异体肿瘤mRNA电转染DC瘤苗的特异性抗骨肉瘤免疫学效应

Anti-osteosarcoma effects of autologous dendritic cells electrotransfected with allogeneic mRNA in rats

目的应用肿瘤细胞来源的mRNA转染至树突状细胞(dendritic cells,DCs)已逐渐成为一种颇具潜能的抗肿瘤免疫治疗新方法。但目前针对异体肿瘤来源的mRNA转染DCs的瘤苗研究尚不够系统明晰。本研究旨在探讨异体大鼠骨肉瘤细胞mRNA电转染树突状细胞瘤苗在荷瘤动物模型诱导的特异性抗肿瘤免疫学效应。方法提取UMR108细胞mRNA的步骤分为2步,首先采用Trizol法提取肿瘤细胞总RNA,继而采用磁珠法纯化mRNA。采用电穿孔的方法将mRNA转染至SD大鼠骨髓单核细胞来源的DCs制备瘤苗,通过荷瘤动物模型观察其特异性抗肿瘤效应。结果异体骨肉瘤mRNA转染DCs瘤苗可诱导针对自体肿瘤特异性的CTLs效应,且该效应可以被抗MHC-I抗体加以抑制。在体内预防保护作用和主动免疫治疗实验中,均有70%的异体mRNA-DC瘤苗接种大鼠可以有效地抵抗肿瘤细胞攻击而得以长期存活。结论该研究验证了异体肿瘤细胞来源的mRNA可以对特异性肿瘤治疗提供抗原信息,尤其是以DC为基础的免疫治疗中优势明显,可能为很多尚缺少特异性抗原的肿瘤患者,提供了更多的治疗选择。

Vaccination with dendritic cells(DCs) transfected with tumor-derived mRNA antigen has emerged as a promising strategy for generating protective immune responses in mammals.However,the integration of allogeneic osteosarcoma mRNA and autologous DCs has not been fully examined.This study was designed to investigate the antitumor effects of tumor vaccine produced by autologous DCs transfected with allogeneic osteosarcoma mRNA through electroporation in tumor-bearing rats model.In the present study,extraction of rat tumor-mRNA was performed as a two-step procedure.First,total-RNA was extracted by use of Trizol,and then mRNA purification was performed by use of polyT-coated magnetic beads.Then we transfected the allogeneic tumor mRNA to SD rat bone marrow-derived DCs through electroporation.The tumor vaccine was applied to tumor-bearing rat model and the specific antitumor effects of the tumor vaccine were observed.We found that the immunization using autologous DCs electrotransfected with allogeneic osteosarcoma mRNA induced specific CTL responses significantly(P < 0.05) and the cytotoxic activity was confirmed in cold target inhibition assays and using mAbs blocking MHC class I molecules.In vivo preimmunized and active therapeutic experiments demonstrated that 70% of the rats immunized with allogeneic osteosarcoma mRNA-transfected DCs were typically able to reject tumor challenge and remained tumor-free.In conclusion,we demonstrated that allogeneic tumor mRNA,isolated from rat osteosarcoma cell line,could be applied to produce tumor vaccine inducing specific antitumor effects,especially in DC-based immunotherapy strategy.This study also provides the foundations for an effective and broadly applicable treatment of a wide range of cancer indications for which tumor-associated antigens have not been identified.