胰腺癌MUC1-VNTR核酸疫苗的构建和体外转染

目的 探索胰腺癌MUC1-VNTR核酸疫苗的构建，以进一步研究对胰腺癌的治疗作用。方法 首先对VNTR编码基因进行了重组设计，氨基端插入人单核细胞趋化蛋白Ⅰ信号肽基因序列，起始码前插入KOZAK促真核翻译序列。将人工合成的重组人VNTR基因定向克隆入真核表达载体pcDNA3，1／Myc-his（＋）A质粒的MCS中。将通过测序鉴定的含有准确插入序列的pcDNA3．1-VNTR／Myc-his（十）A重组质粒转染C087细胞，进行体外转染实验和Westernblot检测VNTR在细胞内外的表达。结果自转化平板筛选的阳性克隆通过测序鉴定，pcDNA3．1-VNTR／Myc-his（＋）A重组质粒含有完整的阅读框架和目的基因。重组质粒可在C087细胞内外表达VNTR，以胞内为主。所表达的VNTR分子量比人工合成的VNTR多肽大。结论 自行构建的胰腺癌MUC1-VNTR核酸疫苗序列准确，可在哺乳动物细胞中表达VNTR。     

新型胰腺癌MUC1 DNA疫苗的免疫原性研究

目的 研究所构建的新型胰腺癌MUC1 DNA疫苗的免疫原性.方法 采用三种优化策略共构建4个重组质粒,接种免疫C57BL/6J (H 2b)雌性小鼠.每2周加强免疫一次,共免疫3次.每次免疫前及处死小鼠前检测抗体和细胞因子.最后一次免疫结束后2周取脾,体外经VNTR合成肽刺激培养后进行杀伤性T淋巴细胞(cytotoxic T lymphocyte,CTL)杀伤功能分析.结果 pIRES2-EGFP3VNTR组、pIRES2-EGFP-3VNTR-CI-144组、pIRES2-EGFP-3VNTR-mIL-18组、pIRES2-EGFP-3VNTR-CI-144-mIL-18组的CTL特异杀伤率显著高于空载体对照组和生理盐水对照组,三重优化构建的质粒pIRES2-EGFP-3VNTR-mIL-18的CTL特异性杀伤率高于双重优化构建和单重优化构建的质粒.各优化构建质粒组的抗MUC1抗体OD值均显著高于对照组(P＜0.05),但组间差异无统计学意义.各优化构建质粒组的外周血IFN-γ均高于对照组(P＜0.05),且以含有IL-18优化策略的两组为高(P＜0.05).结论 所构建的优化重组质粒免疫小鼠后均可诱发抗原特异的CTL应答和抗体应答.C1-144和IL-18可增强疫苗的免疫原性.     

建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究

目的构建胰腺癌黏液蛋白核芯肽连续重复序列(MUC1-VNTR)核酸疫苗,研究疫苗所诱生的免疫应答。方法将重组人VNTR基因定向克隆入真核表达载体pcDNA3．1／Myc-his(+) A质粒的多克隆位点中,构建胰腺癌MUC1-VNTR核酸疫苗。C57BL／6(H-2b)小鼠2次免疫2周后取血检测抗VNTR抗体,取脾细胞体外以VNTR合成肽特异刺激培养6 d后进行细胞毒性T淋巴细胞 (CTL)杀伤试验。结果疫苗组小鼠脾细胞CTL的特异性杀伤率(34．8±3．1)％显著高于载体对照组[(9．2±0．8)％,P<0．01]和空白对照组[(6．1±0．6)％,P<0．01]。CTL对未经VNTR合成肽孵育的靶细胞EL4-VNTR杀伤较低(P<0．01)。VU 3C6可抑制CTL对经VNTR合成肽孵育的靶细胞E14-VNTR+的杀伤活性(P<0．01)。疫苗组小鼠血清抗VNTR抗体等效浓度(2324±238)μg/ml 高于载体对照组(1896±533)μg／ml和空白对照组(1736±142)μg／ml,P<0．01。结论本实验构建的胰腺癌MUC1—VNTR核酸疫苗免疫C57BL／6小鼠后可诱生VNTR特异的CTL应答和抗体应答。     

MUC1 DNA疫苗治疗胰腺癌的研究进展

胰腺癌为常见的恶性肿瘤，发病率逐年上升，但预后很差。由于难以早期诊断，胰腺癌诊断明确时往往已属于局部进展期，肿瘤浸润肠系膜上动静脉、门静脉或者出现了肝转移而失去根治切除的机会。全世界范围内胰腺癌的平均切除率不足120％。不可切除的胰腺癌患者中位生存期是3～6个月，可切除患者的预后也不佳，术后5年生存率大约是10％-19％，中位生存期是12-18个月。     

壳聚糖作为基因递送载体的MUC1基因疫苗治疗胰腺癌的实验研究

【摘要】 目的 研究以壳聚糖作为基因递送载体的MUC1基因疫苗诱导小鼠产生特异性体液及细胞免疫应答的作用,为壳聚糖联合MUC1基因疫苗用于胰腺癌的免疫治疗提供初步实验依据。方法 将30只小鼠平均分3组,分别接种壳聚糖-MUC1质粒、MUC1质粒及空质粒,通过ELISA法检测小鼠血清MUC1抗体生成情况,通过LDH释放法测定CTL对Capan-2细胞的杀伤活性。结果 接种壳聚糖-MUC1质粒后,小鼠血清抗MUC1抗体水平及CTL对Capan-2细胞的杀伤率均明显升高,与其它2组相比有显著性差异(P<0.05)。 结论 壳聚糖作为一种基因递送载体的MUC1基因疫苗能够有效诱导小鼠产生抗MUC1特异性抗体及产生杀伤MUC1+细胞的CTL,为壳聚糖联合MUC1基因疫苗用于胰腺癌的免疫治疗提供了初步的实验基础。     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

目的 构建免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗.方法 在PeDNA 3.1-VNTR/Myc-his(+)A质粒靶基因之后插入乙型肝炎病毒核心抗原C144基因,构建MUC1-VNTR-C144 DNA疫苗.45只C57BL/6小鼠随机分3组,VC组接种PeDNA 3.1-VNTR-C144/Myc-his(+)A质粒,V组单纯接种PeDNA 3.1-VNTR/Myc-his(+)A质粒,C组单纯接种pcDNA3.1-C144/Myc-his(+)A质粒.4周后加强免疫1次.结果 VC组小鼠脾细胞毒性T淋巴细胞(CTL)的特异性杀伤率(46.7±8.2)%显著高于V组(34.8±3.1)%和C组(12.9±1.2)%(E/T=80/1,P<0.01).而CTL对未经VNTR合成肽孵育的靶细胞EIA-VNTR杀伤率较低(P<0.01),VU 3C6可抑制CTL对经VNTR合成肽孵育的靶细胞EIA-VNTR+的杀伤活性(P<0.01),表明具有VC组诱生的CTL应答依然保持VNTR特异性.VC组小鼠血清抗VNTR抗体等效浓度(2810.2±308.3)mg/L高于V组(2323.5±238.3)mg/L和C组(2130.9±138.5)mg/L,差异有统计学意义(P<0.01).结论 增强型胰腺癌MUC1-VNTR-C144 DNA疫苗所诱生的VNTR特异的CTL应答和抗体应答显著增强.     

胰腺癌生物治疗的新靶点:MUC1

胰腺癌预后很差，消耗着巨大的社会卫生资源。目前对胰腺癌的治疗仍缺乏有效的治疗手段。65％胰腺癌病人在诊断明确后半年内死亡，约90％的病人一年内死亡。探索新的有效的治疗方法，是目前急需解决的重要课题。生物治疗，尤其是肿瘤疫苗，作为具有临床应用潜力和价值的治疗手段，近年来开始得到了深入的研究。其关键是选择有效的治疗靶点，即寻找合适的肿瘤相关抗原（tumor associate antigen，TAA）。     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.     

免疫增强型胰腺癌MUC1-VNTR-C144 DNA疫苗的构建

Objective To construct new MUC1-VNTR-C144 DNA vaccine for pancreatic cancer and study both CTL responses and antibody responses specific to VNTR enhanced by the C144 gene. Methods DNA encoding the 144 amino acids of the N-terminus of HBV core gene was cloned and then inserted into the recombinant plasmid pcDNA3.1-VNTR/Myc-his( + ) A. C57BL/6 mice were randomly immunized intramusscularly into anterior tibialis muscle with 100 μg plasmid DNA encoding VNTR ( V group,n = 15) or VNTR/CI44 (VC group,n = 15). Mice injected with the plasmid pcDNA3.1-C144/ Myc-his( + ) A vector (C group, n = 15 ) were used as controls. Four weeks later, all mice were injected a-gain with the same solution as before. Two weeks after the last immunization, spleen cells were obtained and analyzed for cytotoxic activity 6 days after in vitro stimulation by the synthesized VNTR polypeptide (Calcein AM assay). Blood was collected for the ELISA assay of anti-VNTR antibodies. Results The specific CTL killing rate against VNTR polypeptide induced in the VC group (46.7±8.2)% was signifi-cantly higher than in the V group (34.8±3.1 ) % and the C group ( 12.9±1.2) % ( P < 0.01 ) ,indicating the C144 gene enhances the CTL response induced by the vaccine. However,CTL lysed much more VNTR positive EIA than VNTR negative EIA ( P < 0.01 ), while the anti-VNTR antibody VU 3C6 significantly restrained such activity ( P <0.01 ) ,which indicated the CTL response was specific to VNTR. The equiva-lent concentration of specific antibody against VNTR in the VC group (2810.2±308.3 ) mg/L was signif-icantly higher than the V group (2323.5±238.3) mg/L and the C group (2130.9±138.5) mg/L (P < 0.01 ),which implied that the C144 gene could enhance the antibody response induced by the vaccine. Conclusion The VNTR specific CTL response and antibody response induced in mice immunized with new MUCl-VNTR-C144 DNA vaccine for pancreatic cancer can be enhanced significantly by the C144 gene.