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。     

mIL-21基因治疗小鼠肿瘤及其机制的初步研究

建立小鼠Sp2/0皮下肿瘤模型,以我们构建并鉴定过的真核表达质粒peDNA3.1/mIL-21在瘤体内直接注射,考察mIL-21基因治疗对小鼠Sp2/0肿瘤的疗效。结果显示pcDNA3.1/mIL-21质粒治疗对小鼠皮下Sp2/0肿瘤有一定的生长抑制作用,CTL和NK细胞毒活性明显增强,IFN-γ分泌升高显著,但抗体无明显升高。表明mIL-21基因治疗的抗肿瘤效应,可能和细胞免疫功能增强有关。     

肿瘤特异性T杀伤细胞系的建立及其生物学特性

<正> 用小鼠T淋巴细胞性克隆瘤细胞LAC-1免疫同系SW_1小鼠,诱导对该瘤株特异性T杀伤细胞,并采用TCGF条件培液在体外扩大培养,建立体外长期培养肿瘤特异性T杀伤细胞系。实验结果表明:经体内3～4次免疫的小鼠脾脏、腹腔淋巴结细胞均对LAC-1癌细胞有明显的特异性杀伤效应,并随效/靶比例增高,杀     

B16F10/ESAT-6-GPI-IL-21瘤苗安全性及抗肿瘤效应研究

目的:评估B16F10/ESAT-6-GPI-IL-21瘤苗用于C57BL/6小鼠的安全性及其诱导的抗肿瘤免疫效应。方法:应用免疫荧光、FCM检测瘤苗细胞ESAT-6-GPI的表达情况;以Western blot方法检测瘤苗细胞IL-21的表达情况;动物实验检测瘤苗体内应用的安全性;制备荷瘤鼠术后免疫模型,评价瘤苗免疫效果。结果:B16F10/ESAT-6-GPI-IL-21瘤苗细胞表面有靶抗原ESAT-6-GPI表达,并分泌IL-21。于C57BL/6小鼠皮下接种2×105个B16F10/ESAT-6-GPI-IL-21瘤苗细胞,60天内未见致瘤,但能够诱导小鼠产生有效的抗肿瘤免疫效应。结论:B16F10/ESAT-6-GPI-IL-21瘤苗致瘤性明显下降,低剂量应用具有安全性,能够诱导小鼠产生有效的抗肿瘤免疫效应。     

艾氏腹水瘤全细胞瘤苗诱导的特异性杀伤活性

目的研究艾氏腹水瘤全细胞瘤苗诱导的特异性杀伤活性。方法用40g/L的多聚甲醛处理的艾氏腹水瘤细胞作为全细胞瘤苗免疫Balb/c小鼠,建立瘤苗小鼠模型。用HE染色法对亲本肿瘤细胞皮下再次攻击形成的肿瘤结节进行病理学观察;用51Cr释放法测定瘤苗免疫组、荷瘤组、正常组小鼠脾细胞,对亲本艾氏腹水瘤细胞和Sp2/0细胞的杀伤活性。结果HE染色显示,经瘤苗免疫后亲本肿瘤细胞皮下再次攻击形成的肿瘤结节中,瘤细胞几乎完全坏死,同时在坏死部位有大量炎细胞浸润、纤维母细胞和小血管增生;而对照组则无以上现象。效靶比为200∶1时,免疫小鼠脾细胞体外杀伤亲本艾氏腹水瘤细胞的杀伤率(％)为(42.3±3.2)％,显著高于荷瘤组的(12.1±2.3)％、正常组的(6.1±1.1)％和对Sp2/0细胞的杀伤率(8.8±0.4)％(P均∨0.05)。结论艾氏腹水瘤全细胞瘤苗可诱导机体产生特异性杀伤活性。     

小鼠白介素-12基因转染对EL细胞在小鼠体内成瘤性的抑制作用

目的探讨逆转录病毒(RV)载体介导的小鼠白介素12(mIL-12)融合基因转染,对低免疫原性的小鼠T细胞淋巴瘤细胞EL4体内生长的影响.方法用共培养法导入基因,以PCR法检测基因的导入.以脾细胞增殖法测定mIL-12的生物学活性.观察导入mIL-12基因的肿瘤细胞EL4/12于小鼠皮下接种后的成瘤性.对预先接种野生型肿瘤细胞EL4的小鼠,用60Co照射的肿瘤疫苗EL4/12接种于瘤周,观察瘤苗的抗肿瘤作用等.结果在EL4/12细胞中,用PCR可特异地检测到mIL-12p35和P40亚基的cDNA片段.5×105EL4/12细胞48h表达的mIL-12达(10.2±3.2)ng.EL4/12细胞在小鼠体内的成瘤性明显下降.EL4/12瘤苗治疗组约50%的小鼠可长期无瘤生存,而对照组小鼠则在短期内全部成瘤死亡.部分长期生存的小鼠产生了有效的抗肿瘤免疫,能耐受野生型肿瘤细胞的二次攻击.与对照组相比较,疫苗治疗组中其余小鼠的成瘤时间延迟(P＜0.01),小鼠存活时间延长(P＜0.01);死亡时肿瘤体积小(P＜0.05),形成的肿瘤有完整包膜,瘤周和肿瘤内部有较多的淋巴细胞和浆细胞浸润等.结论转染mIL-12基因能抑制EL4肿瘤细胞的成瘤性.mIL-12基因修饰的肿瘤疫苗对野生型肿瘤细胞具有治疗作用.     

小鼠白介素—12基因转染对EL4细胞在小鼠体内成瘤性的抑制作用

目的探讨逆转录病毒(RV)载体介导的小鼠白介素12(mIL-12)融合基因转染,对低免疫原性的小鼠T细胞淋巴瘤细胞EL4体内生长的影响。方法用共培养法导入基因,以PCR法检测基因的导入。以脾细胞增殖法测定mIL-12的生物学活性。观察导入mIL-12基因的肿瘤细胞EL4/12于小鼠皮下接种后的成瘤性。对预先接种野生型肿瘤细胞EL4的小鼠,用60Co照射的肿瘤疫苗EL4/12接种于瘤周,观察瘤苗的抗肿瘤作用等。结果在EL4/12细胞中,用PCR可特异地检测到mIL-12p35和p40亚基的cDNA片段。5×105EL4/12细胞48h表达的mIL-12达(10.2±3.2)ng。EL4/12细胞在小鼠体内的成瘤性明显下降。EL4/12瘤苗治疗组约50%的小鼠可长期无瘤生存,而对照组小鼠则在短期内全部成瘤死亡。部分长期生存的小鼠产生了有效的抗肿瘤免疫,能耐受野生型肿瘤细胞的二次攻击。与对照组相比较,疫苗治疗组中其余小鼠的成瘤时间延迟(P∨0.01),小鼠存活时间延长(P∨0.01);死亡时肿瘤体积小(P∨0.05),形成的肿瘤有完整包膜,瘤周和肿瘤内部有较多的淋巴细胞和浆细胞浸润等。结论转染mIL-12基因能抑制EL4肿瘤细胞的成瘤性。mIL-12基因修饰的肿瘤疫苗对野生型肿瘤细胞具有治疗作用。     

结核分枝杆菌MPT64重组母牛分枝杆菌免疫学特性

目的:研究结核分枝杆菌MPT64重组母牛分枝杆菌疫苗免疫学特性.方法:用分泌表达MPT64的重组母牛分枝杆菌疫苗免疫BALB/c小鼠,ELISA法检测免疫小鼠的特异性抗体滴度和抗体亚类.分离免疫小鼠脾淋巴细胞,检测淋巴细胞增殖、IFN-γ和IL-12产生水平、CD4+细胞和CD8+细胞数、脾淋巴细胞特异性CTL杀伤效应.毒株攻击后对脾脏细菌负荷计数.结果:MPT64重组母牛分枝杆菌疫苗免疫可诱导小鼠高水平的体液免疫应答,免疫小鼠脾淋巴增殖明显,IFN-γ和IL-12含量增加,CD4+和CD8+细胞百分比明显增加,CTL杀伤效应明显,对MTB H37Rv攻击后有一定的保护作用.结论:MPT64重组母牛分枝杆菌疫苗可诱导小鼠有效的体液和细胞免疫应答,有可能作为新型TB疫苗候选.     

免疫重建小鼠抗红白血病的作用研究

目的:观察在免疫重建过程中应用白血病瘤苗对C57BL/6小鼠一般状况、抵御FBL-3细胞攻击及小鼠保护性免疫功能的作用.方法:用同系小鼠脾脏淋巴细胞对照射后的免疫缺陷鼠进行免疫重建,同时用灭活FBL-3细胞皮下注射免疫C57BL/6小鼠,7天后用FBL-3细胞皮下攻击小鼠,观察小鼠的一般状况、成瘤率、瘤块生长情况;通过ELISA法测定外周血IFN-γ含量,以评估细胞免疫作用的强弱;通过病理切片了解瘤块中心及周围细胞浸润情况.结果:免疫重建 瘤苗可以有效提高单纯应用瘤苗所产生的保护性免疫功能,IFN-γ含量明显升高;用FBL-3细胞攻击,成瘤小鼠瘤块生长缓慢,瘤周单个核细胞浸润明显,瘤块内肿瘤细胞坏死明显,生存期明显延长.结论:在免疫重建的同时应用瘤苗,可以加强瘤苗的保护性免疫作用,为临床应用瘤苗治疗时机提供实验依据.     

胃癌细胞总RNA修饰树突状细胞的肿瘤疫苗效果

目的应用肿瘤细胞RNA修饰小鼠树突状细胞(dendrticcell,DC)的肿瘤疫苗免疫小鼠,研究其抗肿瘤免疫作用.方法CD11c磁珠抗体标记小鼠脾单个核细胞悬液,磁性细胞分选器(MACS)分选出CD11c+的DC,短期培养,以肿瘤细胞总RNA脉冲DC,以此瘤苗皮下注射免疫615小鼠两次,间隔1周,最后1次免疫后7d,实验鼠皮下接种5×105小鼠前胃癌细胞(MFC).结果经免疫后的小鼠具有较强抗肿瘤免疫,RNA脉冲DC的瘤苗免疫后肿瘤生长受到明显抑制,其外周血和脾细胞中NK活性均明显升高,肿瘤特异性CTL也明显升高.结论以肿瘤RNA作为抗原脉冲DC可诱导较强的抗肿瘤免疫.     

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.     

小鼠IL-18基因修饰瘤苗的抗白血病作用研究

我们在前期工作中成功制备了IL-18基因修饰的白血病疫苗,为了探讨IL-18基因修饰瘤苗的体内抗白血病作用,实验采用L1210小鼠淋巴细胞白血病模型,在小鼠体内接种IL-18基因修饰瘤苗,观察瘤苗对L1210细胞致瘤性的影响及免疫保护作用,并进一步对其抗白血病作用机制进行了探索。结果显示,IL-18基因修饰瘤苗能够明显延长荷瘤小鼠存活时间,大部分小鼠达到长期生存,且长生存小鼠用野生型L1210细胞二次攻击后大多数仍能长期生存,表明IL-18基因修饰瘤苗有显著的抗白血病作用,并可诱导小鼠产生免疫记忆和免疫保护。机制探讨发现,接种IL-18基因修饰瘤苗后,小鼠脾脏淋巴细胞对L1210肿瘤细胞的CTL及NK细胞杀伤活性明显高于对照组(P<0.05),提示IL-18基因修饰瘤苗能够显著增强抗肿瘤CTL和NK细胞反应。接种瘤苗可使小鼠IFN-γ水平升高,但与对照相比无统计学意义,提示IFN-γ可能在IL-18基因修饰瘤苗诱导的抗肿瘤免疫应答中作用不大。     

Augmentation of anti-tumor activity by immunization with Mycobacterium tuberculosis (Tbc) and tuberculin-coupled tumor cells

The anti-tumor effect of immunization with heat-killed Mycobacterium tuberculosis (Tbc) and Tuberculin (PPD)-coupled syngeneic tumor cells was examined in vivo. Three tumor cell lines were employed. Immunization of Tbc-primed BALB/c mice with PPD-coupled syngeneic Meth-A tumor cells displayed a potent anti-tumor effect on viable Meth-A cells inoculated subcutaneously. Neither PPD-coupled LLC (Lewis Lung Carcinoma) cells nor sonicated PPD-coupled Meth-A cells were capable of immunizing these mice. PPD-coupled syngeneic whole tumor cells were indispensable for induction of this tumor-specific resistance. Immunization of Tbc-primed C3H/He mice with PPD-coupled syngeneic MH134 tumor cells did not elicit anti-tumor activity against MH134, but additional pretreatment of mice with cyclophosphamide brought on an anti-tumor effect. Antimetastatic reactivity was investigated in C57BL/6 mice bearing LLC, with a reduction in metastases noted. This antimetastatic effect was observed even when the mice were immunized with PPD-coupled LLC cells three days after removal of the initial tumor. Immunization with Tbc and PPD-coupled Meth-A cells together with intraperitoneal administration of murine or rat interleukin 2 (IL 2) further augmented anti-Meth-A resistance. Murine IL 2 further inhibited tumor growth during the early stage, while rat IL 2 showed an anti-tumor effect throughout the course of tumor growth.     

IL-5 receptor positive B cells, but not eosinophils, are functionally and numerically influenced in mice carrying the X-linked immune defect

Mouse IL-5 (mIL-5) acts on B cells and eosinophils to inducegrowth and differentiation through the mIL-5 specific receptor(mIL-5R). The functional high-affinity mIL-5R is a heterodimercomposed of and ß chains. We investigated the expressionof mIL-5R and the responsiveness of B cells and eosinophilsto mIL-5 In X-linked immunodeficient (xld) mice. mIL-5R expressionanalyzed by using mAbs specific for and ß chainsrevealed that xld B cells had fewer mIL-5R⁺mIL-5Rß⁺than BALB/c B cells. In particular, a decrease in the numberof peritoneal mIL-5R⁺ B cells among Ly-1 B cells (known as B-1cells) was remarkable. Furthermore, the frequency of precursorsof mIL-5 responsive B cells in xld mice was 100-fold lower thanthat of BALB/c mice. Interestingly, sorted mIL-5R+ peritonealB cells from xld mice displayed a low response to mIL-5. Intraperltonealinjection of mIL-5 into BALB/c mice induced polyclonal IgM productionand an increase in the number of eosinophils. The same regimenfailed to induce an increase in the same parameters in xld mice.However, xld mice showed mIL-5-induced eoslnophilla in peripheralblood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injectedxld mice expressed both and ß chains of mIL-5, andresponded to mlL-5 with prolonged in vitro survival.     

pHSP65疫苗增强结核杆菌特异性T细胞产生IFN-γ

为提高BCG的免疫效果,以BCG进行初次免疫,比较pHSP65基因疫苗和BCG疫苗加强免疫的效果,寻找新型结核疫苗的初免-加强免疫策略。于第0周皮下接种BCG,第6、8周给予pHSP65滴鼻免疫或BCG加强免疫,末次免疫后2周检测全身及肺脏局部IFN-γ的产生。与BCG初免/BCG加强免疫相比,经pHSP65基因疫苗滴鼻加强免疫后,ELISPOT结果显示脾脏和肺局部分泌IFN-γ的淋巴细胞数量显著增加,流式检测显示IFN-γ+CD4+T细胞数量显著增加,ELISA结果证实淋巴细胞IFN-γ的分泌显著增高,提示经BCG初次免疫后,以pHSP65 DNA滴鼻加强免疫可显著增强全身和肺局部T淋巴细胞IFN-γ的产生,具有良好的抗结核感染潜能。     

Autoimmune response induced by dendritic cells exerts anti-tumor effect in murine model of leukemia

An autoimmune response can be induced with the application of dendritic cells (DCs), offering a viable tumor vaccine for cancer immunotherapy. Previous studies have shown that DC-based tumor vaccines in animal tumor models can inhibit tumor growth and induce autoantibodies transiently without clinical or histological features of autoimmunity. The present study aimed to investigate the role of immune response induced by dendritic cells-based therapy, especially anti-ds DNA antibodies in tumor inhibition. In this study, high titers of anti-ds DNA antibodies were induced after injecting syngeneic dendritic cells into BALB/c mice. In addition, mice immunized with DCs showed the inhibition of RL ♂ 1, BALB/c leukemia cell line, tumor growth, and prolonged survival times of tumor mice but no significant difference was found in specific CTL response and NK cell activity when compared to those of the control group. Anti-ds DNA monoclonal antibodies can recognize RL ♂ 1 cells but not normal cells by FACS analysis. Monoclonal anti-ds DNA antibody was demonstrated to be able to lyse tumor cells via complement mediated reaction in vitro and also exhibits the anti-tumor effects when the antibody was injected into tumor-implanted mice. The data suggested that immunization with dendritic cells can induce autoimmune response, which might exert anti-tumor activity in vivo.     

Systemic injection of TLR1/2 agonist improves adoptive antigen-specific T cell therapy in glioma-bearing mice

Adoptive immunotherapy is an attractive strategy for glioma treatment. However, some obstacles still need be overcome. In this study, GL261-bearing mice treated with adoptively transferred antigen-specific T cells and systemic injection of bacterial lipoprotein (BLP), a TLR1/2 agonist, got a long-term survival and even immune protection. By analyzing adoptive T cells, it was found that BLP maintained T cell survival, proliferation and anti-tumor efficacy in the brains of tumor-bearing hosts. Moreover, tumor microenvironment was modified by up-regulating IFN-γ-secreting CD8+ T cells and down-regulating MDSC, which might be related with high CXCL10 and low CCL2 expression. In addition, TLR2 deficiency abrogated therapeutic effect with increased MDSC accumulation and decreased IFN-γ-secreting CD8+ T cells in the brains. Thus, the systemic injection of BLP could improve the adoptive T cell therapy by maintaining T cell persistence, modifying the tumor microenvironment and even inducing systemic anti-tumor immunity, which might offer a clinically promising immunotherapeutic strategy for glioma.     

Vaccination with recoverin,a cancer-associated retinopathy antigen,induces autoimmune retinal dysfunction and tumor cell regression in mice

Recoverin (Rec)-specific CTL present in peripheral blood recognize Rec-expressing tumor cells of patients with cancer-associated retinopathy (CAR), a paraneoplastic retinopathy syndrome. To evaluate the effects of Rec on retina and cancer cells, we generated an experimental mouse model, tested the induction of Rec-specific anti-tumor CTL, and analyzed retinal function using electroretinogram (ERG) in these animals. We observed a Rec-specific CTL response in BALB/c mice and significant growth inhibition of Rec-expressing syngeneic MethA fibrosarcoma cells in vivo. R64 (AYAQHVFRSF) peptide, derived from Rec that induces anti-tumor CTL in humans, produced anti-tumor effects in BALB/c mice. Furthermore, elevated anti-Rec antibodies correlated with decreased ERG amplitudes in Rec, Rec-expressing tumor and R64-treated mice. These data suggest that Rec contains amino acid sequences which cause retinal dysfunction, but they also induce anti-tumor CTL and tumor regression. These observations describe initial characterization of the CAR mouse model, a necessary step in developing new insight into immunological mechanisms of paraneoplastic syndromes and tumor immunity for potential immunotherapeutic approaches to cancer.