To enhance anti-amyloid-beta （Aβ） antibody generation and induce a Th2 immune response, we constructed a new DNA vaccine p（Aβ3-10 ）10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a mo...To enhance anti-amyloid-beta （Aβ） antibody generation and induce a Th2 immune response, we constructed a new DNA vaccine p（Aβ3-10 ）10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant. In this study, we administered this adjuvant intramus-cularly to female C57BL/6J mice at 8-10 weeks of age. Enzyme linked immunosorbent assay was used to detect the titer of serum anti-Aβ antibody, isotypes, and cytokines in splenic T cel s. A 3-（4,5-cimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to detect the prolifera-tion rate of splenic T cel s. Brain sections from a 12-month-old APP/PS1 transgenic mouse were used for detecting the binding capacities of anti-Aβ antibodies to Aβ plaques. The p（Aβ3-10）10-C3d-p28.3 vaccine induced high titers of anti-amyloid-βantibodies, which bound to Aβplaques in APP/PS1 transgenic mouse brain tissue, demonstrating that the vaccine is effective against plaques in a mouse model of Alzheimer’s disease. Moreover, the vaccine elicited a pre-dominantly IgG1 humoral response and low levels of interferon-γ in ex vivo cultured splenocytes, indicating that the vaccine could shift the cel ular immune response towards a Th2 phenotype. This indicated that the vaccine did not elicit a detrimental immune response and had a favorable safety profile. Our results indicate that the p（Aβ3-10）10-C3d-p28.3 vaccine is a promising immunothera-peutic option for Aβvaccination in Alzheimer’s disease.展开更多
目的探讨不同细胞因子鸡尾酒诱导的树突状细胞(DC)疫苗对裸鼠移植瘤的抗肿瘤作用。方法从38名外周血干细胞移植术供者的外周血中分离单个核细胞(PBMC),诱导成未成熟DC,将人肺腺癌细胞A549总RNA电转染入DC,分别用传统细胞因子鸡尾酒(IL-6...目的探讨不同细胞因子鸡尾酒诱导的树突状细胞(DC)疫苗对裸鼠移植瘤的抗肿瘤作用。方法从38名外周血干细胞移植术供者的外周血中分离单个核细胞(PBMC),诱导成未成熟DC,将人肺腺癌细胞A549总RNA电转染入DC,分别用传统细胞因子鸡尾酒(IL-6、IL-1β、TNF-α、PGE2)和改良细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、PGE2、polyⅠ∶C、CpG ODN)刺激DC成熟。致敏DC与自体T细胞混合培养,诱导产生抗原特异性细胞毒T淋巴细胞(CTL),用流式细胞术鉴定T细胞表型,ELISA法检测IFN-γ、TNF-α、IL-4、IL-5和IL-10的分泌。建立肺腺癌A549荷瘤裸鼠模型,随机分为空白对照组、未成熟组、传统组和改良组(n=5),于肿瘤接种d15、d22、d29给予相应CTL治疗,测量肿瘤体积和重量,免疫组化法和Western blotting检测肿瘤组织COX-2、VEGF、Bcl-2和Bax蛋白表达水平。结果1)CD3+CD8+T细胞表达率(%):未成熟组、传统组和改良组分别为35.00±3.24 vs 57.10±2.69 vs 63.98±1.96(P<0.05);2)IFN-γ浓度(pg/mL):3组分别为160.12±42.01 vs 263.17±55.30 vs 1 034.30±253.07(P<0.05);TNF-α浓度(pg/mL):3组分别为72.72±9.13 vs 65.20±8.03 vs 295.89±123.80(P<0.05);IL-10浓度(pg/mL):3组分别为7.26±1.76 vs 31.06±4.19 vs 44.01±12.12(P<0.05);3组IL-4、IL-5含量均未检测出,可见改良组T细胞分泌Th1型细胞因子IFN-γ和TNF-α的能力较未成熟组和传统组明显增强;3)经CTL免疫治疗后,裸鼠肿瘤体积(mm3):3组分别为512±33 vs 345±63 vs203±52(P<0.05),且改良组抑瘤率最大,为69.62%;4)改良组DC诱导的CTL明显上调Bax的表达,下调COX-2、VEGF和Bcl-2的表达。结论改良细胞因子鸡尾酒诱导肺腺癌细胞总RNA转染的DC疫苗能诱导Th1型免疫应答及产生有效的抗原特异性CTL,对肺腺癌裸鼠移植瘤具有良好的抑制作用。展开更多
目的探讨OVA基因修饰的树突状细胞疫苗(DC-OVA)在肿瘤模拟抗原OVA(ovabumin)的裸鼠肺癌模型体内抗肺肿瘤的效应。方法裸鼠右肺原位接种携带OVA的Lewis肺癌细胞(Lewis lung cancer cell-OVA,LLC-OVA)悬液,建立裸鼠肺癌模型。获取DC,经基...目的探讨OVA基因修饰的树突状细胞疫苗(DC-OVA)在肿瘤模拟抗原OVA(ovabumin)的裸鼠肺癌模型体内抗肺肿瘤的效应。方法裸鼠右肺原位接种携带OVA的Lewis肺癌细胞(Lewis lung cancer cell-OVA,LLC-OVA)悬液,建立裸鼠肺癌模型。获取DC,经基因转导制备DC-OVA疫苗。Western印迹法检测OVA蛋白的表达。获取T细胞,进而制备DC疫苗系统(DC-OVA/T)。CCK8试剂检测DC-OVA刺激后T细胞的增殖。模型建立2周后,尾静脉注射DC-OVA/T。Micro-CT扫描荷瘤裸鼠的胸部,观察肺部影像。H-E染色观察肺部及肿瘤组织形态结构。IHC方法观察脾、淋巴结、肿瘤组织中CD8的表达;观察肿瘤组织中SOX2、BMI1的表达。ELISA法检测血清中IL-12水平。结果 Western印迹法在样本中检测出清晰的OVA条带。DC-OVA强有效地刺激了T细胞的增殖(P<0.05)。DC-OVA/T治疗4周,疫苗组裸鼠肺micro-CT未见明显肿瘤阴影;取材,裸眼可见疫苗组病灶减小,H-E染色显示疫苗组的肿瘤组织较少,肺组织形态较完整。疫苗组裸鼠的脾、淋巴结、肿瘤组织内CD8阳性表达均明显高于对照组(P<0.05,P<0.05,P<0.01),肿瘤组织中的SOX2、BMI1阳性表达明显低于对照组(P<0.05,P<0.05);血清中的IL-12含量较对照组明显升高(P<0.001)。结论基因修饰的DC疫苗在裸鼠体内具有强大的抗肺癌效应,为临床治疗肺癌提供了新的实验依据。展开更多
基金supported by the National Natural Science Foundation of China,No.30471927
文摘To enhance anti-amyloid-beta （Aβ） antibody generation and induce a Th2 immune response, we constructed a new DNA vaccine p（Aβ3-10 ）10-C3d-p28.3 encoding ten repeats of Aβ3-10 and three copies of C3d-p28 as a molecular adjuvant. In this study, we administered this adjuvant intramus-cularly to female C57BL/6J mice at 8-10 weeks of age. Enzyme linked immunosorbent assay was used to detect the titer of serum anti-Aβ antibody, isotypes, and cytokines in splenic T cel s. A 3-（4,5-cimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to detect the prolifera-tion rate of splenic T cel s. Brain sections from a 12-month-old APP/PS1 transgenic mouse were used for detecting the binding capacities of anti-Aβ antibodies to Aβ plaques. The p（Aβ3-10）10-C3d-p28.3 vaccine induced high titers of anti-amyloid-βantibodies, which bound to Aβplaques in APP/PS1 transgenic mouse brain tissue, demonstrating that the vaccine is effective against plaques in a mouse model of Alzheimer’s disease. Moreover, the vaccine elicited a pre-dominantly IgG1 humoral response and low levels of interferon-γ in ex vivo cultured splenocytes, indicating that the vaccine could shift the cel ular immune response towards a Th2 phenotype. This indicated that the vaccine did not elicit a detrimental immune response and had a favorable safety profile. Our results indicate that the p（Aβ3-10）10-C3d-p28.3 vaccine is a promising immunothera-peutic option for Aβvaccination in Alzheimer’s disease.
文摘目的探讨不同细胞因子鸡尾酒诱导的树突状细胞(DC)疫苗对裸鼠移植瘤的抗肿瘤作用。方法从38名外周血干细胞移植术供者的外周血中分离单个核细胞(PBMC),诱导成未成熟DC,将人肺腺癌细胞A549总RNA电转染入DC,分别用传统细胞因子鸡尾酒(IL-6、IL-1β、TNF-α、PGE2)和改良细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、PGE2、polyⅠ∶C、CpG ODN)刺激DC成熟。致敏DC与自体T细胞混合培养,诱导产生抗原特异性细胞毒T淋巴细胞(CTL),用流式细胞术鉴定T细胞表型,ELISA法检测IFN-γ、TNF-α、IL-4、IL-5和IL-10的分泌。建立肺腺癌A549荷瘤裸鼠模型,随机分为空白对照组、未成熟组、传统组和改良组(n=5),于肿瘤接种d15、d22、d29给予相应CTL治疗,测量肿瘤体积和重量,免疫组化法和Western blotting检测肿瘤组织COX-2、VEGF、Bcl-2和Bax蛋白表达水平。结果1)CD3+CD8+T细胞表达率(%):未成熟组、传统组和改良组分别为35.00±3.24 vs 57.10±2.69 vs 63.98±1.96(P<0.05);2)IFN-γ浓度(pg/mL):3组分别为160.12±42.01 vs 263.17±55.30 vs 1 034.30±253.07(P<0.05);TNF-α浓度(pg/mL):3组分别为72.72±9.13 vs 65.20±8.03 vs 295.89±123.80(P<0.05);IL-10浓度(pg/mL):3组分别为7.26±1.76 vs 31.06±4.19 vs 44.01±12.12(P<0.05);3组IL-4、IL-5含量均未检测出,可见改良组T细胞分泌Th1型细胞因子IFN-γ和TNF-α的能力较未成熟组和传统组明显增强;3)经CTL免疫治疗后,裸鼠肿瘤体积(mm3):3组分别为512±33 vs 345±63 vs203±52(P<0.05),且改良组抑瘤率最大,为69.62%;4)改良组DC诱导的CTL明显上调Bax的表达,下调COX-2、VEGF和Bcl-2的表达。结论改良细胞因子鸡尾酒诱导肺腺癌细胞总RNA转染的DC疫苗能诱导Th1型免疫应答及产生有效的抗原特异性CTL,对肺腺癌裸鼠移植瘤具有良好的抑制作用。
文摘目的探讨OVA基因修饰的树突状细胞疫苗(DC-OVA)在肿瘤模拟抗原OVA(ovabumin)的裸鼠肺癌模型体内抗肺肿瘤的效应。方法裸鼠右肺原位接种携带OVA的Lewis肺癌细胞(Lewis lung cancer cell-OVA,LLC-OVA)悬液,建立裸鼠肺癌模型。获取DC,经基因转导制备DC-OVA疫苗。Western印迹法检测OVA蛋白的表达。获取T细胞,进而制备DC疫苗系统(DC-OVA/T)。CCK8试剂检测DC-OVA刺激后T细胞的增殖。模型建立2周后,尾静脉注射DC-OVA/T。Micro-CT扫描荷瘤裸鼠的胸部,观察肺部影像。H-E染色观察肺部及肿瘤组织形态结构。IHC方法观察脾、淋巴结、肿瘤组织中CD8的表达;观察肿瘤组织中SOX2、BMI1的表达。ELISA法检测血清中IL-12水平。结果 Western印迹法在样本中检测出清晰的OVA条带。DC-OVA强有效地刺激了T细胞的增殖(P<0.05)。DC-OVA/T治疗4周,疫苗组裸鼠肺micro-CT未见明显肿瘤阴影;取材,裸眼可见疫苗组病灶减小,H-E染色显示疫苗组的肿瘤组织较少,肺组织形态较完整。疫苗组裸鼠的脾、淋巴结、肿瘤组织内CD8阳性表达均明显高于对照组(P<0.05,P<0.05,P<0.01),肿瘤组织中的SOX2、BMI1阳性表达明显低于对照组(P<0.05,P<0.05);血清中的IL-12含量较对照组明显升高(P<0.001)。结论基因修饰的DC疫苗在裸鼠体内具有强大的抗肺癌效应,为临床治疗肺癌提供了新的实验依据。
