高效非病毒基因载体的构建及重组树突状细胞应用于肿瘤的免疫治疗
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摘要
目的：构建筛选高效非病毒基因转染载体,用于携载报告基因及目的基因转染树突状细胞(Dendritic Cell, DC)。通过理化性质和生物学手段评价非病毒转染载体的转染效率、安全性及转染机制,并在小鼠模型上评价以基因转染DC为疫苗预防黑色素瘤生成的效果。
方法：1、合成和表征β-聚氨酯(Polyβ-amino ester, PBAE),壳聚糖-聚醚酰亚胺(Chitosan-Polyetherimide,CP)及精胺-右旋糖酐(Spermine-Dextran,SD)三种非病毒基因转染材料；2、孵育法制备非病毒基因转染材料/DNA纳米复合物,并对各种材料/DNA复合物进行表征,通过携载虫荧光素酶报告基因质粒或绿荧光蛋白EGFP质粒等报告基因对肿瘤细胞株进行常规转染；3、进一步利用上述制备的携载有报告基因质粒的材料/DNA纳米复合物转染DC,利用流式细胞技术及荧光显微成像技术考察纳米复合物转染效率、利用MTT法考察复合物细胞毒性,利用共聚焦显微成像技术考察复合物入核入胞情况,从而筛选出适于DC转染的安全高效载体；4、利用上述材料/DNA复合物包裹肿瘤相关抗原基因及细胞性趋化因子受体基因共转染DC,考察该DC疫苗免疫预防抑制小鼠黑色素实体瘤效果；5、利用所筛选的载体材料携载肿瘤相关抗原基因及细胞性趋化因子受体基因转染DC,利用Transwell方法考察基因重组DC体外游走迁移情况,并通过细胞示踪结合载体荧光成像技术考察经转染DC体内游走迁移情况。
结果：成功合成PBAE、CP与SD等非病毒基因转染材料。通过孵育法成功制备PBAE,CP和SD的基因复合纳米粒。TEM观察表明SD/DNA复合物是结构均一类球形复合纳米粒。通过携载报告基因对细胞株进行转染结果表明三种材料载体基因复合物对于肿瘤细胞株均有良好的转染活性。荧光显微镜及激光共聚焦显微镜结果表明经三种非病毒载体携载的DNA均能成功进入DC胞浆及细胞核。三种非病毒基因转染材料携载EGFP报告基因转染实验结果表明,与市售试剂Lipofectamine2000 (Lipo)/DNA复合物相比,CP与SD的基因复合纳米粒对DC具有较佳转染效率及生物安全性,对于后者,其入胞抑制实验结果显示SD/DNA在入胞抑制剂NaN3及MβCD存在下入胞量减少,证明其入胞途径为能量依赖性的小窝-脂筏介导的内吞途径。经SD携载趋化性细胞因子受体CCR7质粒复合物转染的DC在体外迁移实验中较Lipo基因复合物转染DC显示出更高的迁移活性；进一步,在体内迁移实验中亦显示出更明显的淋巴趋向归巢现象。在ex vivo实验中,经SD携载的黑色素瘤抗原gp100质粒转染的DC疫苗具有良好的免疫抑制黑色素实体瘤效果,而经CP携载的gp100抗原质粒转染的DC疫苗免疫抑瘤活性较差。进一步的DC疫苗免疫实验中,经SD携载抗原质粒gp100和CCR7质粒转染的DC疫苗组产生了显著的抑瘤效果并提高荷瘤小鼠生存率。
结论：成功合成PBAE,CP与SD三种非病毒基因转染材料,其对肿瘤细胞株具有高效安全的转染效率。经筛选SD携载的DNA纳米复合物,对于DC具有良好的转染活性,转染目的基因后能够提高DC的抗原表达能力及迁移活性,体内免疫预防实验证明经SD转染的DC疫苗免疫对预防和治疗癌症具有很好的应用潜力。
Objective:Construction of efficient non-viral gene transfer vectors for DC gene delivery. Characterization of physical and chemical properties of these novel non-viral vector, and evaluaton of transfection efficiency and safety of materials/DNA nanocomplex in DCs. Evaluation of DC gene vaccine immunotherapy effect against melanoma cells in vivo.
Methods:1.Synthesis and characterization of different non-viral gene transfer materials; 2.Preparation and characterization of various material/DNA nanocomplexes by incubation. After tranfection on cell lines by different material/DNA nanocomplexes, Spermine-Dextran(SD) was selected as the relatively better non-toxic and efficient non-viral gene transfer material for DCs.3.Transfection efficiency, cytotoxicity and mechanisms of distribution in cytoplasm and nucleus were all investigated in dendritic cells (DC) by using SD/DNA nanocomplexes; 4.By using SD/DNA complex, DCs were co-transfected with plasmid DNA encoding tumor-associated antigen and chemokine. Then we studied the transferred DCs migration behavior in vitro and in vivo. Finally, we examined vaccine protection treament effect in mice beared melanoma solid tumor.
Results:After incubation, Spermine-Dextran (SD) and plasmid DNA can form homogeneous spherical composite nanoparticle complexes. The incubated complex shows relatively high transfect efficiency and low cytotoxicity in DCs. Endocytosis results showed DCs uptook SD/DNA into cytoplasm through energy dependendent lipid-raft mediated endocytosis pathway. After 4 hours transfection, plasmid DNA can enter into the nucleus. The SD/CCR7 plasmid complex transfection of DCs transfected by SD/CCR7 plasmid complexes appeared high migration activity in vivo. In ex vivo experiments, DCs were co-transfected by plasmids encoding gp100, a melanoma antigen peptide, and CCR7, a chemokine receptor. It showed good anti-tumor therapeutic effect after vaccination of the engineered DCs, and the survival of tumor-bearing mice was increased largely.
Conclusion:SD/DNA nano-complex is a novel efficient non-viral gene transfer vector with low cytotoxicity for transfection of DC. DCs engineered by these complexes have a good vaccine effect for tumor immunotherapy and have a good clinical therapy potential
方法：1、合成和表征β-聚氨酯(Polyβ-amino ester, PBAE),壳聚糖-聚醚酰亚胺(Chitosan-Polyetherimide,CP)及精胺-右旋糖酐(Spermine-Dextran,SD)三种非病毒基因转染材料；2、孵育法制备非病毒基因转染材料/DNA纳米复合物,并对各种材料/DNA复合物进行表征,通过携载虫荧光素酶报告基因质粒或绿荧光蛋白EGFP质粒等报告基因对肿瘤细胞株进行常规转染；3、进一步利用上述制备的携载有报告基因质粒的材料/DNA纳米复合物转染DC,利用流式细胞技术及荧光显微成像技术考察纳米复合物转染效率、利用MTT法考察复合物细胞毒性,利用共聚焦显微成像技术考察复合物入核入胞情况,从而筛选出适于DC转染的安全高效载体；4、利用上述材料/DNA复合物包裹肿瘤相关抗原基因及细胞性趋化因子受体基因共转染DC,考察该DC疫苗免疫预防抑制小鼠黑色素实体瘤效果；5、利用所筛选的载体材料携载肿瘤相关抗原基因及细胞性趋化因子受体基因转染DC,利用Transwell方法考察基因重组DC体外游走迁移情况,并通过细胞示踪结合载体荧光成像技术考察经转染DC体内游走迁移情况。
结果：成功合成PBAE、CP与SD等非病毒基因转染材料。通过孵育法成功制备PBAE,CP和SD的基因复合纳米粒。TEM观察表明SD/DNA复合物是结构均一类球形复合纳米粒。通过携载报告基因对细胞株进行转染结果表明三种材料载体基因复合物对于肿瘤细胞株均有良好的转染活性。荧光显微镜及激光共聚焦显微镜结果表明经三种非病毒载体携载的DNA均能成功进入DC胞浆及细胞核。三种非病毒基因转染材料携载EGFP报告基因转染实验结果表明,与市售试剂Lipofectamine2000 (Lipo)/DNA复合物相比,CP与SD的基因复合纳米粒对DC具有较佳转染效率及生物安全性,对于后者,其入胞抑制实验结果显示SD/DNA在入胞抑制剂NaN3及MβCD存在下入胞量减少,证明其入胞途径为能量依赖性的小窝-脂筏介导的内吞途径。经SD携载趋化性细胞因子受体CCR7质粒复合物转染的DC在体外迁移实验中较Lipo基因复合物转染DC显示出更高的迁移活性；进一步,在体内迁移实验中亦显示出更明显的淋巴趋向归巢现象。在ex vivo实验中,经SD携载的黑色素瘤抗原gp100质粒转染的DC疫苗具有良好的免疫抑制黑色素实体瘤效果,而经CP携载的gp100抗原质粒转染的DC疫苗免疫抑瘤活性较差。进一步的DC疫苗免疫实验中,经SD携载抗原质粒gp100和CCR7质粒转染的DC疫苗组产生了显著的抑瘤效果并提高荷瘤小鼠生存率。
结论：成功合成PBAE,CP与SD三种非病毒基因转染材料,其对肿瘤细胞株具有高效安全的转染效率。经筛选SD携载的DNA纳米复合物,对于DC具有良好的转染活性,转染目的基因后能够提高DC的抗原表达能力及迁移活性,体内免疫预防实验证明经SD转染的DC疫苗免疫对预防和治疗癌症具有很好的应用潜力。
Objective:Construction of efficient non-viral gene transfer vectors for DC gene delivery. Characterization of physical and chemical properties of these novel non-viral vector, and evaluaton of transfection efficiency and safety of materials/DNA nanocomplex in DCs. Evaluation of DC gene vaccine immunotherapy effect against melanoma cells in vivo.
Methods:1.Synthesis and characterization of different non-viral gene transfer materials; 2.Preparation and characterization of various material/DNA nanocomplexes by incubation. After tranfection on cell lines by different material/DNA nanocomplexes, Spermine-Dextran(SD) was selected as the relatively better non-toxic and efficient non-viral gene transfer material for DCs.3.Transfection efficiency, cytotoxicity and mechanisms of distribution in cytoplasm and nucleus were all investigated in dendritic cells (DC) by using SD/DNA nanocomplexes; 4.By using SD/DNA complex, DCs were co-transfected with plasmid DNA encoding tumor-associated antigen and chemokine. Then we studied the transferred DCs migration behavior in vitro and in vivo. Finally, we examined vaccine protection treament effect in mice beared melanoma solid tumor.
Results:After incubation, Spermine-Dextran (SD) and plasmid DNA can form homogeneous spherical composite nanoparticle complexes. The incubated complex shows relatively high transfect efficiency and low cytotoxicity in DCs. Endocytosis results showed DCs uptook SD/DNA into cytoplasm through energy dependendent lipid-raft mediated endocytosis pathway. After 4 hours transfection, plasmid DNA can enter into the nucleus. The SD/CCR7 plasmid complex transfection of DCs transfected by SD/CCR7 plasmid complexes appeared high migration activity in vivo. In ex vivo experiments, DCs were co-transfected by plasmids encoding gp100, a melanoma antigen peptide, and CCR7, a chemokine receptor. It showed good anti-tumor therapeutic effect after vaccination of the engineered DCs, and the survival of tumor-bearing mice was increased largely.
Conclusion:SD/DNA nano-complex is a novel efficient non-viral gene transfer vector with low cytotoxicity for transfection of DC. DCs engineered by these complexes have a good vaccine effect for tumor immunotherapy and have a good clinical therapy potential
引文
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