加载野生型p53基因鼠树突细胞的抗肿瘤作用

目的 观察加载了野生型p53基因的树突细胞(DC)对不同位点p53基因突变肿瘤得庖咧瘟谱饔?方法通过锥虫蓝染色、同种异体混合白细胞反应及流式细胞仪检测DC细胞表面分子,评估腺病毒(Ad)-p53感染DC是否影响DC的免疫功能.以Ad或转导了野生型p53基因的Ad分别感染骨髓De(Ad-DC和Ad-p53-DC)后,静脉注射免疫C57BL/6小鼠各5只,分离脾细胞,采用标准6 h51 Cr释放试验测定其诱导不同肿瘤细胞系(MethA、D459和P815)细胞毒性T淋巴细胞(CTL)的杀伤活性;效应细胞(Ad-p53-DC免疫后的小鼠脾细胞)和靶细胞(Ad-p53-P815和D459)孵育时分别加入抗CD4抗体或抗CD8抗体,观察CTL的活性变化.使用MethA和D459肿瘤细胞系得到不同的荷瘤鼠,于肿瘤形成前后分别使用Ad-p53-DC免疫,Ad-DC对照,当实体瘤三维直径之和＞20 cm时处死小鼠,用生存曲线评估Ad-p53-DC免疫的预防或治疗作用.结果 (1)Ad-p53-DC免疫诱导的抗Ad-p53-P815、D459和MethA的CTL反应(效应细胞:靶细胞=50:1)分别为(27.8±3.4)%、(23.5±2.7)%及(58.3±9.2)%,与Ad-DC免疫诱导的反应[(9.3±1.8)%、(4.6±1.0)%及(23.5 ±3.7)%]相比,差异有统计学意义(td值分别为5.79、3.68、5.02,均P＜0.05).Ad-p53-DC免疫小鼠T淋巴细胞与靶细胞Ad-p53-P815或D459的CTL活性,抗CD4组[(59.8 ±4.6)%、(18.9±2.4)%]与无抗体组[(64.4±6.3)%、(22.2±3.0)%]相比,差异无统计学意义(td值分别为0.84、0.91,均P＞0.05),而抗CD8组[(26.7±2.8)%、(6.1±1.2)%]差异有统计学意义(td值分别为9.03、7.67,均P＜0.05).抗CD8组与抗CD4组比较,差异有统计学意义(td值分别为8.79、9.18,均P＜0.05).(2)Ad-p53-DC和Ad-DC静脉注射2次免疫小鼠后,分别以D459细胞或MethA肉瘤细胞荷瘤20只小鼠.在Ad-p53-DC免疫组分别有14只和16只小鼠肿瘤的生长得到完全抑制,与Ad-DC免疫组比较差异有统计学意义(x2值分别为6.72、5.86,P＜0.05).皮下接种小鼠D459后,Ad-p53-DC免疫治疗组的小鼠肿瘤生长速度比Ad-DC组延缓2周左右,二组比较差异有统计学意义(x2 值为9.48,P＜0.05).结论 Ad-p53-DC可诱导抗MethA、P815和D459靶细胞的由CD8+T淋巴细胞介导的CTL反应,并抑制鼠体内肿瘤细胞的形成和生长.

Anti-tumor immune response of dendritic cells transfected with wild-type p53 gene

Objective To explore the anti-tumor immune responses of dendritic cells (DCs) loaded with intact wild-type p53 to mice challenged with tumor cells expressing p53 genes with mutations at different sites.Methods Ad-p53-DC immunization function was assessed by the expression of surface molecules and allogeneic MLR.DCs derived from bone marrow were transduced with adenovims or a human wild-type p53 containing recombinant adenovirus (Ad-DC and Ad-p53-DC)and immunized C57BL/6 mice.Splenocytes were separated and cell cytotoxicity was measured against tumor cells expressing mutant 003 ( MethA,D459 and PS15)in a standard 6-h5t Cr-release assay.Effector and target cells were incubated in the presence of anti-CD4 or anti-CD8 antibody.Ad-p53-DC was immunized in control Ad-DC before or after mice were challenged with either D459 tumor or with MethA sarcoma cells to observe whether immune response would provide tumor protection.Results Immunization with Ad-p53-DC developed significantly higher substantial CTL responses against Ad-p53-P815,D459 and MethA cells (effectors:target cells = 50:1 ),(27.8±3.4)%,(23.5±2.7)%,(58.3±9.2)% than with Ad-DC(9.3±1.8)%,(4.6±1.0)%,(23.5±3.7) % (td = 5.79,3.68,5.02,all P ＜ 0.05 ).In Ad-p53-DC immunized mice,anfi-CD8 antibody blocked the cytotoxicity against Ad-p53-PS15 (26.7±2.8)% or D459(6.1 ± 1.2)%,but not anti-CD4 antibody [(59.8± 4.6) %,( 18.9±2.4 ) %,td = 8.79 or 9.18,all P ＜ 0.05].Ad-p53-DC immunization provided complete tumor protection in 80% of mice challenged with D459 and in 70% of mice challenged with MethA,while none protected in Ad-DC immunization group(x2 = 6.72,5.86,all P ＜0.05).Treated with Ad-p53-DC after D459 inoculation subcutaneously,mice were killed due to the bulky tumor more than 2 weeks later than the mice in the Ad-DC treatment group during 7 week observation ( X2 = 9.48,P ＜ 0.05 ).Conclusion DCs tranfected with 100 MOI Ad-p53 induced intense CTL responses against P815,D459 and MethA.This CTL response is mediated by CDs+ T ceils.Treatment with Ad-p53-DC significantly developed tumor immunology and slowed the growth of established tumors.