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     

B7-1基因转染小鼠EL-4淋巴瘤诱导带瘤宿主抗瘤效应的体内研究

本文研究了IL-2、IL-4、IL-6基因转染后B16黑色素瘤细胞表面MHCⅠ类抗原及ICAM-1的表达水平,并探讨了其在CTL诱导过程中的作用.结果表明,IL-2、IL-4、IL-6基因转染的B16黑色素瘤细胞表面MHCⅠ类抗原及ICAM-1表达均高于野生型B16黑色素瘤细胞及转染对照质粒的B16黑色素瘤细胞.体内接种后,小鼠脾脏CTL活性明显增强,CTL培养体系中IFN-γ、TNF-α的分泌水平也增高.在CTL诱导体系中加入抗ICAM-1单抗可以抑制CTL的活化,加入抗MHCⅠ类分子单抗后可使CTL的活化完全阻断.这提示细胞因子基因转染可能通过使肿瘤细胞表面MHCⅠ类抗原、ICAM-1的表达增加,从而增强瘤苗的免疫原性.     

GM-CSF/IL-4基因修饰的瘤苗不同的免疫途径诱导不同免疫反应的观察

对细胞因子基因修饰瘤苗的研究表明,IL-2、IL-4、IFN-γ、GM-CSF等一系列细胞因子基因以不同载体转入肿瘤细胞制成瘤苗后皮下免疫小鼠,均可增强机体抗肿瘤免疫力,其机制可能是由于瘤苗分泌的细胞因子促进了免疫细胞对肿瘤抗原的识别、提呈及对肿瘤细胞的杀伤能力.有文献报道,逆转录病毒介导的GM-CSF和IL-4共同转染瘤苗可以有效激发机体抗肿瘤免疫力,为探讨不同途径瘤苗免疫后机体的免疫反应,我们采用皮下、腹腔、脾内、静脉四种途径接种GM-CSF、IL-4基因双重转染的小鼠红白血病细胞FBL-3瘤苗,发现免疫途径不同,所激发的免疫应答类型不同,诱导机体生成的免疫力不同,提示某些瘤苗应用时应选择适当的免疫途径.     

AFP基因转染的树突状细胞的生物学特性及靶向性CTL细胞毒作用

目的:观察基因转染的树突状细胞生物学特性及靶向性CTL对肿瘤细胞的杀伤作用。方法:用重组IL-4和GM-CSF诱导扩增小鼠骨髓来源的单个核细胞,将表达AFP基因的重组腺病毒转染DC(dendriticcells,DC),构建基因转染的DC瘤苗,并免疫C57BL/6小鼠。用流式细胞术(FCS)检测转染前后DC细胞表面分子MHCI、MHCII、LFA、ICAM、B7.1、和B7.2等的变化。取免疫小鼠脾细胞诱导细胞毒性CTL,LDH非放射性细胞毒性试验检测其对不同肿瘤细胞的杀伤作用。用ELISA法检测CTL诱导过程中TNF-α和IFN-γ分泌变化规律。结果:AFP基因转染后的DC分子表面高表达MHCI、MHCII、LFA、ICAM、B7.1、和B7.2等分子,尤其是MHCII、ICAM和LFA分子,与转染前比较具有显著性差异(P<0.05)。与对照组比较,AFP抗原诱导的靶向性CTL对AFP分泌性肿瘤细胞具有更强的杀瘤活性,且在CTL诱导过程中TNF-α和IFN-γ分泌水平较对照组也有显著性差异。结论:AFP基因重组腺病毒修饰的DC能表达高水平的MHCI、MHCII、LFA、ICAM、B7.1、和B7.2等分子,为CTL进一步活化提供了共刺激信号;用该瘤苗免疫C57BL/6J小鼠,能诱导出针对AFP抗原的靶向性CTL,从而实现对AFP分泌性肿瘤细胞的特异性杀伤作用。     

GM-CSF和多种细胞因子基因转导系统及其抗肿瘤免疫效应的研究

为了增强免疫基因治疗瘤菌诱导机体抗肿瘤系统免疫效应,我们应用逆病毒中介基因转移技术,将IL-2、IL-4、IL-6、IL-7、IFN-α、B7-1、TNF-α、GM-CSF等多种细胞因子基因导入肿瘤细胞,证实了这些基因修饰的肿瘤细胞.由于多种细胞因子基因的导入,诱导宿主抗肿瘤系统免疫能力明显增强.在此基础上,我们为了开发增强宿主免疫的肿瘤基因治疗新方案,构建了可以表达双价细胞因子GM-CSF、TNF-α、IL-4的逆病毒载体的基因修饰瘤苗.通过接种放射线灭活的分泌GM-CSF和IL-4细胞因子的肿瘤细胞即细胞因子肿瘤疫苗,来诱导有效地抗肿瘤系统免疫功能.应用构建的双价逆病毒载体和逆病毒中介基因转移技术,对肺癌、脑肿瘤小鼠模型进行了双价细胞因子基因瘤苗的抗肿瘤免疫增强效果和     

重组痘苗病毒介导的IL-2基因对小鼠Lewis肺癌的体内药效学研究

目的应用重组痘苗病毒介导的IL-2对小鼠Lewis肺癌直接进行局部转染,并对其疗效进行观察和评价.方法本项研究采用缺陷型重组痘苗病毒为载体,经同源重组,筛选出IL-2基因的rW-IL-2,体外转染小鼠Lewis肺癌,对其疗效进行观察.结果短期内即可检出IL-2的表达,并且免疫原性也得到提高.用该载体以in vivo方式原位转染荷瘤小鼠局部;激活了小鼠体内的CTL、LAK及NK细胞,显著抑制了小鼠肿瘤的生长,部分小鼠未能形成肿瘤.结论提示重组痘苗病毒为载体介导的IL-2基因治疗具有良好的前景.     

腺病毒介导的IL—2基因及B7—1基因联合转染G422细胞致瘤原性和免疫原性的变化

目的研究联合细胞因子基因转染的D422胶质母细胞瘤细胞体内致瘤原性和免疫原性的变化,为胶质瘤的免疫基因治疗打下基础.方法IL-2基因和B7-1基因转染的G422细胞1×105皮下和脑内接种,观察肿瘤生长速度和荷瘤小鼠的存活期,2周取脾脏,检测NK、LAK和CTL的杀伤活性.结果IL-2和B7-1基因联合转染的G422细胞,皮下接种后肿瘤生长明显减慢,脑内接种动物存活期明显延长,NK、LAK和CTL的杀伤活性增强.结论IL-2基因和B7-1基因联合转染的G422细胞,致瘤原性下降,免疫原性增强,能有效激活机体特异性与非特异性抗肿瘤免疫反应.     

4-1BBL修饰小鼠结肠癌疫苗体外诱导抗肿瘤免疫反应的研究

背景与目的:研究4-1BBL(4-1BB Ligand,即CD137配体)转基因小鼠结肠癌细胞瘤苗体外诱导细胞毒性T淋巴细胞(Cytotoxic T lymphocytes,CTL)特异性杀伤活性及刺激淋巴细胞产生细胞因子的作用.材料与方法:采用脂质体介导法将真核表达质粒pMKITneo/4-1BBL导入小鼠结肠癌colon26细胞,经G418筛选后获得4-1BBL高表达克隆,用丝裂霉素C(MMC)处理后,制成肿瘤细胞瘤苗,与经体外诱导的同系小鼠CTL共同培养,测定对CTL特异性杀伤活性及对脾细胞产生细胞因子(IL-2、IL.-4和IFN-γ)的影响.结果:转染4-1BBL的colon26细胞高表达4-1BBL蛋白,将该细胞经MMC处理后制成的瘤苗,与野生型colon26细胞相比,对CTL特异性杀伤亲本肿瘤细胞的作用明显增强(P＜0.01),但CTL对非亲本肿瘤细胞的杀伤作用无明显影响(P＞0.05);该瘤苗在体外能显著增强脾细胞分泌细胞因子(IL-2、IL-4和IFN-γ)的能力(P＜0.01).结论:4-1BBL转基因小鼠结肠癌细胞瘤苗能有效诱导抗结肠癌免疫反应.     

米托蒽醌处理B16F10-ESAT-6-gpi/IL-21瘤苗特征及其诱导的抗肿瘤免疫反应初探

背景与目的:蒽环类药物处理可使肿瘤细胞免疫原性增加。本文旨在分析米托蒽醌(mitoxantrone,MIT)处理的B16F10-ESAT-6-gpi/IL-21瘤苗特征,初步探讨该瘤苗诱导的抗肿瘤免疫反应。方法:MIT处理B16F10-ESAT-6-gpi/IL-21瘤苗后,用吖啶橙/嗅化乙啶(AO/EB)染色法观察瘤苗细胞形态,流式细胞仪(FCM)检测其粒度及凋亡比例,荧光显微镜观察瘤苗凋亡后细胞膜表面结核杆菌早期分泌靶抗原6KD(ESAT-6)的表达情况,蛋白质印迹法(Western blot)检测瘤苗经MIT处理后IL-21的表达。瘤苗免疫小鼠后,FCM检测了补体依赖的细胞毒性(complement dependent cytotoxicity,CDC)及细胞毒T细胞(cytotoxicT lymphocyte,CTL)活性。结果:经MIT处理后,瘤苗停止分裂,细胞逐渐增大,数日内可保持生物活力,并表达IL-21。瘤苗细胞凋亡后,ESAT-6成点簇状分布于胞膜表面。MIT处理的瘤苗能诱导小鼠产生抗肿瘤免疫应答,免疫鼠血清和CD8+T细胞可分别通过CDC和CTL杀伤野生型B16F10细胞。结论:MIT处理的B16F10-ESAT-6-gpi/IL-21瘤苗失去增殖能力,但仍能表达IL-21且具有免疫原性,能诱导小鼠产生抗肿瘤免疫反应。     

用表达IL-2的重组腺病毒瘤体内注射诱导肿瘤消退及免疫反应

IL-2能通过增强CTL细胞的杀伤活性、诱导LAK细胞及激活TIL等具有抗肿瘤作用。而腺病毒以其扩增快、转染范围广,以及不整合入感染细胞的基因组等优点适于临床应用。本文作者将携带IL-2基因的重组腺病毒AdCA IL-2转入肿瘤细胞后种入小鼠体内或直接在肿瘤部位进行瘤体内注射,检测了IL-2的表达,并观察了对肿瘤生长的影响。     

ANTI-METASTATIC EFFECT OF ONCOLYSATES FROM MURINE MELANOMA CELLS TRANSFECTED WITH RECOMBINANT VACCINIA VIRUS ENCODING HUMAN IL-2

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Enhanced antitumor immune responses of IL-2 gene-modified tumor vaccine by combination with IL-1 and low dose cyclophosphamide.

To enhance the antitumor immunity induced by IL-2 gene-modified tumor vaccine, we proposed a combined protocol to treat tumor-bearing mice using IL-2 gene-modified tumor vaccine in combination with IL-1 and low-dose Cyclophosphamide(Cy). After treatment with IL-2 gene-modified B16 melanoma cell vaccine alone, the pulmonary metastases of tumor-bearing mice were reduced and their survival time was prolonged. The anti-metastases effect was improved when the vaccine was used in combination with IL-1 or low-dose Cy. The best therapeutic effect was achieved when the IL-2 gene-modified vaccine was combined with IL-1 and low-dose Cy. The cytotoxicity of the splenic CTL, NK, and the levels of IL-2, TNF secreted by splenocytes increased after tumor-bearing mice were treated with the IL-2 gene-modified tumor vaccine. The above antitumor immune functions were augmented more significantly when IL-1, low-dose Cy were used in combination with IL-2 genemodified tumor vaccine. These results demonstrated that the IL-2 gene modified vaccine could exert more potent anti-metastases effects when it is combined with IL-1 or/and low-dose Cy by activating the specific and non-specific antitumor immune responses more effectively.     

Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine

Background

Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine.

Methods

The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis.

Results

We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub-enriched proteins vaccine showed a significant inhibitory effect on in vivo growth of homologous tumor, as well as allogeneic tumor, compared with Ub-depleted proteins and tumor cell lysate. Tumor growth was regressed after three times of vaccination with Ub-enriched proteins in contrast to other groups.

Conclusion

These results indicated that Ub-enriched proteins isolated from tumor cells may have a potential as a potent vaccine for immunotherapy against cancer.     

Anti-angiogenic effect of Biophytum sensitivum is exerted through its cytokine modulation activity and inhibitory activity against VEGF mRNA expression, endothelial cell migration and capillary tube formation

Angiogenesis is a crucial step essential for the growth, progression and metastasis of solid tumors. Substances produced by inflammatory cells, such as cytokines play an important role in the stimulation and progression of angiogenesis. In this study we investigated the anti-angiogenic effect of Biophytum sensitivum, using in vivo as well as in vitro models. In vitro antiangiogenic activity was studied using B16-F10 melanoma cell-induced capillary formation in C57BL/6 mice. Intraperitoneal administration of the extract at a concentration of 50 mg/kg significantly inhibited the tumor directed capillary formation induced by melanoma cells. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, GM-CSF and the direct endothelial cell proliferating agent, VEGF. Administration of Biophytum extract could differentially regulate these cytokine's elevation. The differential elevation is further evidenced by the increased production of IL-2 and tissue inhibitor of metalloprotease-1 (TIMP-1) in the B16-F10 injected, extract treated animals. The extract of B. sensitivum at non-toxic concentrations (1 microg/ml, 5 microg/ml and 10 microg/ml) inhibited the VEGF-induced vessel sprouting in rat aortic ring assay. Moreover, B. sensitivum was able to inhibit the VEGF-induced proliferation, cell migration and capillary-like tube formation of primary cultured human endothelial cells. Furthermore B. sensitivum showed inhibitory effect on VEGF mRNA levels in B16-F10 melanoma cells. Hence the observed antiangiogenic activity of the plant B. sensitivum is exerted through its cytokine modulation activity and inhibitory activity against VEGF mRNA expression.     

Extraction of immunogenic and suppressogenic antigens from variants of B16 melanoma exhibiting low or high metastatic potentials

This investigation examined the effect of soluble antigen prophylaxis on s.c. and metastatic growth of B16 variants which demonstrated either a low or high propensity to colonize the lungs (B16-F1 and B16-F10, respectively) of syngeneic C57BL/6J mice. The two variants share a tumor-associated antigen, since immunization with crude butanol extracts (CBEs) of B16-F1 cells protected hosts against s.c. challenge with either B16-F1 or B16-F10 cells. CBE from B16-F10 (CBE-F10) were unable to engender a measurable immune response against s.c. challenge with either tumor variant. Pretreatment with 100 to 300 micrograms CBE from F1 cells was also effective in reducing the outgrowth of experimentally induced B16-F1 or B16-F10 pulmonary foci. However, mice immunized with 100 micrograms CBE-F10 bore significantly more pulmonary tumors than did phosphate-buffered saline-treated controls. The enhancing and protective activities were specific for the B16 tumor and could be adoptively transferred 24 hr prior to tumor challenge by i.p. injection of 5 X 10(7) spleen cells from CBE-immunized mice. The enhancing activity in the CBE-F10 immune spleen cell population was abolished by depletion of adherent cells onto plastic. Adoptive transfer of the CBE-F10-immune adherent cell population did not affect metastatic growth, suggesting that, in this experimental system, the adherent population was not an efferent suppressor and could not recruit host elements to effect suppression. Indeed, spleen cell-mixing experiments demonstrated that only immune adherent cells combined with immune nonadherent cells could partially reconstitute the tumor growth-enhancing potential of the unfractionated CBE-F10-immune spleen cell population.     

Effect of retinoic acid on tumor-mediated immunologic alterations in mice bearing a variant of the B16 melanoma

This investigation examined the effect of retinoic acid on tumor progression and immunological status of mice bearing the B16-F10 melanoma (previously selected for high lung-colonizing capacity). Tumor cells were implanted s.c. in syngeneic C57BL/6 mice, half of which were treated with beta-all trans retinoic acid (RA). Although RA failed to exhibit direct toxicity on this variant at the concentration used, the immunologic aberrations induced by the tumors were diminished by i.p. RA administration (at 45 micrograms twice/week for 3 weeks). In mice bearing B16-F10 tumors, tumor burdens were decreased from 2.9% of body weight to 1.6%. The mitogenic responses of splenic lymphocytes to concanavalin A (ConA) were increased in tumor-bearing mice following this RA treatment. The presence of these tumor cells decreased the absolute number of CD4- and CD8-positive splenic lymphocytes. Following RA treatment, the CD8-positive population was increased in tumor-bearing mice, while the CD4+ population was not significantly altered. Since previous studies indicated that plasma membrane fragments (or vesicles) could alter lymphocyte distributions and proliferative capacities, the in vitro shedding of membrane fragments from B16-F10 tumor cells was assayed and observed to be decreased after continuous treatment of cultures with 10(-6) M RA for 21 days. Membrane shedding from B16-F10 cells was inhibited by 48.5% following RA treatment. Based on these in vivo and in vitro results, we suggest that RA treatment may diminish tumor growth by decreasing tumor-induced immunosuppressive events.     

Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase sense and antisense genes.

Murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for the prepro form of urokinase in an SV40 expression vector (plasmid pSV2-uPA), and cells expressing high amounts of the human urokinase gene product were selected for by an enzyme-linked immunosorbent assay specific for human high molecular weight urokinase. Southern analysis showed one of the cell lines (clone 7) had incorporated 150 copies of the pSV2-uPA plasmid into its genomic DNA. The human urokinase synthesized by the pSV2-uPA-transfected murine B16 cells was found to be glycosylated and did not bind to the murine cell surface urokinase receptor sites. In an in vivo assay that measures metastasis from a primary tumor (spontaneous metastatic assay), clone 7 cells showed an increased ability to metastasize (12 of 12 mice showed metastatic tumors), while control cells showed a lower ability to metastasize (only 2 of 11 mice showed metastatic tumors). In a second in vivo assay, which measures only the steps of the metastatic migration process during which tumor cells extravasate from the blood and then grow into pulmonary tumors (lung colonization assay), a significant multifold increase in the ability to form lung tumors was shown by the high human urokinase-secreting B16-F1 cells. In B16-F10 cells incorporating an antisense sequence to preprourokinase (plasmid pSV1-ASuPA-265) and secreting significantly decreased amounts of murine urokinase, a corresponding significant decrease in lung colonization was observed. These results provide direct experimental support for a role of secreted (non-surface-bound) urokinase in the colonization steps of the metastatic process. Furthermore, the data indicate that the higher lung colonization ability of the B16-F10 line than of the B16-F1 line is primarily based on the quantitative differences in their abilities to produce urokinase.     

THE HUMORAL ANTITUMOR RESPONSES INDUCED BY IL-4 GENE-MODIFIED TUMOR VACCINE

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Inhibition of murine melanoma experimental metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor

Recombinant desulfatohirudin (r-hirudin), a highly specific inhibitor of thrombin, was examined to determine whether it would inhibit production of experimental lung metastasis by B16-F10 melanoma cells. In in vitro assays using mouse plasma, the high level of procoagulant activity in B16-F10 cells was significantly inhibited by r-hirudin in a dose-dependent manner. From 15 to 120 min after s.c. administration into C57BL/6 mice, r-hirudin (10 mg/kg) markedly prolonged clotting time in a time course pattern that directly correlated with that of blood distribution of 125I-labeled r-hirudin. The production of experimental lung metastasis by B16-F10 cells was significantly inhibited by r-hirudin administered s.c. at time points ranging from 120 min before to 60 min after tumor cell inoculation with the most significant effects found in mice given r-hirudin 15 or 2 min before the i.v. injection of tumor cells. The organ distribution of [125I]IdUrd-labeled tumor cells demonstrated a clear difference in the lungs of mice treated with r-hirudin and the lungs of control mice, and these differences directly correlated with the number of lung tumor colonies found 3 weeks later. The inhibition of lung metastasis was not due to direct antitumor effects of r-hirudin. These results suggest that inhibition of coagulation events by r-hirudin significantly inhibit experimental lung metastasis during a critical time of 60 min after the entry of tumor cells into the circulation.