转染肿瘤细胞总RNA的树突状细胞联合CIK细胞抗小鼠肝癌作用的实验研究

目的探讨转染小鼠肝癌H22细胞总RNA的树突状细胞(DC)疫苗体外抗小鼠肝癌的免疫作用。方法提取小鼠四肢长骨骨髓,在rmGM-CSF和rmIL-4体外刺激下增殖分化为DC。制备小鼠脾淋巴细胞,在体外经rmIFN-γ、anti-CD3、rmIL-2和rmIL-1b诱导成为细胞因子诱导的杀伤性细胞(CIK细胞)。制备肝癌H22细胞总RNA,体外转染DC,流式细胞仪检测转染前后的DC表面分子的表达情况。将转染H22细胞总RNA的DC和CIK细胞混合培养制备成为效应细胞,LDH释放法测定效应细胞对小鼠H22细胞、S180细胞的杀伤活性。结果经H22细胞总RNA转染后的DC,其MHCⅠ类分子、MHCⅡ类分子及CD83、CD86表达率明显增高,CD14表达率降低(P<0.05)。转染H22细胞总RNA的DC激活的CIK细胞对H22细胞的杀伤率高于其对S180细胞的杀伤率(P<0.05)。转染H22细胞总RNA的DC激活的CIK细胞对H22细胞的杀伤率高于其他各组效应细胞对H22细胞的杀伤率(P<0.05)。结论转染H22细胞总RNA的DC激活的CIK细胞对H22细胞具有高效而特异性的体外抗小鼠肝癌免疫作用。

Study on Anti-mouse Hepatocellular Carcinoma Effect of Cytokine-induced Kill Cells Activated by Dendritic Cells Transfected With Mouse Hepatocellular Carcinoma Total RNA in vitro

Objective To investigate anti-mouse hepatocellular carcinoma(HCC) effect of cytokine-induced killer cells(CIK) activated by dendritic cells(DC) transfected with mouse HCC total RNA in vitro.Methods The DC precursors and DC harvested from the bone marrow were incubated with recombinant murine granulocyte marcophage-colony stimulating factor(rmGM-CSF) and recombinant murine interleukin-4(rmIL-4) in vitro.Splenocytes were isolated from mouse spleen.Nonadherent splenocytes were induced to CIK by recombinant murine IFN-gamma(rmIFN-γ),anti-mouse CD3(anti-CD3),recombinant murine interleukin-2(rmIL-2),recombinant murine IL-1b(rmIL-1b)in vitro.Tumor-derived total RNA extracted from actively growing H22 cells was mixed with DC to transfect DC in vitro.The phenotypes of DCs(DCs transfected with total RNA or DCs non-transfected with total RNA) were analyzed by flow cytometry.Dendritic cells and CIK cells were co-cultured as effector cells.Mouse H22 cells and S180 cells were used as target cells in the LDH release methods to determine their cytotoxic activity.Results The expressions of MHC-Ⅰ,MHC-Ⅱ,CD83,CD86 were up-regulated and the expressions of CD14 was down-regulated after DC was transfected with tumor total RNA.DC transfected total RNA of H22 cells achieved higher cytotoxicity on H22 cells than on S180 cells(P<0.05).CIK cells activated by DC transfected total RNA of H22 cells achieved the highest cytotoxicity on H22 cells in all groups(P<0.05).Conclusion CIK cells activated by DC transfected total RNA of H22 cells present efficient and specific anti-mouse immune response of HCC on H22 cells in vitro.