树突状细胞疫苗联合胸腺肽α1对人源化免疫重建裸鼠结肠癌生长的抑制效应

目的观察胸腺肽αl（Ted）联合结肠癌细胞裂解物致敏树突状细胞（LyDCs）对人源化免疫重建裸鼠结肠癌的免疫治疗效应。方法常规DCs负载结肠癌细胞裂解物制备LyDCs疫苗，流式细胞仪（FCM）检测Tαl体外刺激前、后的LyDCs表型。HT-29结肠癌裸鼠模型成瘤后，经尾静脉注射人外周血T淋巴细胞6×10^6个／只，2d后，FCM检测裸鼠外周血人源性CD4^＋、CD8^＋T细胞；将人源化免疫重建裸鼠分为3组，分别用LyDCs＋Tctl、LyDCs和生理盐水皮下免疫注射治疗；治疗后7d，体外观察各组裸鼠脾脏淋巴细胞的肿瘤杀伤作用及IFN-γ、IL4分泌水平，实验结束时，观察LyDCs联合Tctl对荷瘤裸鼠的体内抑瘤作用。结果LyDCs的表型HLA．DR、CD80、CD86、CD83较刺激前明显上调；免疫重建裸鼠均检测到人源性CD4^＋、CD8^＋T细胞；LyDCs＋Tctl组裸鼠脾脏T淋巴细胞的肿瘤杀伤作用与LyDCs组比较差异有统计学意义（P〈0．01），LyDCs＋Ted组T细胞的IFN-γ分泌水平与LyDCs组比较差异有统计学意义（P〈0．01）；接种HT-29细胞58d后，LyDCs＋Tαl组、LyDCs组抑瘤率分别为60．41％、37．20％，两组之间抑瘤效应比较差异有统计学意义（P〈0．01），两组的抑瘤效应与对照组比较分别（P〈0．01）。结论Tαl能增强LyDCs诱导的CD4^＋，Th1细胞反应和CTLs杀伤效应，对DCs疫苗的抗癌免疫治疗功效具有明显的放大作用。     

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.     

胎儿来源的树突状细胞体外诱导抗前列腺癌效应的实验研究

目的研究胎儿来源树突状细胞(DC)体外诱导抗前列腺癌的特异性细胞免疫效果。方法从胎儿骨髓、肝脏获得单个核细胞,经重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)和重组人肿瘤坏死因子-α(rhTNF-α)诱导产生DC。50%~70%硫酸铵饱和沉淀法获取前列腺癌细胞DU145含热休克蛋白(HSP)成分的细胞溶解物,以该抗原负载DC,激活胎脾细胞产生肿瘤特异性细胞毒性T淋巴细胞(CTL)。MTT法检测CTL对DU145、PC3和EJ细胞的杀伤效应。结果胎儿骨髓、肝脏可诱导出功能成熟的DC,高表达CD1 a、CD86、HLA-DR和CD83。负载DU145抗原的DC可诱导产生CD8+CTL。CD8+T细胞表型由转化前的(14.09±2.46)%变为转化后的(62.76±2.64)%。对DU145细胞有明显细胞毒作用,显著高于对EJ细胞杀伤效应(P<0.01)。结论含HSP成分的肿瘤细胞溶解物负载胎儿来源DC,体外可诱导出特异性抗肿瘤免疫应答。     

IL-12基因修饰树突状细胞体外诱导免疫杀伤肝癌细胞

目的探讨IL-12基因修饰树突状细胞(DC)体外诱导免疫杀伤肝癌细胞的效能及其机制。方法IL-12基因修饰肝癌病人外周血DC(DC-IL-12),ELISA法检测DC-IL-12培养上清中IL-12和IFN-γ水平,以冻融HepG2所获得的肿瘤相关抗原(TAA)刺激DC-IL-12,MTT法检测TAA负载的DC-IL-12刺激同源T淋巴细胞增殖分化能力,细胞毒试验检测DC-IL-12诱导的细胞毒T淋巴细胞(CTL)及其上清液对HepG2肝癌细胞株的杀伤作用,ELISA法检测CTL上清液IFN-γ水平。结果DC经IL-12基因修饰后48h分泌高水平IL-12(24·35±5·4)pg/ml及IFN-γ(725±30)pg/ml,均显著高于未转染DC组(P<0·01)。DC-IL-12诱导的CTL及其上清液对HepG2均有显著杀伤作用,杀伤率显著高于未转染DC组,分别为(82·43±8·7)%vs(57·4±4·3)%和(76·45±8·5)%vs(18·23±5·3)%(P<0·01),DC-IL-12诱导的CTL上清液IFN-γ水平显著高于未转染DC组,分别为(1125·0±32·7)pg/ml、(281·3±14·7)pg/ml(P<0·01)。结论IL-12基因修饰增强DC体外诱导自体T淋巴细胞产生特异性抗肝癌免疫,其机制与IL-12基因修饰促进DC分泌IL-12、增强T淋巴细胞活化及分泌IFN-γ密切相关。     

肺癌抗原负载的树突细胞体外诱导细胞毒性T淋巴细胞的杀瘤作用

目的探讨不同方式肺癌抗原负载的树突状细胞(DC)在抗肿瘤免疫中的作用。方法取肺癌患者外周静脉血体外诱导和培养DC,分别以肺癌细胞总RNA转染DC(转染组)、肺癌细胞融合DC(融合组)和肺癌细胞冻融抗原负载DC(冻融组),以未负载抗原的DC(未负载组)和T细胞组作为对照,比较各组(每组n=6)DC诱导的细胞毒性T淋巴细胞(CTL)对肺癌细胞A_(549)的杀伤率(%)。结果转染组、融合组、冻融组、未负载组和T细胞组的杀伤率分别为(73．2±5．9)%、(61．6±6．2)%、(55．3±6．9)%、(22．3±6．1)%和(19．8±6．3)%(P＜0．05)；其中转染组、融合组和冻融组高于未负载组和T细胞组(P＜0．05)；转染组和融合组之间差异无统计学意义(P＞0．05),但两者均大于冻融组(P＜0．05)。RNA转染DC所需的肿瘤细胞数仅是冻融及融合方式的1/5和1/6。结论3种肺癌抗原负载DC方式均有诱导效应细胞杀伤靶细胞的增效作用,而以肺癌细胞总RNA转染方式为佳。     

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.     

MUC1-VNTR基因修饰树突细胞对胰腺癌细胞株体外杀伤效应的研究

目的:通过体外杀伤试验,研究转染黏液蛋白核心肽-连续重复序列(MUC1-VNTR)基因的树突细胞(DC)诱导的细胞毒T淋巴细胞(CTL)对人胰腺癌细胞株的特异杀伤效应.方法:选取入淋巴细胞A抗原(HLA-A2)阳性健康人外周血单个核细胞,体外诱导成熟DC,脂质体介导MUC1-VNTR基因转染入DC(DC-VNTR),以转染空质粒pcDNA3.1(DC-pcDNA3.1)及Lipofectamine处理之DC(DC-Lipo)作为对照,用Western印迹法检测MUC1-VNTR的表达,体外刺激同源T细胞,Elispot检测DC-VNTR诱导分泌IFN-γ和Gramzyme B之CTL能力,细胞毒试验检测DCVNTR诱导的CTL对Capan2和AsPC-1胰腺癌细胞株的杀伤作用.结果:Western印迹法检测到MUC1-VNTR的表达;DC-VNTR诱导的分泌IFN-γ、Gramzyme B的CTL数高于用DC-pcDNA3.1及DC-Lipo作对照者(P＜0.05);DCVNTR诱导的CTL对HIJA-A2、MUC1均阳性的Capan2细胞有显著杀伤效应.且显著高于DC-pcDNA3.1组及DC-Lipo组(P＜0.05).该CTL对HLA-A2阴性、MUC1阳性的AsPC-1细胞无明显杀伤效应.结论:以MUC1-VNTR基因转染的DC可以诱导具特性的CTL,并能特异地杀伤Capan2胰腺癌细胞株.     

逆转录病毒携带白细胞介素-12转染树突状细胞体外诱导特异免疫杀伤肝癌细胞

目的探讨逆转录病毒携带白细胞介素(IL)12转染树突状细胞(DC)体外诱导免疫杀伤肝癌细胞的效能及其机制。方法感染指数(MOI)为100,含IL12的重组逆转录病毒修饰肝癌患者外周血DC(DCIL12),酶联免疫吸附试验法(ELISA)检测DCIL12(5×103个/ml)培养上清中IL12和γ干扰素(IFNγ)水平,以冻融肝癌细胞株HepG2(1×107个/ml)所获得的肿瘤相关抗原(TAA)刺激DCIL12,MTT法检测TAA负载的DCIL12刺激同源T淋巴细胞(1×105个/ml)增殖分化能力,细胞毒试验检测DCIL12诱导的细胞毒T淋巴细胞(CTL)及其上清液对HepG2肝癌细胞株杀伤作用,ELISA法检测CTL上清液IFNγ水平。结果DC经IL12基因修饰后48h分泌高水平IL12(24.35±5.40)ng/L及IFNγ(725±30)ng/L,均显著高于未转染DC组(P<0.01及P<0.05)。DCIL12诱导的CTL及其上清液对HepG2均有显著杀伤作用,杀伤率显著高于未转染DC组,分别为(82.43±8.70)%vs(57.4±4.3)%(P<0.01)和(76.45±8.50)%vs(18.23±5.30)%(P<0.01),DCIL12诱导的CTL上清液IFNγ水平显著高于未转染DC组,分别为(1125.0±32.7)ng/Lvs(281.3±14.7)ng/L(P<0.01)。结论IL12基因修饰增强DC体外诱导自体T淋巴细胞产生特异性抗肝癌免疫,其机制与IL12基因修饰促进DC分泌IL12、增强T淋巴细胞活化及分泌IFNγ密切相关。     

细胞因子信号传导抑制因子-1在树突状细胞介导T细胞分化中的作用

目的 观察细胞因子信号传导抑制因子-1(SOCS1)在不同诱导方法下树突状细胞(DC)的表达及其对T细胞刺激活性的影响.方法 1000 U/ml重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和500 U/ml白细胞介素(IL)-4体外诱导的人外周血单个核细胞来源的DC,经1mmol/L丁酸钠、1 mg/L脂多糖(LPS)、细胞因子鸡尾酒法诱导成熟,分别以流式细胞仪、酶联免疫吸附试验(ELISA)、混合淋巴细胞反应(MLR)检测DC表面标志、IL-12、IL-10分泌和刺激淋巴细胞增殖能力.Western blot和实时荧光定量聚合酶链反应(Real-time PCR)分别检测各组SOCS1蛋白水平及mRNA的表达.结果 丁酸钠诱导的不成熟DC的SOCS1蛋白表达最高(1.61±0.22,P＜0.05);其SOCS1 mRNA表达差异倍数(1.58±0.11)显著高于成熟组DC(0.99±0.04、1.27±0.05,P＜0.05).高表达SOCS1的DC刺激淋巴细胞增殖的能力(1.53±1.04),IL-12分泌量(142.79 ±15.61)较成熟DC显著降低(P＜0.05);而IL-10分泌(3.29±0.21)较成熟DC比较显著升高(P＜0.05).结论 SOCS1是调节DC介导的T细胞分化的关键因子.     

白细胞介素-2基因修饰树突状细胞体外增强其诱导的抗肝癌免疫反应

目的探讨腺病毒介导的白细胞介素(IL)-2基因修饰能否使抗原冲击的树突状细胞(DC)在体外诱导出更强的抗肝癌免疫反应。方法IL-2的重组腺病毒载体体外转染经肝癌细胞株HepG2冻融抗原致敏的DC(AdIL-2-HepG2/DC),FACS分析AdIL-2-HepG2/DC表面分子的表达,酶联免疫吸附试验法(ELISA)检测IL-2水平,3H-TdR掺入法检测T淋巴细胞增殖分化能力,噻唑蓝(MTT)法检测细胞毒性T淋巴细胞(CTL)效应。结果AdIL-2-HepG2/DC能高水平的表达CD1a(61.7±8.1)%,CD11c(72.9±6.2)%,CD80(81.1±7.1)%,CD86(76.4±6.8)%以及HLA-DR(90.6±6.4)%,并分泌较高水平的IL-2(6.78±0.18)mq/L。AdIL-2-HepG2/DC能非常显著地刺激自体T细胞增殖(CPM值为21 878±1089),当靶细胞为HepG2时,其诱导的CTL杀伤活性(74.5±3.8)%显著高于其他各组,并且其杀伤能力与效应细胞数量成正比。结论AdIL-2- HepG2/DC可以增强体外诱导的特异性抗肝癌免疫反应。     

树突状细胞与胃癌细胞融合的肿瘤疫苗研究

目的以肿瘤细胞与树突状细胞(dendriticcells,DCs)相融合使融合细胞能将肿瘤抗原递呈给T细胞,并提供T细胞激活所必需的第二信号,借此激活机体的抗肿瘤免疫。方法通过PEG法将鼠源性胃癌细胞MFC与小鼠DC进行融合(T/DC),融合后经50Gy电子线照射,以1×106肿瘤疫苗于小鼠皮下注射免疫2次,间隔1周后接种,后一次免疫后1周于小鼠皮下接种5×105MFC胃癌细胞。肿瘤细胞接种3周后处死实验动物,测量肿瘤大小、外周血NK活性和脾细胞CTL活性。结果肿瘤接种后一周对照组小鼠皮下均有肿瘤生长,而T/DC免疫组均无皮下可触及的肿瘤(10/10),10天后T/DC免疫组才有4/10只鼠皮下出现可触及的肿瘤,3周后对照组肿瘤体积明显大于T/DC免疫组。经T/DC免疫后小鼠生存期明显较对照组延长,对照组于肿瘤接种后2周开始有小鼠衰竭死亡,至肿瘤接种后3周对照组仅有3只存活,T/DC免疫组有8只鼠存活,并仍有3只无肿瘤生长。经T/DC免疫后,小鼠NK活性和肿瘤特异性CTL活性均明显提高,T/DC免疫组和对照组MFC特异性CTL活性分别为30.09%和7.12%。结论肿瘤细胞鄄DC融合的肿瘤疫苗可通过加强抗原递呈激活肿瘤特异性CTL而具明显的抗肿瘤活性。