加载野生型p53基因鼠树突细胞的抗肿瘤作用  

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

作　　者：刘红菊[1] 辛建保[1] 李卓亚[3] 熊先智[1] 陶晓南[1] 胡豫[2] 

机构地区：[1]华中科技大学同济医学院附属协和医院呼吸科,武汉430022 [2]华中科技大学同济医学院附属协和医院血液内科,武汉430022 [3]华中科技大学基础医学院免疫学系

出　　处：《中华结核和呼吸杂志》2008年第12期902-907,共6页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：留学回国人员科研启动基金资助项目（20071108）

摘　　要：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基因突变肿瘤的免疫治疗作用。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）-p63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DC（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6h^51Cr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CD3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〉20cm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-053-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（t。值分别为0．84、0．91，均P〉0．05），而抗CD。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�[ Abstract] 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 adenovirus 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 p53 （ MethA, IM59 and P815）in a standard 6-h51Cr-release assay. Effector and target ceils 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 1M59 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-PS15, IM59 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, anti-CDs antibody blocked the cytotoxicity against Ad-p53-PS15 （26. 7 ±2. 8）% or IM59（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 19459 and in 70% of mice challenged with MethA, while none protected in Ad-DC immunization grouP（χ^2= 6. 72, 5. 86,aU P 〈0. 05）. Treated with Ad-p53-DC after IM59 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 （ χ^2 = 9.48, P 〈 0. 05 ）. Conclusion DCs tranfected w

关 键 词：树突细胞 基因 p53 细胞毒性 免疫 

分 类 号：R686[医药卫生—骨科学]