CpG寡聚脱氧核苷酸增强树突状细胞疫苗诱导的特异性抗前列腺癌作用

目的 观察CpG寡聚脱氧核苷酸(CpG-ODN)对截短的人前列腺特异性膜抗原(tPSMA)基因修饰的树突状细胞(DCs)诱导的抗前列腺癌效应.方法 利用复制缺陷性腺病毒AdEasy-1系统,通过基因重组技术构建Ad-tPSMA及Ad-eGFP.将Ad-tPSMA和Ad-eGFP感染鼠源性DCs,用含10μg/L粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素(IL)-4和含10%胎牛血清(FCS)的RPMI 1640培养基诱导培养6 d后,然后再添加1 mg/L的CpG-ODN体外培养1 d,最后添加1 mg/L的脂多糖(LPS)继续培养1 d,使其成熟.流式细胞仪检测DCs细胞表型,CCK-8法检测混合淋巴细胞反应T细胞增殖能力,酶联免疫吸附试验(ELISA)试剂盒检测T细胞分泌细胞因子[IL-2、干扰素(INF)-γ]的影响,CCK-8试剂检测T细胞特性杀伤靶细胞活性.结果 CpG-ODN促进Ad-tPSMA感染的DCs(CpG/DCs-Ad-tPSMA)高表达MHC Ⅱ(83.8±3.7)%、CD80(79.8±5.6)%和CD86(78.3 ±2.8)%,且刺激同种异体T细胞增殖能力明显高于DCs-Ad-tPSMA组、DCs-Ad-eGFP组和未转染的DCs组(P＜0.05);培养上清中细胞因子IL-2(179.64±2.72)ng/L和INF-γ(1581.75±28.61)ng/L,表达水平均明显高于DCs-Ad-tPSMA组、DCs-Ad-eGFP组和未转染的DCs组(P＜0.05);CpG/DCs-Ad-tPSMA组诱导RM-1-tPSMA细胞特异性杀伤率为(55.3±1.2)%,明显高于DCs-Ad-tPSMA组(40.7±1.4)%、DCs-Ad-eGFP组(12.7±1.2)%和未转染的DCs组(10.8±1.7)%(P＜0.05).结论 CpG-ODN能增强PSMA基因修饰的DCs疫苗诱导的特异性抗前列腺癌效应.

Abstract：

Objective To observe cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODN) for the anti-prostate cancer effect induced by dendritic cells (DCs) transduced with recombinant adenovirus vector bearing truncated human prostate specific membrane antigen (tPSMA) gene. Methods Replication deficient adenovirus AdEasy-1 system was used to construct recombination adenovirus Ad-tPSMA and Ad-eGFP. Mouse derived DCs were transduced with Ad-tPSMA and Ad-eGFP, and cultured for 6 days in the presence of 10 μg/L GM-CSF and IL4 in RPMI 1640 containing 10% FCS. After culture with 1 mg/L CpG-ODN for 1 day, DCs were further matured with lipopolysaccharide (LPS) at a concentration of 1μg/L for 1 day. The phenotype of DCs was analyzed by using flow cytometry. T cells proliferation stimulated by DCs in allogeneic mixed lymphocyte reactions and the level of interleukin (IL)-2, interferon (IFN)-γ were detected by ELISA kit, and cytotoxic CTL activity induced by DCs was tested by CCK-8 assay. Results CpG-ODN up-regulated the expression of MHCⅡ (83. 8 ±3. 7)% , CD80 (79. 8 ±5. 6)% and CD86 (78. 3 ±2. 8)% in Ad-tPSMA-tranduced DCs (CpG/DCs-Ad-tPSMA). T cells proliferation stimulated by CpG/DCs-Ad-tPSMA was significantly higher than that in DCs control group, DCs-Ad-tPSMA group and DCs-Ad-eGFP group (P ＜0.05). CpG/DCs-Ad-tPSMA induced increased IL-2 (179. 64 ±2. 72) ng/L, and INF-7 (1581.75 ±28. 61) ng/L expression levels as compared to DCs control group, DCs-Ad-tPSMA group, and DCs-Ad-eGFP group (P＜0. 05), and induced more outstanding RM-1-tPSMA cell specific cytotoxic rate (40.7 ±1.4)%than DCs control group (10. 8 ± 1.7) % , DCs-Ad-tPSMA group (40.7 ± 1.4)% , and DCs-Ad-eGFP group (12.7 ± 1. 2)% (P＜0.05). Conclusion CpG-ODN can promote specific anti-prostate cancer induced by DCs modified with recombinant adenovirus vector bearing tPSMA gene.

CpG-ODN promotes specific anti-prostate cancer induced by dendritic cells vaccine

Objective To observe cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODN) for the anti-prostate cancer effect induced by dendritic cells (DCs) transduced with recombinant adenovirus vector bearing truncated human prostate specific membrane antigen (tPSMA) gene. Methods Replication deficient adenovirus AdEasy-1 system was used to construct recombination adenovirus Ad-tPSMA and Ad-eGFP. Mouse derived DCs were transduced with Ad-tPSMA and Ad-eGFP, and cultured for 6 days in the presence of 10 μg/L GM-CSF and IL4 in RPMI 1640 containing 10% FCS. After culture with 1 mg/L CpG-ODN for 1 day, DCs were further matured with lipopolysaccharide (LPS) at a concentration of 1μg/L for 1 day. The phenotype of DCs was analyzed by using flow cytometry. T cells proliferation stimulated by DCs in allogeneic mixed lymphocyte reactions and the level of interleukin (IL)-2, interferon (IFN)-γ were detected by ELISA kit, and cytotoxic CTL activity induced by DCs was tested by CCK-8 assay. Results CpG-ODN up-regulated the expression of MHCⅡ (83. 8 ±3. 7)% , CD80 (79. 8 ±5. 6)% and CD86 (78. 3 ±2. 8)% in Ad-tPSMA-tranduced DCs (CpG/DCs-Ad-tPSMA). T cells proliferation stimulated by CpG/DCs-Ad-tPSMA was significantly higher than that in DCs control group, DCs-Ad-tPSMA group and DCs-Ad-eGFP group (P ＜0.05). CpG/DCs-Ad-tPSMA induced increased IL-2 (179. 64 ±2. 72) ng/L, and INF-7 (1581.75 ±28. 61) ng/L expression levels as compared to DCs control group, DCs-Ad-tPSMA group, and DCs-Ad-eGFP group (P＜0. 05), and induced more outstanding RM-1-tPSMA cell specific cytotoxic rate (40.7 ±1.4)%than DCs control group (10. 8 ± 1.7) % , DCs-Ad-tPSMA group (40.7 ± 1.4)% , and DCs-Ad-eGFP group (12.7 ± 1. 2)% (P＜0.05). Conclusion CpG-ODN can promote specific anti-prostate cancer induced by DCs modified with recombinant adenovirus vector bearing tPSMA gene.