表达ESAT-6-gpi和IL-21的B16F10瘤苗免疫鼠伴发自身免疫损伤初探

初步探讨B16F10-ESAT-6-gpi/IL-21瘤苗诱导抗肿瘤免疫的同时伴发自身免疫相关毒性作用。采用Western blot方法检测B16F10-ESAT-6-gpi/IL-21瘤苗免疫小鼠自身抗体产生情况;采用免疫荧光技术检测瘤苗免疫小鼠血清对正常小鼠眼、睾丸、皮肤、肝及肾组织冰冻切片的反应;瘤苗免疫小鼠的生殖功能检测。结果显示B16F10-ESAT-6-gpi/IL-21瘤苗免疫会引起部分C57BL/6小鼠发生鼠毛色素脱失现象,其免疫血清可同皮肤、睾丸等组织正常成分发生抗原抗体反应,瘤苗免疫过的雌雄小鼠合笼后能够正常繁育子代小鼠。实验说明B16F10-ESAT-6-gpi/IL-21瘤苗诱发小鼠产生抗肿瘤免疫反应的同时,部分受试鼠可伴发自身免疫损伤,但对机体并未产生危及生命的严重病理反应。本实验为全面评估B16F10-ESAT-6-gpi/IL-21瘤苗应用价值奠定了基础。     

糖基化磷脂酰肌醇修饰的小鼠IL-21瘤苗抗肿瘤效应及其机制初探

目的 构建糖基化磷脂酰肌醇(glycosyl phosphafidylinositol,GPI)修饰的小鼠IL-21瘤苗,并对此瘤苗的抗肿瘤效应及其机制作初步探讨.方法 通过重叠PCR方法获得IL-21-GPI融合基因并将其插入空载体pcDNA3.1.将鉴定过的重组载体以脂质体法转染B16F10细胞制成瘤苗,细胞间接免疫荧光法及流式细胞仪检测转染瘤细胞膜表面IL-21的表达,通过对小鼠脾细胞的增殖作用鉴定表达的IL-21的生物学活性.将瘤苗接种小鼠后,通过观察小鼠肿瘤体积和生存率分析瘤苗的抗瘤性,并检测了瘤苗免疫鼠的细胞免疫活性.结果 正确构建了pcDNA3.1/IL-21-GPI重组载体,膜表达的IL-21有良好的生物学活性,制备的瘤苗能发挥抗肿瘤效应,其机制与免疫鼠细胞免疫活性增强有关.结论 成功构建了具有抗肿瘤活性的GPI修饰的IL-21瘤苗,为其进一步抗肿瘤免疫治疗研究奠定了基础.     

表达ESAT-6-gpi和IL-21的B16F10瘤苗的构建及其活性鉴定

为构建膜表达糖基化磷脂酰肌醇(GPI)锚定的结核杆菌早期分泌靶抗原6 kD(ESAT-6)和分泌IL-21的B16F10瘤苗并鉴定其活性,利用重叠PCR法构建pIRES-ESAT-6-gpi/IL-21重组质粒,以脂质体转染重组质粒到B16F10细胞,G418筛选出阳性克隆,用RT-PCR、免疫荧光、FCM和Western blot检测瘤苗细胞靶抗原表达,用瘤苗细胞培养上清刺激小鼠CD8+T细胞,检测瘤苗所分泌IL-21的生物学活性。结果表明,pIRES-ESAT-6-gpi/IL-21重组质粒DNA测序正确,B16F10-ESAT-6-gpi/IL-21瘤苗细胞目的基因ESAT-6表达于瘤苗细胞表面,增殖能力未受外源基因导入影响,分泌的IL-21具有生物学活性,为研究膜表达ESAT-6和分泌表达IL-21瘤苗的抗瘤效应奠定了基础。     

GPI修饰的ESAT-6核酸疫苗的构建及其免疫功能初探

目的构建糖基化磷脂酰肌醇(GPI)修饰的结核杆菌早期分泌性抗原靶(ESAT-6)核酸疫苗,初步分析其免疫功能。方法利用重叠PCR法构建pIRES-ESAT-6-gpi重组体,转染B16F10细胞,G418筛选阳性克隆,用RT-PCR、免疫荧光检测转染细胞的ESAT-6抗原表达情况;采集ESAT-6核酸疫苗免疫鼠血清,分别检测抗体滴度和CDC效应,免疫磁珠法分选CD4+和CD8+T细胞,CFSE/7-AAD细胞毒实验分析CTL细胞毒活性。结果测序正确的重组体pIRES-ESAT-6-gpi转染细胞后,ESAT-6表达于细胞膜表面。ESAT-6核酸疫苗免疫血清和CD8+T细胞分别通过CDC效应和细胞毒作用杀伤膜表达ESAT-6的B16F10细胞。结论构建的GPI修饰的ESAT-6核酸疫苗能够诱导免疫鼠产生体液和细胞免疫反应,杀伤膜表达ESAT-6的B16F10细胞,为进一步基于该法构建B16F10瘤苗的抗肿瘤免疫效应及机制研究奠定了基础。     

IL-21 gpi和sGM-CSF共同修饰的瘤苗构建及其抗肿瘤效应

目的:构建膜锚定IL-21和分泌性GM-CSF(sGM-CSF)双表达瘤苗,并对其抗肿瘤效应及其机制作初步探讨.方法:用分子生物学方法构建双表达IL-21 gpi和sGM-CSF重组质粒,将鉴定过的重组质粒以脂质体转染B16F10细胞制成瘤苗,用流式细胞仪检测转染瘤苗IL-21 gpi和sGM-CSF的表达.以瘤苗治疗荷瘤鼠,经观察小鼠肿瘤体积、生存率来分析瘤苗的抗瘤性,并检测了瘤苗治疗鼠的细胞免疫活性.结果:正确构建了pRSC/IL-21 gpi-sGM-CSF重组质粒,转染细胞可很好地表达膜锚定IL-21和分泌性GM-CSF,制备的瘤苗能有效地发挥抗肿瘤效应,其机制与瘤苗治疗鼠的脾细胞增殖活性、NK细胞及CD8+细胞细胞毒活性增强有关.结论:成功构建了具有抗肿瘤活性的膜锚定IL-21和分泌性GM-CSF双表达瘤苗,为进一步抗肿瘤免疫治疗研究奠定了基础.     

沉默HIF-1α表达逆转缺氧诱导肝癌免疫抑制的实验研究

目的:探讨缺氧诱导因子-1(HIF-1α)表达在缺氧诱导肝癌免疫抑制中的作用,以期阐明缺氧致肝癌免疫抑 制的分子机制。方法:构建缺氧C57BL/6 小鼠移植瘤模型,缺氧探针HypoxyprobeTM-1 标记缺氧组织,免疫组化检测缺氧组织 HIF-1α表达,qPCR 检测缺氧组织肿瘤免疫因子表达;慢病毒构建稳定敲减HIF-1α的Hepa1鄄6 小鼠肝癌细胞株,并移植于 C57BL/6 小鼠,6 周后检测移植瘤瘤重,Western blot 检测移植瘤HIF鄄1琢表达,流式细胞仪检测移植瘤中肿瘤浸润免疫细胞 CD4+CD25+ Foxp3+调节性T 细胞表达,qPCR 检测缺氧组织肿瘤免疫因子表达。结果:C57BL/6 小鼠缺氧移植瘤模型构建成 功,缺氧组织HIF鄄1琢高表达,肿瘤抑制因子IFN-γ、IL-12b 和CXCL10 低表达,肿瘤促进因子IL-10、IL-1β和IL-17 高表达;敲减 HIF-1α明显抑制了移植瘤生长,降低了CD4+ CD25+ Foxp3+调节性T 细胞浸润,同时抑制了肿瘤促进因子IL-10、IL-1β和IL-17 表达,促进了肿瘤抑制因子IFN鄄酌、IL鄄12b 和CXCL10 表达。结论:缺氧可诱导肝癌组织HIF鄄1琢表达及免疫抑制,相反,沉默 HIF鄄1琢可抑制移植瘤生长并逆转肝癌免疫抑制,揭示了HIF-1α在缺氧致肝癌免疫抑制中的作用,可能为将来肝癌诊治提供 新思路。     

B7—1基因修饰的肿瘤细胞疫苗抗肿瘤的实验研究

目的：研究B7-1基因修饰的肿瘤细胞作为瘤苗的抗肿瘤作用。方法：通过逆转录病毒载体。将小鼠B7-1基因导入EL-4淋巴瘤细胞中,研究EL-4/B7-1在同系C57BL/6小鼠中的成瘤性及其诱导抗肿瘤免疫的效果。结果：转B7-1基因诱导了CD80的高表达,EL-4/B7-1细胞在小鼠中的成瘤性下降,在肿瘤生长的早期对实验性荷瘤小鼠进行治疗。基因修饰的瘤苗作用明显增强,EL-4/B7-1细胞能有效地保护野生型肿瘤细胞的攻击,射线灭活的EL-4/B7-1瘤苗作用减弱。结论：B7-1基因修饰的肿瘤疫苗可诱导体内的抗肿瘤免疫反应,导致肿瘤的部分根除,为基因修饰的肿瘤疫苗的临床应用提供了实验依据。     

mIL-21基因转染的Sp2/0瘤苗细胞的抗肿瘤机制探讨

目的:探讨小鼠IL-21瘤苗-Sp2/0-mIL-21抗肿瘤效应的机制。方法:用流式细胞术检测Sp2/0-mIL-2瘤苗细胞表面MHC-Ⅰ类分子及CD80分子的表达。以瘤苗细胞接种BALB/c小鼠,以CFSE,7-AAD标记,用流式细胞术检测NK细胞、CTL的细胞毒活性。观察肿瘤组织中淋巴细胞的浸润。用RT-PCR检测肿瘤组织中CXC家族趋化因子I-TAC的表达。结果:与对照组相比,瘤苗细胞膜表面MHC-Ⅰ类分子的表达明显上调。瘤苗接种组小鼠NK细胞、CTL的细胞毒活性明显增强。病理分析发现,肿瘤组织中有较多的淋巴细胞浸润并检测到干扰素诱导的T细胞α趋化因子(I-TAC)的表达上调。结论:肿瘤细胞瘤苗Sp2/0-mIL21可增强小鼠的细胞免疫作用,其抗肿瘤机制可能与T细胞的增殖、活化,促进NK细胞的分化成熟,淋巴细胞向肿瘤组织浸润,以及增强NK细胞和CTL的细胞毒活性有关。     

重组沙门菌表达结核分枝杆菌ESAT-6及其特异性免疫应答分析

目的 分析重组沙门菌表达的结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌性蛋白ESAT-6诱导的特异性免疫应答.方法 将ESAT-6蛋白编码基因导入原核表达载体pYA3333中,构建重组质粒pYA33-esat.通过电穿孔法转化减毒鼠伤寒沙门菌X4550,获得重组菌X4550(33-esat).以每只105CFU剂量的重组菌滴鼻免疫C57BL/6小鼠,间隔18 d,在第2次免疫后10 d取免疫小鼠脾脏、肺脏、肠系膜淋巴结(mesenteric lymph node,MLN)及派伊尔淋巴集结(Peyer's patch,PP)细胞,以ESAT-6多肽作为刺激原,检测特异性的IFN-γ分泌细胞和IL-4分泌细胞.同时,运用CFSE方法榆测了体内抗原特异性CTL效应.结果 经沙门菌表达并运送的Mtb抗原ESAT-6能诱导特异性的免疫应答.在肺脏及PP细胞巾,检测到较高水平的IFN-γ和IL-4分泌细胞,免疫应答以Th1型为主.而在脾脏和MLN中,免疫应答呈现Th1/Th2混合应答.此外,体内CTL试验表明,重组菌能够诱导抗原特异的CTL效应,且特异性杀伤率为69.9%.结论 以滴鼻方式接种重组沙门菌,不仅能够诱导ESAT-6蛋白特异性的细胞免疫应答,还能激发特异的CTL效应,为结核病的防控提供了新的认识.     

小鼠白细胞介素12基因转染及其在黑色素瘤细胞B16F10中的表达

目的:探索小鼠白细胞介素12(mIL-12)基因在小鼠黑色素瘤B16F10细胞中的表达。方法:应用DNA重组技术将mIL-12基因插入pcDNA3.1真核表达载体中, 通过电穿孔转染B16F10细胞, 筛选出阳性细胞克隆后, 应用PCR、RT-PCR及Westernblot技术检测mIL-12基因在B16F10细胞中的整合及表达。结果:在DNA、mRNA及蛋白质3个水平均证实mIL-12基因已转染到B16F10细胞中并表达。结论:mIL-12基因可成功地转染体外培养的B16F10细胞并表达, 为进一步研究IL-12基因修饰的肿瘤细胞的基因瘤苗奠定了基础。     

Antitumor efficacy of viable tumor vaccine modified by heterogenetic ESAT-6 antigen and cytokine IL-21 in melanomatous mouse

The goal of this study was to investigate whether glycosylphosphatidylinositol (GPI)-anchored 6?kDa early secreted antigenic target (ESAT-6) and IL-21-producing B16F10/ESAT-6-GPI-IL-21 viable vaccine would induce antitumor efficacy. Mice were immunized with B16F10/ESAT-6-GPI-IL-21 vaccine and challenged by B16F10 cells 2?weeks later. Antitumor efficacy and mechanisms of the vaccine were analyzed. Vaccination with the viable B16F10/ESAT-6-GPI-IL-21 vaccine resulted in an increase of IFN-γ level and the CD8(+)CTL cytotoxicity, a decrease in TGF-β generation and increase in the expression of miR-200c that serves as melanoma suppressor by directly targeting zinc-finger E-box binding homeobox 1 to inhibit epithelial-mesenchymal transition and block tumor metastasis. The vaccine significantly inhibited the melanoma growth, reduced the lung melanoma nodules, and prolonged the mouse survival compared with the controls. These findings highlighted IL-21 as an immune adjuvant in an engineered viable tumor vaccine to reinforce heterogenetic antigen ESAT-6 immune tolerance break to induce powerful antitumor efficacy in mice.     

Supplementation with RD antigens enhances the protective efficacy of BCG in tuberculous mice

Different combinations of ESAT-6, CFP-10, CFP-21, MPT-64, encoded by RD1 and RD2 of Mycobacterium tuberculosis were evaluated on the basis of antigenicity in PPD positive TB contacts and immunogenicity in C57BL/6J mice immunized with the combination of all four RD antigens. The peripheral blood mononuclear cells of TB contacts showed maximum recognition in response to the combination of ESAT-6+MPT-64 in terms of predominant lymphoproliferation, IFN-gamma levels and the number of responders. On the contrary, the combination of ESAT-6+CFP-21+MPT-64 was found to be most immunogenic based on both T-cell and antibody responses in immunized mice. Prophylactic potential of the selected combinations was assessed as supplementation vaccines to BCG against intravenous challenge with M. tuberculosis in mice. BCG supplementation with the selected combinations resulted in significantly greater protection as compared to BCG alone against experimental tuberculosis and thus appears to be a promising approach to enhance the protective efficacy of the existing vaccine.     

Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens

Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.     

Therapy of established tumour with a hybrid cellular vaccine generated by using granulocyte–macrophage colony-stimulating factor genetically modified dendritic cells

Dendritic cells (DCs) are the most powerful of all antigen-presenting cells and play a critical role in the induction of primary immune responses. DC-based vaccination represents a potentially powerful strategy for cancer immunotherapy. In this study, a new approach for a DC-based melanoma vaccine was described. Splenic DCs from C57BL/6 mice were fused with B16 melanoma cells, and the resultant B16/DC hybrid cells expressed major histocompatibility complex (MHC) molecules - B7 as well as the B16 tumour marker M562 - which were enriched by Ia-mediated positive selection with a MiniMACS column. The fusion rates were 12.7-26.8%. To generate hybrid tumour vaccines with potentially greater potent therapeutic efficacy, we genetically engineered DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to cell fusion. Recombinant adenovirus vector was used to mediate gene transfer into DCs with high efficiency and DCs expressed GM-CSF at 96-138 ng/105 cells/ml 24 hr after GM-CSF gene transfer. GM-CSF gene-modified DCs (DC.GM) exhibited higher expression of B7 and co-stimulatory capacity in mixed lymphocyte reaction (MLR). Fusion of DC.GM with B16 cells generated B16/DC.GM hybrid cells secreting GM-CSF at 59-63 ng/105 cells/ml. Immunization of C57BL/6 mice with the B16/DC hybrid vaccine elicited a specific cytotoxic T-lymphocyte (CTL) response and protected the immunized mice from B16 tumour challenge, reduced pulmonary metastases and extended the survival of B16 tumour-bearing mice. The B16/DC.GM hybrid vaccine was able to induce a CTL response and protective immunity more potently and tended to be therapeutically more efficacious than the B16/DC vaccine. In vivo depletion of T-cell subsets demonstrated that both CD8+ and CD4+ T cells were essential for the therapeutic effects of B16/DC and B16/DC.GM hybrid vaccines. Additionally, other non-specific effector cells may also contribute to tumour rejection induced by the B16/DC.GM hybrid vaccine. These data indicate that a DC-based hybrid tumour vaccine may be an attractive strategy for cancer immunotherapy, and that GM-CSF gene-modified DCs may lead to the generation of hybrid vaccines with potentially increased therapeutic efficacy.     

DNA vaccine encoding ESAT-6 enhances the protective efficacy of BCG against Mycobacterium tuberculosis infection in mice

ESAT-6 is a protein produced by virulent Mycobacterium tuberculosis but absent in Mycobacterium bovis bacillus Calmette-Guerin (BCG) and is considered as a promising vaccine subunit against tuberculosis. The protective efficacy of a new strategy based on the combination of DNA vaccine encoding ESAT-6 (DNA-E6) and BCG immunization was investigated in the present study. BALB/c mice were immunized with DNA-E6, BCG, DNA-E6 plus BCG, vector plus BCG or vector alone. Three weeks after the last immunization, antigen-specific interferon (IFN)-gamma secreted upon stimulation with BCG-PPD or ESAT-6 protein by splenocytes of the different groups was determined by an ELISA. The immunized mice were challenged intravenously with low-dose virulent M. tuberculosis H37Rv and bacterial load of the infected mice was measured in the spleen and lung 6 weeks later. Pathological changes in the lung were observed by haematoxylin-eosin (HE) or acid-fast staining. Our study demonstrated that ESAT-6-specific IFN-gamma was improved in mice vaccinated with DNA-E6 plus BCG, compared with those mice immunized with BCG alone. The combination of DNA-E6 and BCG resulted in the maximum reduction in bacterial load in both the lung and spleen, and only slight pathological changes were detected in the lung. These results suggested that the combination of DNA-E6 and BCG vaccination could be a better strategy against M. tuberculosis infections in human.