胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

目的 构建含有胰腺癌相关抗原黏蛋白(MUC)4(sv12)的腺病毒载体并转染树突状细胞(DC)构建DC疫苗,观察DC疫苗对胰腺癌细胞的免疫效应.方法 NCBI获得MUC4(sv12)mRNA序列,利用片段内酶切位点和重叠聚合酶链反应(PCR)技术获得MUCA(sv12)的编码序列,编码序列克隆到腺病毒载体,构建重组腺病毒(rAd-MUC4).病毒颗粒感染树突状细胞,产生活化的MUCA特异性细胞毒性T淋巴细胞(CTL),乳酸脱氢酶(LDH)释放法和Elispot法分别检测体外杀伤活性及干扰素(IFN)-γ的表达,同时设置空病毒(GFP)组和磷酸盐缓冲液(PBS)组作为阴性对照.结果 成功构建含有胰腺癌相关抗原MUCA(sv12)基因的重组腺病毒,LDH法检测结果:MUCA特异性CTL对HLA-A2+和MUC4+Capan-1细胞的毒性[E/T=10:1时为(13.7±6.0)%,20:1时为(21.4±4.7)%,40:时为(36.1±9.5)%],高于HLA-A2+Mcf-7细胞及MUCA+Bxpe-3细胞(P<0.05).Elispot法检测结果:E/T=20:时,Capan-1细胞的MUCA组斑点数为(139.1±23.3),高于GFP组(66.0±16.4)和PBS组(30.5±4.4),P<0.05;GFP组和PBS组差异无统计学意义.结论 腺病毒介导MUCA基因修饰的DC疫苗,可以体外诱导产生特异性CTL细胞,并获得有效的HLA限制性杀伤效应.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.     

胰腺癌黏蛋白4基因修饰的树突状细胞疫苗构建及对胰腺癌细胞的免疫效应

Objective To obtain the dendritic cells ( DC)-based vaccine modified by adenovirus containing MUC4 gene , and evaluate the anti-tumor efficacy of DC vaccine to pancreatic tumor cells. Meth-ods The mRNA sequence of tumor associated antigen, MUC4, was obtained from NCBI, and MUC4 se-quence was acquired through the restriction enzyme sites and over lap PCR, then subcloned into adenovirus plasmid to create recombinant adenovirus ( rAd-MUC4) . The DCs were infected by rAd-MUC4 virus and then stimulated the lymph cells from the same donor to induce MUC4 specific cytotoxicity T lympbocytes ( CTL) . The efficacy of CTL was analyzed by LDH releasing assay. Elispot was used to detect the IFN-γ release. Results The recombinant adenovirus containing MUC4 ( sv12) gene was obtained. The MUC4-induced CTL could specifically kill the Capan-1 pancreatic tumor cells [ ( 13. 7±6.0)% , ( 21.4± 4. 7)% , (36.1±9. 5)% at ratios of 10: ,20: ,40: ] , higher than MCF-7 and Bxpc-3 cells respectively, P < 0. 05. The spots number of CTL induced by rAd-MUC4 was ( 139.1±23.3) , more than GFP and PBS control group,P<0.05. Conclusion The Muc4 gene modified DC vaccine could induce the proliferation of CTL, which provided a significant cytotoxicity to HLA-matched MUC4 positive tumor cell lines in vitro.