【作者】 王福利；

【导师】 王禾；

【作者基本信息】 第四军医大学 ， 外科学， 2007， 博士

【摘要】 目的:树突状细胞(DC)是目前在肿瘤免疫中功能最强的一种抗原递呈细胞,以其为基础制成的肿瘤疫苗,可多途径诱导机体产生特异性抗肿瘤免疫。但是肿瘤细胞分泌大量的转化生长因子(TGF-β)抑制DC递呈抗原和促进DC凋亡。我们采用前列腺癌TRAMP-C2细胞裂解产物作为抗原加载C57BL/6小鼠骨髓DC,诱导出前列腺癌特异性的DC。修饰TβRII基因,构建逆转录病毒载体,体外基因转染前列腺癌特异性的DC,检测转染后前列腺癌特异性DC的表面抗原、细胞增殖性、分泌功能变化,评价TGF-β不敏感的DC疫苗对TRAMP-C2前列腺癌荷瘤C57BL/6小鼠的免疫治疗作用。方法:1)构建含有显性负相TGF-βII型受体(TβRIIDN)质粒的逆转录病毒:含有TβRIIDN质粒的逆转录病毒转染293包装细胞,32℃下共转染12小时,10% DMEM培养基37℃下孵化过夜,PBS漂洗后同样条件再次转染293细胞24小时,收集上清,获得含有TβRIIDN重组逆转录病毒。2)负载前列腺癌抗原的DC细胞分离、培养及鉴定:将C57BL/6小鼠新鲜骨髓用红细胞去除法获取DC细胞,加入含rhIL-4(1000U/ml)、GM-CSF(1000U/ml)的完全培养基,隔日半量换液,在第6d梯度离心收获非粘附细胞为非成熟的DC细胞。反复冻融获得TRAMP-C2细胞裂解物,裂解物反复刺激非成熟的DC细胞获得负载前列腺癌抗原的成熟DC细胞。于光镜下形态学鉴定,用流式细胞仪检测细胞表型MHC class II,CD40,CD11c,CD80和CD86。3)DC细胞转染,获取TβRIIDN-DC细胞:含有TβRIIDN重组逆转录病毒转染负载前列腺癌抗原的DC细胞5%CO2、37℃下孵化48小时,获得表达TβRIIDN的DC细胞,流式细胞仪检测DC细胞表型变化。4)TβRIIDN-DC细胞体外实验:不同的DC细胞与TGF-β共同培养,流式细胞仪测定表面共刺激分子(CD80/CD86)变化,脱氧胸腺嘧啶苷核素掺入法进行增殖抑制实验,Western blot检测SMAD-2和磷酸化SMAD-2的表达。5)DC疫苗体内抗肿瘤评价:建立前列腺癌TRAMP-C2细胞皮下荷瘤C57BL/6小鼠模型,皮下荷瘤小鼠40只,随机分为4组,每组10只,分别皮下注射TβRIIDN-DC细胞、负载前列腺癌抗原的DC细胞、GFP-DC细胞和磷酸盐缓冲液(PBS),15天后重复注射,酶联免疫吸附(ELISA)测定细胞因子IFN-γ和IL-12变化,观察各组荷瘤小鼠的生存期和肿瘤的体积改变。6)体内CTL活性检测:最后一次疫苗免疫后5 d取其脾脏,分离、活化T淋巴细胞,Cr51释放实验测定肿瘤特异性杀伤T细胞(CTL)对TRAMP-C2细胞的细胞毒作用。黑色素瘤细胞B16-F1作为对照靶细胞。结果:1)流式细胞仪分析CD11c表达,小鼠骨髓来源的DC细胞纯度为90.8%。含有TβRIIDN和GFP的逆转录病毒转染效率分别为92.7%和90.6%。2)流式细胞仪检测不成熟DC细胞、负载前列腺癌抗原的成熟DC细胞、TβRIIDN-DC细胞、GFP-DC细胞的CD86、CD80、CD40、CD11c及MHC-II分子表型改变,成熟DC细胞、TβRIIDN-DC细胞、GFP-DC细胞表型表达明显高于不成熟DC细胞(P<0.01),成熟DC细胞、TβRIIDN-DC细胞、GFP-DC细胞之间细胞表型无明显差别(P>0.05)。说明表达TβRIIDN不影响DC细胞的分子表型。3)负载前列腺癌抗原的成熟DC细胞、TβRIIDN-DC细胞、GFP-DC细胞与5 ng/ml的TGF-β孵育后,Western blot在各组中均可以检测到Smad-2, TβRIIDN-DC细胞中却没有检测到磷酸化的Smad-2。表明TβRII显性负相表达阻断了DC细胞的TGF-β信号通路。4)与TGF-β共同培养72小时后,非转染的成熟DC细胞、GFP-DC细胞和TβRIIDN-DC细胞的脱氧胸腺嘧啶苷核素摄入抑制率分别为62.5%、65.5%和15%,TβRIIDN-DC细胞的脱氧胸腺嘧啶苷核素摄入抑制率与其他各组比较差异有显著统计学意义(P<0.05)。在给予TGF-β的正常条件下,非转染的成熟DC细胞、GFP-DC细胞不能增殖并且15天之内死亡,然而,TβRIIDN-DC细胞继续正常增殖和生长,表明TβRIIDN-DC细胞对TGF-β的抗增殖作用不敏感。5)非转染的成熟DC细胞、GFP-DC细胞和TβRIIDN-DC细胞与TGF-β及肿瘤抗原共同培养7天,流式细胞仪测定表面共刺激分子(CD80/CD86),在TGF-β作用下CD80和CD86在TβRIIDN-DC细胞的表达明显高于其他各组(P<0.01)。6)成功地构建了前列腺癌TRAMP-C2细胞皮下荷瘤C57BL/6小鼠模型,接种非转染的DC细胞、GFP-DC细胞和TβRIIDN-DC细胞的荷瘤小鼠与对照组相比能明显抑制肿瘤生长(P<0.01,P<0.05 and P<0.05,vs.对照组),其中TβRIIDN-DC组抑制效果更明显,有2例肿瘤完全消失。这表明TβRIIDN-DC疫苗有更强的抗肿瘤效果。50天后接种PBS、非转染的DC细胞、GFP-DC细胞和TβRIIDN-DC细胞的荷瘤小鼠生存率分别为0%、20%、30%和80%。Mantel-Haenszel log-rank统计分析表明:TβRIIDN-DC细胞与其他2组相比差别有统计学意义(P<0.01)。这些结果说明TGF-β不敏感的DC细胞能有效提高TRAMP-C2皮下荷瘤小鼠的生存率。7)接种PBS的荷瘤小鼠体内测出IFN-γ和IL-12基础水平,接种非转染的DC细胞、GFP-DC细胞的荷瘤小鼠体内IFN-γ和IL-12水平明显升高,接种TβRIIDN-DC细胞的荷瘤小鼠体内该两种细胞因子水平升高更明显。8)标准的Cr51释放实验测定CTL杀伤活性,非转染的DC细胞、GFP-DC细胞和TβRIIDN-DC细胞处理的荷瘤小鼠CTL对TRAMP-C2细胞有较高的杀伤活性,其中,TGF-β不敏感的DC细胞诱导出最强的CTL杀伤活性(E/T比率为100:1时杀伤活性为85%),对无关的黑色素瘤B16-F1细胞没有杀伤作用。结果表明阻断的TGF-β信号通路能增加肿瘤特异性杀伤活性,提高DC疫苗的效能。结论:1)成功构建了含有TβRIIDN质粒的逆转录病毒;2)采用前列腺癌TRAMP-C2细胞裂解产物作为抗原加载C57BL/6小鼠骨髓DC,诱导出了前列腺癌特异性的DC;3)首次使用修饰后TβRII基因转染前列腺癌特异性的DC,使TβRII显性负相表达,阻断TGF-β信号通路;4)TβRIIDN并不影响转染DC细胞的表型,在TGF-β作用下TβRIIDN-DC表面共刺激分子表达增高;5)TGF-β不敏感的DC细胞能明显提高TRAMP-C2皮下荷瘤小鼠的生存率,明显抑制肿瘤生长,而且有2例肿瘤完全消失。TGF-β不敏感的DC细胞处理的荷瘤小鼠体内IFN-γ和IL-12水平明显升高;6)TGF-β不敏感的DC细胞诱导出最强的CTL杀伤活性,E/T比率为100:1时杀伤活性为85%,CTL具有肿瘤特异性,对无关的黑色素瘤B16-F1细胞没有杀伤作用。更多还原

【Abstract】 ObjectiveDCs are highly potent initiators of the immune response, characterized by their ability to engulf, process, and present antigens to T lymphocytes. In recent years, DC-based anti-tumor vaccines have emerged as promising strategies for cancer immunotherapy. But high levels of TGF-βproduced by cancer cells inhibit the ability of DCs to present antigen, stimulate tumor-sensitized T lymphocytes. C57BL/6 murine bone marrow DCs were pulsed with freeze–thawed TRAMP-C2 tumor lysate and induced prostate cancer specific DCs. We successfully constructed a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN). The tumor lysate-pulsed DCs were rendered TGF-βinsensitive by infecting with a retrovirus containing TβRIIDN, leading to the blockade of TGF-βsignals to members of the Smad family. After transfection we detected surface antigen, cell proliferation, secretion and evaluated the immunotherapy of TGF-β insensitive DC vaccines to TRAMP-C2 tumor-bearing C57BL/6 mice. Materials and methods1. To construct a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN): Pantropic GP293 retroviral packaging cells were seeded at a density of 2.5×106 cells in T-25 collagen I-coated flasks 24 h before plasmid transfection in antibiotic-free 10% Dulbecco’s Modified Eagle Medium. A mixture of 2μg retroviral plasmid and 2μg vesicular stomatitis virus envelope G protein (VSV-G) envelope plasmid was cotransfected in serum-free DMEM using LipofectAMINE-Plus, according to the manufacturer’s protocols. Briefly, cells were transfected for 12 h followed by the addition of an equivalent volume of 10%DMEM and incubation for an additional 12 h. Afterward, the supernatant was aspirated, the cells were rinsed gently in PBS, and 3 ml of fresh 10%DMEM was added each flask. 24 h later, virus-containing supernatant was collected and used to infect target cells.2. Isolation, cultivation and identification of tumor lysate-pulsed DC: Erythrocyte-depleted murine bone marrow cells were obtained from the femurs and tibiae of C57BL/6 mice under aseptic conditions and cultured at 1×106 cells/ml in CM supplemented with 1000 U/ml recombinant murine granulocyte-macrophage-colony-stimulating factor (rmGM-CSF) and 1000 U/ml recombinant murine interleukin-4 (rmIL-4). The medium was replaced on day 2 with additional recombinant cytokines. On day 6, nonadherent DCs were harvested by gradient centrifugation and were further purified with MACS CD11c beads. Immature DCs were pulsed with freeze–thawed tumor lysate to obtain tumor lysate-pulsed DC. Tumor lysate-pulsed DCs were identified under light microscope. Immatured DCs, tumor lysate-pulsed DCs, GFP-transduced DCs and tumor lysate-pulsed TGF-β-insensitive DCs, which were cultured for an additional 18 h, were analyzed by flow cytometry, using a panel of Abs specific for MHC class II, CD40, CD11c, CD80, and CD86.3. Infection of DCs with Retrovirus: Tumor lysate-pulsed DCs were infected with the retrovirus containing TβRIIDN or GFP vector. The cells were rinsed gently in 5%CO2, 37℃for 48 hours to get TβRIIDN DCs. The immunophenotype of TβRIIDN DCs were analyzed by flow cytometry.4. Experiment of TβRIIDN DCs in vitro: Different DCs were cultured with TGF-β. The surface co-stimulatory molecules (CD80/CD86) were analyzed by flow cytometry. The antiproliferative effects of TGF-βwere observed by thymidine incorporation assay. Smad-2 and phosphorylated Smad-2 were detected by Western blot.5. Antitumor Analyses in Vivo: TRAMP-C2 tumors were established in mice. 40 C57BL/6 tumor-bearing mice were divided into four groups randomly and inoculated s.c. with nontransduced DCs, tumor lysate-pulsed TGF-β-insensitive DCs, GFP-transduced DCs, or PBS. The vaccination was repeated on day 15. Serum levels of IFN-γand IL-12 were determined by enzyme-linked immunoabsorbant assay (ELISA). Tumor growth and mouse survival were monitored daily post-inoculation. 6. Cytotoxicity T Lymphocyte Assays: Splenic cells were obtained from the tumor-bearing mice 5 days after the final vaccination and cocultured with X-ray (40 Gy)-irradiated TRAMP-C2 cells (2×105) in 24-well plates for 4 days. The activated T-cells were harvested and used as effector cells against 51Cr-loaded TRAMP-C2 target cells. An irrelevant cancer cell line, mouse melanoma cell line, B16-F1 was used as a nonspecific control.Results:1. Erythrocyte-depleted murine bone marrow cells were freshly isolated from the femurs and tibiae of C57BL/6 mice. The purity of bone marrow–derived DC, determined through analysis on CD11c staining by flow cytometry, was 90.8%. Tumor lysate-pulsed DCs were infected with the TβRIIDN-containing or GFP control retrovirus. The infection efficiency was determined by flow cytometry analysis. They were 92.7% and 90.6%, respectively, for the T?RIIDN- containing and the GFP control retroviruses.2. DC surface molecules MHC class II, CD40, CD11c, CD80 and CD86, were analyzed and compared among immatured DCs, tumor lysate-pulsed DCs, GFP-transduced DCs and TβRIIDN-transduced DCs by flow cytometry. The expression of CD86, CD80, MHC class II and CD40 on tumor lysate-pulsed DCs, GFP-transduced DCs and TβRIIDN-transduced DCs was higher than those on immatured DCs (P<0.01). No obvious differences were observed among tumor lysate-pulsed DCs, GFP-transduced DCs and TβRIIDN-transduced DCs (P>0.05), indicating that the transduction of TβRIIDN did not affect the immunophenotype of DCs.3. Smad-2 and phosphorylated Smad-2 were detected by Western blot analysis after the TβRIIDN-transduced or GFP-transduced DCs were treated with 5 ng/ml TGF-β1. The presence of Smad-2 was detected in all DC groups. But, phosphorylated Smad-2 was only detected in nontransduced and GFP-transduced DCs in response to TGF-β1; absence of phosphorylated Smad-2 in TβRIIDN- transduced DCs confirmed that TGF-βsignal transduction was blocked by the presence of the TβRIIDN.4. The inhibitory rate of TGF-βon thymidine uptake was compared among the TβRIIDN-transduced DCs, GFP-transduced, and nontransduced DCs after the addition of TGF-β1 for 72 h. TGF-β1 showed a dramatic antiproliferative effect on the established nontransduced and GFP-transduced DCs, inhibiting uptake by a mean of 62.5% and 65.5% respectively. Whereas the mean inhibitory rate of thymidine uptake by TβRIIDN-transduced DCs was 15%, the resistance to the antiproliferative effects of TβRIIDN-transduced DCs was statistically significant when compared with the other groups (P<0.05). Importantly, when cells were maintained under normal growth conditions in the presence of TGF-β1, the nontransduced and GFP-transduced DCs failed to proliferate and died within 15 days. TβRIIDN-transduced DCs, however, continued to proliferate and grow normally, showing significant resistance to the antiproliferative effects of TGF-β1.5. Nontransduced DCs, GFP-transduced DCs and TβRIIDN- transduced DCs were cultured with freeze–thawed tumor lysate and 10 ng/mL TGF-β1 for 7 days. The surface co-stimulatory molecules CD80 and CD86, were analyzed by flow cytometry. As expected, expression of CD86 and CD80 was higher on TβRIIDN-transduced DCs than on nontransduced DCs and GFP-transduced DCs (P<0.01) in the presence of TGF-β1.6. To assess the antitumor effect of the TβRIIDN-transduced DC vaccine in vivo, TRAMP-C2 tumors were established in C57BL/6 mice. A suspension of nontransduced DCs, GFP-transduced DCs, or TβRIIDN-transduced DCs was injected into tumor-bearing mice (n = 10/group). PBS was used as a negative control. These experimental groups were designed to evaluate whether blocking TGF-βsignaling alters the efficacy of DC vaccine in inducing anti-tumor immune responses and mortality of the tumor-bearing mice. Immunization with TβRIIDN-transduced DCs, GFP-transduced DCs or nontransduced DCs significantly suppressed the growth of the tumor (P<0.01, P<0.05 and P<0.05, vs. control, respectively), with the TβRIIDN-transduced DCs showing the more significant inhibitory effect. Complete tumor regression occurred in 20% of TRAMP-C2-tumor-bearing mice that were treated with TβRIIDN-transduced DCs. These results indicated that the tumor lysate-pulsed TGF-β-insensitive DC vaccine had the strongest anti-tumor effect in all the vaccination groups. Another four groups of ten mice were used to evaluate the survival rate after 50 days. Results showed that survival rate of the untreated, GFP-vector control and nontransduced DCs treated mice was 0%, 20%and 30% respectively, while the survival of the TβRIIDN-transduced DCs treated cohort was 80%. Statistical analysis by using the Mantel-Haenszel log-rank test indicated a significant difference between the TGF-β-insensitive-DC and the other two control groups (P<0.01). The result demonstrate that the TGF-β-insensitive DC vaccine was effective in improving the survival rate in mice bearing TGF-β-secreting tumors.7. In animals injected with PBS, there was a basal level of IFN-γand IL-12. In animals received nontransduced DCs or GFP-transduced DCs, there was a significant increase in levels of both cytokines. A further increase in serum IL-12 and IFN-γwas observed when these cells were rendered insensitive to TGF-? (the T?RIIDN-transduced DCs group), suggesting the increase of activated immune cells in these hosts.8. The ability of CTLs to lyse TRAMP-C2 cells was assayed in vitro using a standard 51Cr release assay. Compared with the PBS-vaccinated group, mice treated with TβRIIDN-transduced DCs, GFP-transduced DCs, or non-transduced DCs all showed a higher cytotoxicity against the TRAMP-C2 cells. The most potent TRAMP-C2-specific splenic CTL response was induced by the TGF-?-insensitive DCs in the tumor bearer (85% killing activity at an effector:target cell ratio of 100:1). No apparent lysis was observed against irrelevant B16-F1 cells. This result indicates that tumor-specific cytolysis is generated by blocking TGF-βsignaling, which enhances the efficacy of DC vaccines. Conclusion1. 1. We successfully constructed a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN).2. C57BL/6 murine bone marrow DCs were pulsed with freeze–thawed TRAMP-C2 tumor lysate and induced prostate cancer specific DCs.3. The tumor lysate-pulsed DCs were rendered TGF-βinsensitive by infecting with a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN), leading to the blockade of TGF-βsignals to members of the Smad family.4. Expression of TβRIIDN did not affect the phenotype of transduced DCs. Expression of CD86 and CD80 was higher on TβRIIDN-transduced DCs than on nontransduced DCs and GFP-transduced DCs (P<0.01) in the presence of TGF-β1.5. TβRIIDN-transduced DCs suppressed tumor growth and increased survival rate of TRAMP-C2 tumor-bearing mice. Furthermore, complete tumor regression occurred in 2 vaccinated mice. TβRIIDN-transduced DCs induced higher IFN-γand IL-12 level in vivo.6. The most potent TRAMP-C2-specific splenic CTL response was induced by the TGF-?-insensitive DCs in the tumor bearer (85% killing activity at an effector:target cell ratio of 100:1). No apparent lysis was observed against irrelevant B16-F1 cells.更多还原