法：采用基因重组技术构建VEGFR-2胞外区基因表达载体pcDNA3.1-VR-2， DNA序列分析证实后，脂质体法转染COS-7细胞， Western blotting检测其VEGFR蛋白表达；以氯化钙法将pcDNA3.1-VR-2转化减毒沙门菌SL3261，制备口服型VEGFR-2 DNA疫苗。口服型疫苗免疫C57BL/6小鼠，ELISA法检测免疫后小鼠外周血VEGFR-2抗体水平，MTT法检测小鼠脾淋巴细胞对小鼠血管内皮细胞的体外杀伤活性。结果：成功制备口服型VEGFR-2 DNA疫苗。ELISA法检测显示，疫苗组小鼠抗体滴度随着免疫时间和免疫次数的增加而增高，在免疫6周后产生了高水平抗VEGFR-2 IgG类抗体(1.07±0.018)，明显高于生理盐水组(0.14±0.033)和质粒对照组(0.14±0.038)，差异均有统计学意义（F=853.17，P=0.000）。MTT法检测显示，疫苗组小鼠脾淋巴细胞对靶细胞MS1的杀伤率随着效靶比的增加而增加，在效靶比8∶1时CTL活性(89.38±1.51)明显高于生理盐水组(15.17±2.54)和空质粒对照组(12.99±4.31)，差异有统计学意义(F=315.42,P=0.000)。结论：成功制备了口服型VEGFR-2 DNA疫苗，该疫苗可激活小鼠特异性的细胞免疫和体液免疫，为进一步抗肿瘤研究奠定了基础。     

口服DNA疫苗pcDNA3.1+/flk-1(n1-7)抗小鼠结肠癌肝转移

背景与目的：血管内皮生长因子（vascular endothelial growth factor，VEGF）及其主要受体血管内皮生长因子受体-2（vascular endothelial growth factor receptor-2，VEGFR-2）在肿瘤血管生成过程中起着重要作用。VEG—FR-2在鼠中为flk-1，本研究以减毒沙门氏菌SL3261为载体制备了抗肿瘤血管生成口服DNA疫苗pcDNA3．1＋／flk-1（n1-7），研究该疫苗抗小鼠结肠癌肝转移的作用，并探讨其可能的作用机制。方法：RT—PCR法扩增鼠VEGFR-2胞外cDNA全长序列flk-1（n1-7），构建重组质粒pcDNA3．1＋／flk-1（n1-7）。将重组质粒转化减毒沙门氏菌SL3261，制备成以SL3261为载体的口服DNA疫苗。将18只小鼠随机分组，口服接种疫苗两周后，用CT-26细胞建立结肠癌肝转移动物模型，15d后处死小鼠，体视学方法计数肝脏的肿瘤结节数目。流式细胞仪检测小鼠外周血CD4^＋细胞和CD8^＋细胞变化。结果：构建成重组质粒pcDNA3．1（＋）／flk-1（n1-7），制备成以SL3261为载体的口服DNA疫苗pcD—NA3．1（＋）／flk-1（n1-7）。疫苗组小鼠肝脏内的肿瘤转移病灶明显低于对照组，P〈0．05。结论：成功构建了重组质粒pcDNA3．1（＋）／flk-1（n1-7），制备成以SL3261为载体的口服DNA疫苗pcDNA3．1（＋）／flk-1（n1-7）。这种疫苗能抑制小鼠结肠癌的肝脏转移。     

基于TEM-8新型颗粒性疫苗对结肠癌小鼠模型免疫治疗的作用

目的:用基于TEM-8(tumor endothelialmarker-8)真核表达的颗粒性疫苗免疫荷结肠癌小鼠,探讨该疫苗的免疫治疗作用。方法:用阳离子肽[K]16-tat49-57与带有负电荷的真核表达质粒结合成颗粒性疫苗,用透射电镜和免疫印迹法鉴定,酶联免疫斑点试验(ELISPOT)检测疫苗所诱导的特异性CTL分泌IFN-γ,并对荷瘤Balb/c小鼠进行免疫治疗后,观察荷瘤体积和小鼠生存率,用免疫组化方法(抗CD31)观察肿瘤组织血管生长情况,并计算肿瘤微血管密度。结果:在电荷比为r=2的制备条件下,所构建的颗粒性疫苗进行电镜扫描,显示疫苗能形成颗粒,转染COS-7细胞后可有效介导TEM-8蛋白表达。ELISPOT检测显示在体内疫苗能有效激发抗原特异性CTL效应,可诱导CTL产生IFN-γ等细胞因子,肿瘤治疗实验证实,颗粒性疫苗能有效抑制肿瘤的生长速度,提高荷瘤小鼠的生存率,与对照组相比差异有统计学意义,P<0.05。结论:基于TEM-8的新型抗肿瘤血管生成疫苗能有效激发出特异性CTL应答,并在荷瘤小鼠模型中有效抑制肿瘤血管生成,提高荷瘤小鼠的生存率,为新型结肠癌治疗性疫苗的设计提供了新思路。     

口服VEGFR2 DNA疫苗抗小鼠Lewis肺癌血管生成的实验研究

背景与目的血管内皮生长因子(VEGF)及其主要受体血管内皮生长因子受体-2(VEGFR2)在肿瘤新生血管和肿瘤基质形成过程中起着重要作用。本研究的目的是观察口服VEGFR2 DNA疫苗抗C57BL/6小鼠Lewis肺癌皮下移植瘤生长的作用,并探讨其可能的作用机制。方法将重组DNA疫苗对小鼠进行免疫,通过观察小鼠Lewis肺癌皮下移植瘤大小,记录各组小鼠离体肿瘤湿重,检测移植瘤微血管密度(MVD)及血液CD3 、CD8 T细胞水平,评价重组疫苗的抑瘤作用。结果疫苗组、空质粒组、生理盐水组的MVD分别为1.75±1.07、6.89±2.52、7.57±3.75,肿瘤湿重分别为(2.05±1.32)、(4.83±1.47)、(5.12±1.02)g,疫苗组与其它两组比较,差异均有统计学意义(P<0.05)。接种肿瘤后,疫苗组CD3 T细胞仍维持较高水平,其它两组明显下降(P<0.05);疫苗组CD8 T细胞水平较其它两组明显升高(P<0.05)。结论口服VEGFR2DNA疫苗对小鼠Lewis肺癌皮下移植瘤的生长具有较强的抑制作用。该疫苗可能是通过杀伤肿瘤内皮细胞、抑制血管生成而起到抗肿瘤生长的作用。     

表达HPV16 E6和E7蛋白的非复制型重组痘苗病毒诱发的抗肿瘤免疫反应

目的　观察表达人乳头瘤病毒 (HPV) 16E6和E7蛋白的非复制型重组痘苗病毒的抗肿瘤免疫效果。方法　以重组痘苗病毒NTVJmE6E7免疫C5 7BL/ 6小鼠 ,检测特异性的细胞毒性T淋巴细胞 (CTL)活性 ;经免疫后的小鼠以TC 1肿瘤细胞攻击 ,观察免疫保护效果 ;荷瘤小鼠切除肿瘤后接种重组痘苗病毒 ,观察肿瘤复发情况。结果　以重组痘苗病毒NTVJmE6E7免疫小鼠 ,可诱导产生针对TC 1细胞的特异性的CTL反应 ;加强免疫后的小鼠能耐受 1× 10 4TC 1细胞的攻击 ;以重组痘苗病毒NTVJmE6E7免疫肿瘤术后小鼠 ,能有效地预防肿瘤复发。结论　非复制型重组痘苗病毒NTVJmE6E7可作为HPV16的相关肿瘤及其癌前病变免疫治疗的候选疫苗。     

颅脑

表达VEGFR-2的减毒鼠伤寒沙门氏菌疫苗诱导特异性抗胶质瘤血管免疫应答；脑星形细胞瘤MRI定量参数评价血管内皮生长因子表达的价值；133例经单鼻孔蝶窦入路治疗垂体腺瘤的经验；神经导航系统在颅内病变活检术中的应用；磁共振成像3种增强新技术在颅内肿瘤诊断中的应用；经鼻内镜颅底病变的外科手术；神经内镜下经鼻蝶窦入路垂体腺瘤切除术（附36例报告）；核磁共振脑功能成像指导切除脑功能区肿瘤（附6例报告）；功能区胶质瘤的术中直接电刺激判断核心手术技术。[编者按]     

腺病毒介导IL-2基因转染的瘤苗体内抗肿瘤作用

目的 :探讨重组腺病毒介导的 IL- 2基因转染的瘤苗的体内抗肿瘤作用及其免疫学机制。方法 :应用腺病毒介导的鼠 IL- 2基因转染 CT2 6小鼠结肠癌细胞 ,灭活后用作瘤苗治疗荷瘤小鼠 ,观察皮下肿瘤生长及其存活期。采用乳酸脱氢酶释放法检测荷瘤小鼠脾细胞 CTL、L AK、NK细胞的杀伤活性。结果 :鼠 IL- 2基因转染瘤苗治疗能显著抑制荷瘤小鼠皮下肿瘤生长并明显延长其存活期 (P<0 .0 1)。体内免疫功能检测表明 ,鼠 IL- 2基因转染疫苗治疗组小鼠脾细胞 CTL 活性、L AK活性和 NK活性显著高于对照组 (P<0 .0 1)。结论 :腺病毒介导鼠 IL- 2基因转染的瘤苗体内具有较强的抗肿瘤效应 ,其机制可能是提高了荷瘤小鼠特异性和非特异性抗肿瘤免疫反应     

血管内皮生长因子2型受体胞外1～3区核酸疫苗的构建及对H22肿瘤生长的抑制作用

背景与目的:大多数实体肿瘤的生长转移都需要新生血管的支持,血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)在肿瘤的血管生成中居中心地位,而flk1(fetalliverkinase1)是VEGF发挥作用的关键。阻断VEGF同flk1的结合可望达到抑制肿瘤生长的目的。我们构建了小鼠flk1胞外1～3区核酸疫苗,并检验疫苗对肝癌细胞H22小鼠移植瘤生长的抑制作用。方法:RT-PCR法克隆flk1胞外1～3区基因片段,将片段插入质粒pcDNA3.1(+),构建核酸疫苗pcDNA3.1(+)-flk1-Domain1-3;脂质体转染COS7细胞,Westernblot法检测flk1-Domain1-3的真核表达;采用标准4h51Cr释放实验检验疫苗免疫小鼠特异性CTL。30只小鼠分为疫苗接种组,pcDNA3.1(+)接种组和生理盐水接种组。股四头肌肌肉丰厚处接种10天后再皮下接种H22细胞,记录肿瘤的生长情况、体积、湿重、微血管密度、小鼠死亡时间,观察疫苗对小鼠H22皮下移植瘤生长的抑制作用。结果:PCR扩增出1252bp大小的基因片段,测序证明所获基因序列阅读框架与GenBank所公布的flk1胞外1～3区一致。Westernblot显示转染疫苗的细胞中有分子量约为44kDa的蛋白表达,与flk1胞外1～3区蛋白大小一致;51Cr释放实验提示,疫苗可引起针对内皮细胞的特异性CTL反应。疫苗对肿瘤的预防作用实验发现,肿瘤出现时     

胃癌MG7-Ag模拟表位口服DNA疫苗的研制

目的　利用减毒鼠伤寒沙门氏菌研制胃癌MG7 Ag模拟表位的口服DNA疫苗 ,并观察其对小鼠的免疫效能及保护作用。方法　构建MG7 Ag模拟表位和通用性辅助性T细胞表位融合基因的真核表达载体。将真核表达载体转入减毒鼠伤寒沙门氏菌得到模拟表位的口服DNA疫苗。以 1× 10 8cfu疫苗菌口服免疫C5 7BL/6J小鼠 ,以携带空载体的沙门氏菌和PBS口服作为对照。以ELISA法检测小鼠血清中抗MG7 Ag抗体的滴度 ,以H TDR掺入法检测小鼠脾淋巴细胞对人工合成的MG7 Ag抗原肽刺激的增殖能力。同时 ,用表达MG7 Ag的小鼠艾氏腹水瘤细胞进行肿瘤攻击 ,观察疫苗对小鼠的保护作用。结果　口服疫苗可诱导小鼠产生MG7抗体 ,但各组小鼠脾淋巴细胞体外刺激增殖实验差异无显著性。肿瘤攻击 2周后 ,疫苗免疫组 7只小鼠中有 2只未见肿瘤形成 ,而对照组 4只小鼠则全部成瘤。结论　胃癌MG7 Ag模拟表位的口服DNA疫苗具有免疫原性 ,可以诱导小鼠产生抗肿瘤免疫 ,并具有一定保护作用     

A combination of flk1-based DNA vaccine and an immunomodulatory gene (IL-12) in the treatment of murine cancer

AIM: The aim of this study was to investigate the antivasculature effects and the antitumor effects of combining attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) with plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. METHODS: Mouse models of Gl261 glioblastoma were treated with combining orally given attenuated Salmonella typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IL-12 (mIL-12) gene. The volumes of tumors were observed. Cytolytic T lymphocyte (CTL) response was measured by a 4-hour 51Cr release assay, vessle density and tumor cell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNEL staining. RESULTS: Compared to mice receiving single agent therapy, received either oral immunization flk1-based vaccine only or the therapeutic gene-IL-12 plasmid DNA only or those in the control group, the combination therapy groups developed a strong CTL response and showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization and cell proliferation in these mice. CONCLUSIONS: The therapy of attenuated Salmonella typhimurium vaccine strain encoding flk1 combined with the interleukin-12 gene has significant synergistic effect against tumors.     

异种血管内皮生长因子基因重组T7噬菌体疫苗对小鼠Lewis肺癌的抑制作用

背景与目的:肿瘤的生长、转移和新生血管生成有密切关系,血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)是已知作用最强、最专一的促血管生成因子。本实验采用噬菌体展示技术,构建人血管内皮生长因子重组T7噬菌体疫苗,并检验疫苗对小鼠Lewis肺癌的抑制作用。方法:用反转录聚合酶链反应(RT-PCR)从人肺癌组织克隆出人VEGF165基因,将人VEGF165基因与T7Select10-3b噬菌体基因重组,构建T7Select10-3b_VEGF噬菌体疫苗,制备噬菌体效价达到1×1013pfu/ml。将C57BL/6J小鼠随机分为3组,T7-VEGF疫苗组10只、空白噬菌体组(T7)10只、生理盐水组(NS)10只。将各组样品与等体积弗氏佐剂混合,各组小鼠每只每周分别免疫1×1012pfu/200!l重组噬菌体疫苗、空白噬菌体、生理盐水,共免疫4周。接种Lewis肺癌细胞,观察肿瘤的生长情况和小鼠状态.接种肿瘤14天后,处死小鼠,剥离肿瘤称重,检测小鼠血清中特异性抗VEGF抗体滴度、肿瘤微血管密度(microvesseldensity,MVD)。结果:T7-VEGF疫苗组,T7组、生理盐水组肿瘤均重分别为:(0.543±0.259)g、(0.982±0.359)g、(1.169±0.460)g。疫苗组肿瘤重量明显小于生理盐水组(P<0.01)。疫苗组血清产生抗VEGF抗体滴度为1∶1000。疫苗组、空白噬菌体组、生理盐水组MVD分别为:8.5±0.8,16.4±1.3,18.5±1.6。疫苗组MVD明显小于生理盐水组。结论:异种血管内皮生长因子基因重组T7噬菌体疫苗可干扰机体对自身VEGF的免疫耐受,可产生较高水平的特异性抗VEGF抗体,并具有抑制小鼠Lewis肿瘤生长的效应。     

地方株HPV16E7基因疫苗诱发的小鼠免疫反应

目的评价地方株HPV16E7基因疫苗诱导的免疫反应。方法从先期构建的地方株HPV16E7基因重组质粒pGEM-T-E7经限制性核酸内切酶酶切,获得E7基因亚克隆到真核表达载体pcDNA3.1(-)中,构建地方株pcDNA-E7基因疫苗,转染CHO细胞,通过IFA试验验证E7蛋白的表达。用pcDNA-E7肌注方法免疫6周龄BALB/c小鼠,同时设pcDNA空载体为阴性对照,于第1天、第15天和第43天共免疫3次,用IFA和MTT法分析基因免疫在小鼠体内诱导的免疫应答。结果pcDNA-E7免疫小鼠后能检测出特异的抗体,而原载体免疫后不能产生特异抗体;但2组刺激淋巴细胞增殖的能力差异不显著。结论地方株HPV16E7基因疫苗可诱导产生小鼠免疫反应。     

Human sodium iodide symporter gene adjunctive radiotherapy to enhance the preventive effect of hMUC1 DNA vaccine

We demonstrate the use of combination therapy to overcome the limitations of cancer DNA vaccines by adding radioiodine gene therapy in an animal cancer model. We established a stable cell line (CT26/hMUC1-hNIS-Fluc: CMNF) expressing the hMUC1, hNIS and Fluc genes using a retro- and lentivirus system. The survival rates (%) of CMNF cells were determined using clonogenic assays after (131)I treatment. After i.m. immunization to 4 groups of Balb/c mice (pcDNA3.1, pcDNA3.1+(131)I, pcDNA3-hMUC1+PBS and pcDNA3-hMUC1+(131)I groups) with pcDNA3-hMUC1 or pcDNA3.1 once a week for 2 weeks, 1 x 10(5) CMNF cells were injected s.c. into the right thighs of mice in each group. Twenty-one days after tumor transplantation, (131)I was administered i.p. to the pcDNA3.1+(131)I and pcDNA3-hMUC1+ (131)I groups. Tumor progression was monitored in the 4 groups by bioluminescent and scintigraphic imaging and by taking caliper measurements. Tumor masses were extracted and weighted at 39 days post-tumor challenge. We confirmed that CMNF cells highly express hMUC1, hNIS and Fluc by FACS, (125)I uptake, and luciferase assay. The survival rates of CMNF were markedly reduced to (14.6 +/- 1.5)% after (131)I treatment compared with the survival rates of parental cells (p < 0.001). Tumor growth inhibition was significant only in the pcDNA3-hMUC1+ (131)I group at 39 days post challenge. Tumor masses in pcDNA3-hMUC1+ (131)I group were smaller than those of the other groups. This study shows that the weak preventive effects of cancer DNA vaccine can be overcome by radioiodine gene therapy utilizing sodium iodide symporter.     

A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication

A novel strategy achieved the eradication of lung tumor metastases by joint suppression of angiogenesis in the tumor neovasculature and induction of tumor cell apoptosis. This was accomplished by CTLs induced by a DNA vaccine encoding secretory chemokine CCL21 and the inhibitor of apoptosis protein survivin, overexpressed by both proliferating endothelial cells in the tumor vasculature and tumor cells. Oral delivery of this DNA vaccine by doubly attenuated Salmonella typhimurium (dam(-) and AroA(-)) to such secondary lymphoid organs as Peyer's patches in the small intestine, elicited marked activation of antigen-presenting dendritic cells, and an effective CD8(+)T cell immune response against the survivin self-antigen. This resulted in eradication or suppression of pulmonary metastases of non-small cell lung carcinoma in both prophylactic and therapeutic settings in C57BL/6J mice. Moreover, the suppression of angiogenesis induced by the vaccine did not impair wound healing or fertility of treated mice. It is anticipated that such novel DNA vaccines will aid in the rational design of future strategies for the prevention and treatment of cancer.     

An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express alpha-fetoprotein

Antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In the present study, we first established a useful method to facilitate in vivo monitoring of attenuated S. typhimurium uptake using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. We found that the ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. We further examined the therapeutic efficacy of vaccination using attenuated S. typhimurium carrying the mouse alpha-fetoprotein (AFP) gene against a cancer line CT26-murine alpha-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. Attenuated S. typhimurium oral DNA vaccine was found to promote protective immunity against both CT26-mAFP and Hepa1-6 tumor cells growth. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development.