PML-RARα-hIL-2DNA疫苗诱导BALB／C小鼠的细胞和体液免疫应答

目的分析PML-RARα融合基因疫苗诱导小鼠特异体液和细胞免疫应答情况。方法用成功构建的重组质粒（pIRES-PML-RARα-hIL-2）免疫小鼠后，分别利用ELISA法检测小鼠血清中INF-γ生成情况；LDH释放法检测小鼠脾细胞特异性CTL细胞针对APL细胞株NB4的杀伤率；用间接免疫荧光法检测血清中抗PML-RARα抗体。结果免疫后第7周时，小鼠血清中INF-γ含量、抗PML-RARα抗体及脾脏CTL细胞针对NB4的杀伤率均高于对照组。结论所构建的pIRES-PML-RARα-hIL-2重组质粒可诱导小鼠产生了特异性体液和细胞免疫应答。     

癌胚抗原与IL-2真核双表达质粒的构建及体外表达

目的：构建癌胚抗原（CEA）与白细胞介素2（IL－2）的真核双表达质粒，并检测其在体外的表达。方法：利用分子生物学技术，将CEA基因片段和IL－2基因片段连接于真核双表达质粒pIRES中，测定序列后，用脂质体包裹转染细胞，采用ELISA双抗体夹心法检测CEA和IL－2的表达。结果：核酸序列测定证实本实验所构建的质粒正确，质粒上所接入的CEA和IL－2的碱基序列与标准序列的同源性分别为99．8％和99．9％。该双表达质粒在体外转染细胞内后可表达CEA和IL－2分子。结论：实验所构建的pIRES-CEA-IL－2双表达质粒能在体外同时表达CEA和IL－2分子，说明本质粒具有在细胞水平同时表达两种生物大分子的活性，这一结果为其以后在整体动物水平的实验研究奠定了基础。     

癌胚抗原乳酸菌疫苗影响小鼠细胞免疫效果的实验观察

目的:对癌胚抗原乳酸菌疫苗在体内的细胞免疫效果进行评价.方法:将疫苗对小鼠进行免疫后检测T淋巴细胞亚群以及细胞因子IFN-γ、IL-2,观察小鼠的免疫效果.结果:除乳酸菌菌体本身能诱发一定的细胞免疫反应外,CEA表面展示组(pSec:Leiss-cea 2-lcsB)对机体产生了显著的免疫刺激.结论:本研究进一步证明表面展示的CEA能够有效的诱发免疫反应,为乳酸菌CEA口服疫苗的应用打下了基础.     

载体对癌胚抗原DNA疫苗在小鼠体内表达CEA及诱导体液免疫应答的影响

目的：比较用pcDNA3和pCI-neo做载体构建的癌胚抗原（CEA）真核表达质粒在小鼠体内表达CEA及诱导体液免疫应答的差别。方法 将两套重组质粒分别肌注BABL／c小鼠，ELISA法检测二者在小鼠体内表达CEA及抗－CEA水平。结果 pCI-CEA免疫的小鼠体内表达CEA量高于pcDNA3-CEA组，二者OD值分别为0．65和0．35；抗体量的表达前者OD值为0．89，后者为0．70，比较差异有显著性（P＜0．05）。结论 表达载体的结构对抗原的表达及抗体的产生具有重要作用。     

载体对癌胚抗原DNA疫苗在小鼠体内表达CEA及诱导体液免疫应答的影响

目的　比较用pcDNA3和pCI-neo做载体构建的癌胚抗原 (CEA)真核表达质粒在小鼠体内表达CEA及诱导体液免疫应答的差别。方法　将两套重组质粒分别肌注BABL/c小鼠 ,ELISA法检测二者在小鼠体内表达CEA及抗 -CEA水平。结果　以pCI -CEA免疫的小鼠体内表达CEA量高于pcDNA3-CEA组 ,二者OD值分别为 0 65和 0 35 ;抗体量的表达前者OD值为 0 89,后者为0 70 ,比较差异有显著性 (P <0 .0 5 )。结论　表达载体的结构对抗原的表达及抗体的产生具有重要作用     

带免疫佐剂CEA基因疫苗的构建及其稳定同步表达的研究

背景与目的：基因疫苗是目前肿瘤免疫基因治疗研究的热点。目前癌胚抗原阳性肿瘤的治疗效果不佳,本研究拟利用质粒pVAX1构建带有免疫佐剂白细胞介素2同步表达的癌胚抗原(carcinoembryonic antigen,CEA)基因疫苗,探讨一种新的肿瘤生物治疗方法。方法：利用RT-PCR从患者肿瘤组织中钓取CEA目的基因cDNA;运用分子克隆技术,通过内部核糖体进入位点(IRES),将CEA cDNA和hIL-2 cDNA连接于质粒pVAX1中,采用化学发光免疫法和ELISA双抗体夹心法分别检测CEA抗原和hIL-2因子的表达;同时用蛋白质分析软件分析和预测目的抗原的特性。结果：测序结果证实本实验所构建的重组质粒插入DNA序列无错配,质粒上所接入的CEAcDNA与GenBank公布的M29540和M17303的序列99．8％同源,经蛋白质分析软件预测,其抗原性、跨膜结构片段、信号肽位点和二、三级结构特征与同源癌胚抗原基本一致。hIL-2 cDNA与母本序列100％同源。检测结果说明该重组质粒在体外CHO细胞株中可同步表达CEA和hIL-2分子。结论：从肿瘤组织中成功地获取了CEA cDNA,所构建的pVCEA2重组质粒能在体外细胞中同步表达CEA和IL-2蛋白分子,这为进一步体内实验研究基因治疗CEA阳性肿瘤提供了新的可能途径。     

肺癌细胞致敏树突状细胞疫苗的体内外抗肿瘤作用

目的:研究以NCI-H460肺癌细胞致敏的树突状细胞(Dendritic cells,DCs)疫苗的抗肿瘤作用。方法:将IL-18基因转染的NCI-H460细胞及未转染NCI-H460细胞与DC融合作为转染融合DC组及融合DC组疫苗,以NCI-H460细胞RNA冲击的DC为冲击DC组疫苗,以未冲击的DC为DC组疫苗。以MTT法检测4组DC疫苗刺激T细胞增殖的作用,用ELISA法测定DC疫苗上清液中IL-12的含量,用LDH法测定DC疫苗对NCI-H460细胞的杀伤作用。裸鼠皮下接种NCI-H460细胞及DC疫苗,观察成瘤时间及裸鼠存活情况;荷瘤裸鼠瘤内注射DC疫苗,比较肿瘤体积。结果:4组DC疫苗均能刺激T细胞增殖,刺激作用强度为转染融合DC组>融合DC组>冲击DC组>DC组;4组均能分泌IL-12,转染融合DC组IL-12的分泌量高于融合DC组,冲击DC组高于DC组;转染融合、融合、冲击DC和DC组疫苗对NCI-H460细胞的杀伤率分别为79.73%、50.68%、35.81%及4.05%。转染融合组裸鼠移植瘤成瘤时间[(12.82±2.85)d]长于冲击DC组[(8.52±1.97)d](P<0.05)和DC组[(8.33±1.63)d](P<0.01);移植瘤内注射疫苗后的肿瘤体积比较,转染融合组<融合组<冲击DC组相似文献   

TCRγ独特型DNA疫苗诱导特异性免疫反应的实验研究

目的：观察TCRγ独特型DNA疫苗诱导BALB／c小鼠的抗人类Jurkat淋巴肿瘤免疫反应的情况。方法：碱裂解法大量提取质粒，制备DNA疫苗。选用16只BALB／c小鼠随机分为pcDNA3组、pcDNA3／TCRγV1组，双侧股四头肌内注射疫苗免疫，于第0、2、4周各免疫1次，共3次。每次免疫前及免疫开始后至第8周取鼠血，用间接免疫荧光法检测小鼠血清中抗体生成情况；RT-PCR法检测重组质粒在小鼠肌肉中的mRNA表达。结果：pcDNA3／TCRγV1组小鼠血清中全部产生了特异性抗独特型抗体，抗体滴度在第4周开始增高，第6周时达到高峰(1：160)。在pcDNA3／TCRγV1组小鼠中用RT—PCR法检测到了重组质粒在肌肉中的mRNA表达。结论：TCRγV1独特型DNA疫苗可以诱导小鼠产生特异性抗淋巴瘤细胞独特型抗体。     

CEA mRNA转染的成熟树突状细胞体外诱导特异性抗肿瘤作用

目的:将体外转录的癌胚抗原(carcinoembryonic antigen,CEA)mRNA转染入成熟树突状细胞(dendritic cell,DCs),观察其体外诱导的特异性抗肿瘤作用。方法:GM-CSF、IL-4、TNF-α体外诱导成熟DCs并用流式鉴定。构建pcDNA3.1-CEA载体,体外转录为CEA mRNA,电穿孔法将CEA mRNA转染入DCs。流式细胞术检测转染后DCs中CEA蛋白的表达;MTT法检测DCs刺激T细胞增殖能力;LDH法检测DCs体外诱导的CTL的特异性抗肿瘤作用;ELISA法检测诱导的CTL上清中IFN-γ的水平。结果:CEA mRNA转染后DCs细胞内CEA蛋白显著高于对照组(83.32%vs3.34%,P<0.01)。CEA mRNA转染组DCs在效靶比为1∶10时,刺激T细胞增殖作用最强,明显高于未转染组[(19.11±1.89)%vs(15.59±0.70)%,P<0.05]。CEAmRNA转染组DCs能产生CEA特异性的CTL效应,在效靶比为5∶1、10∶1、20∶1和40∶1时杀伤率分别为[(5.42±0.87)%、(14.09±1.13)%、(27.16±0.72)%、(32.49±0.84)%,P<0.01],而未转染组和对照靶细胞均无杀伤作用。转染组DCs诱导的CTL上清中IFN-γ分泌量显著高于未转染组[(141.73±28.61)vs(9.45±4.63)pg/ml,P<0.01]。结论:CEAmRNA转染的成熟DCs体外能产生特异性抗肿瘤作用,为研制CEA RNA-DCs疫苗提供了实验依据。     

猪endoglin胞外段DNA疫苗诱导抗小鼠结肠癌免疫反应

背景与目的:研究表明,endoglin是肿瘤血管生成的标志性分子,用抗endoglin单克隆抗体被动免疫治疗可以有效抑制肿瘤生长,而异种DNA疫苗可以打破自身同源分子的免疫耐受。因此,本研究用猪endoglin胞外段真核表达质粒(ppEDG)作为异种DNA疫苗,了解该DNA疫苗是否具有抑制小鼠结肠癌生长的作用,并探讨其作用的免疫学机理。方法:观察免疫治疗后的荷瘤小鼠肿瘤体积和生存情况,用免疫组化方法(抗CD31)观察肿瘤组织血管生长并计算肿瘤微血管密度,用Westernblot和ELISA方法检测荷瘤小鼠免疫治疗后的血清是否含有抗自身endoglin的抗体以及抗体亚型,用ELISPOT方法检测荷瘤小鼠脾脏中分泌抗自身endoglin抗体的B淋巴细胞。结果:肿瘤接种18天后,ppEDGDNA疫苗主动免疫治疗组肿瘤体积明显小于空质粒(e-p)对照组及生理盐水对照组(P<0.05),存活时间明显延长(P<0.001)。ppEDG组、e-p组和生理盐水组肿瘤组织微血管密度计数分别为19.2±4.5、76.9±14.4和81.4±16.9(P<0.001)。治疗组小鼠血清中含有抗自身endoglin的抗体,抗体亚型主要为IgG1和IgG2b。三组小鼠分泌抗自身endoglin抗体的B淋巴细胞数分别为82.5±14.1、3.6±1.3和4.7±2.0(P<0.001)。结论:ppEDGDNA疫苗诱导抗自身endoglin抗体产生,从而抑制肿瘤血管生成和肿瘤生长。     

癌胚抗原与γ-干扰素核酸疫苗对机体特异性细胞免疫的激活作用

目的检测以双表达质粒pIRES为载体构建的带有全序列癌胚抗原(carcinoembry-onic antigens,CEA)基因和γ-干扰素(interfer-on-γ,IFN-γ)基因的核苷酸疫苗对机体特异性抗肿瘤免疫反应的激活效果。方法利用分子生物学技术,将CEA基因片段和IFN-γ基因片段连接于真核双表达质粒pIRES中,用肌肉注射方法接种核酸疫苗;检测疫苗在小鼠肌肉组织中的表达及其对小鼠脾细胞CEA特异性细胞免疫反应的激活效果。结果小鼠经肌肉注射质粒后,核酸疫苗可在体内有效表达CEA和IFN-γ分子;小鼠特异性淋巴细胞增殖反应明显增高,并且伴有自然杀伤细胞(natural killercell,NK细胞)活性显著增高,F=15289·67,P<0·001。结论实验所构建的核酸疫苗pIRES-CEA、pIRES-CEA-IFN-γ和pIRES-CEA-IFN-γ等可在小鼠体内高效表达,并表现出良好的细胞免疫原性。     

Immunogenicity and safety of a DNA prime/adenovirus boost vaccine against rhesus CEA in nonhuman primates

Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients.     

A focused immune response targeting the homotypic binding domain of the carcinoembryonic antigen blocks the establishment of tumor foci in vivo

Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA-expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self-form of the CEA Ig V-like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA-mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody-dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients.     

Enhancement of colorectal tumor targeting using a novel biparatopic monoclonal antibody against carcinoembryonic antigen in experimental radioimmunoguided surgery.

Biparatopic CEA, carcinoembryonic antigen (MAb) was newly designed and tested as to whether it enhanced the accuracy of tumor detection by reducing non-specific binding in experimental radioimmunoguided surgery. Biparatopic MAb was prepared by using cross-linking of reduced Fab' fragments from PR1A3 and T84.66. Fifty-nine tumors from 2 human colorectal carcinoma cell lines with high (KM-12c) and low (Clone A) carcinoembryonic antigen (CEA) expression were successfully implanted subcutaneously on the backs of 42 nude mice. Tumors were localized using 125I-labeled MAbs: IgG, F(ab')(2) and Fab' of PR1A3, and biparatopic MAb of PR1A3 and T84.66. Radioactivity counted on a portable radioisotope detector correlated well with that counted on a gamma counter (p < 0.001). Accumulations of radioactivity in control mice without tumorigenesis were the greatest in PR1A3 IgG-pretreated mice and the least in biparatopic MAb-pretreated mice. Tumors of 2 cell lines did not differ in the distribution of radiolabeled MAbs. Localization indices of the tumor in various organs revealed 1.3 to 4.1 in PR1A3 IgG-pretreated mice, 2.4 to 6.6 in fragment MAbs of PR1A3-pretreated mice and 2 to 4.6 in biparatopic MAb-pretreated mice. Silver grains and immune staining were predominantly distributed in tumor cells of all types of MAb-pretreated mice. Sensitivity and specificity of tumor localization by radioimmunoguided surgery (RIGS) were the highest in the biparatopic MAb-pretreated mice (90.9% and 94.5%, respectively) and the least in the PR1A3 IgG-pretreated mice (50% and 72%). The biparatopic MAb using 2 anti-CEA MAbs against different epitopes achieved a great affinity and avidity with accurate localization of colorectal carcinoma in experimental radioimmunoguided surgery.     

Leucocyte adherence inhibition and carcinoembryonic antigen in combination for diagnosis of colorectal cancer

A combination of leucocyte adherence (LAI) and carcinoembryonic antigen (CEA) diagnosed colorectal carcinoma with 91% sensitivity and 68% specificity. Relative operating characteristic (ROC) analysis was used to calculate the cutoff points for optimum detectability in a group of 159 patients with bowel symptoms who were investigated by endoscopy and radiology. Combining LAI and CEA would be likely to reduce the chance of missed diagnosis of colorectal carcinoma when x-ray and colonoscopic findings are equivocal.     

Baseline carcinoembryonic antigen (CEA) serum levels predict bevacizumab‐based treatment response in metastatic colorectal cancer

Carcinoembryonic antigen (CEA) affects tumorigenesis by enhancing tumor cell survival and by inducing tumor angiogenesis. This study aimed to evaluate baseline CEA serum levels to predict bevacizumab‐based therapy effect and survival in patients with metastatic colorectal cancer (mCRC). Two hundred and ninety eight mCRC patients receiving chemotherapy plus either bevacizumab or cetuximab were analyzed in a retrospective study. Disease control (DC), progression‐free survival (PFS), and overall survival were assessed and related to pretreatment CEA serum levels. Patients with baseline CEA serum levels below the statistical median of 26.8 ng/mL (group I) were compared with patients with higher CEA levels (group II). The cetuximab‐based treatment cohort was analyzed for specificity assessment of CEA to predict the anti‐vascular endothelial growth factor effect in mCRC. Baseline CEA serum levels inversely correlated with therapeutic response in patients receiving bevacizumab‐based treatment (disease control rate, 84% vs 60%), inversely correlated with median PFS leading to a median PFS benefit of 2.1 months for patients in group I when compared with group II, as well as inversely correlated with median overall survival (37.5 months vs 21.4 months). In an independent cohort of 129 patients treated with cetuximab‐based therapy, no association of therapeutic response or PFS with CEA serum levels was found. As expected, baseline CEA levels were prognostic for mCRC. These data give first evidence that baseline serum CEA levels might constitute an important predictor for the efficacy of first‐line bevacizumab‐based therapy in patients with mCRC.