沉默树突状细胞恒定链以增强其抗肿瘤作用的体外实验研究  

In vitro anti-tumor effect of invariant chain silenced dendritic cells

作　　者：柯山[1] 陈雪华[1] 蔡劬[1] 李建芳[1] 俞焙秦[1] 顾琴龙[1] 朱正纲[1] 刘炳亚[1] 

机构地区：[1]上海交通大学医学院瑞金医院消化外科研究所,200025

出　　处：《中华普通外科杂志》2007年第4期291-294,共4页Chinese Journal of General Surgery

基　　金：国家自然科学基金资助项目（30471961、30670939）

摘　　要：目的 初步观察用小分子干扰RNA（siRNA）沉默树突状细胞（DCs）恒定链（Ii）后，DCs疫苗的体外抗肿瘤效果。方法 从小鼠骨髓分离骨髓前体细胞，细胞经100ng／ml GM—CSF和100ng／ml IL-4诱导培养6d后，转染针对DCs Ii链特异的Ii—siRNA，转染后加用50ng／ml TNF—α继续诱导细胞成熟48h，然后分别用Western blot检测沉默效果及CCK-8试剂盒检测DCs刺激同种异体淋巴细胞增殖的能力；此外，DCs共转染Ii—siRNA和小鼠胃癌前体细胞MFC的总RNA后，与同种异体淋巴细胞共培养，通过ELISA检测培养上清IFN-γ和IL-4的水平，并收集致敏淋巴细胞进行体外杀伤实验。结果 Ii—siRNA明显抑制DCs Ii的表达。沉默Ii链能够增强DCs的淋巴细胞增殖能力，并促使淋巴细胞向Th1的方向漂移[IFN-γ：（5107±351）pg／ml，IL-4：（65±13）pg／ml，P〈0．05]。淋巴细胞经共转染Ii—siRNA和MFC RNA的DCs激活后，明显而特异地杀伤靶肿瘤细胞（杀伤百分率：66．94％±2．75％，P〈0．05）。结论 通过siRNA沉默DCs的Ii链可能是一种行之有效的增强抗肿瘤免疫的方法。Objective To observe the anti-tumor effect of invariant chain silenced dendritic cells in vitro. Methods Bone marrow-derived DCs were cultured for 6 days in the presence of 100 ng/ml GM-CSF and 100 ng/ml IL-4, then siRNA specific for Ii chain of DCs was transfected by lipefection. DCs were further matured with TNF-α at a concentration of 50 ng/ml for 48 h. The silencing effect of siRNA was evaluated by Western blot. Allogeneic lymphocyte proliferation assay was carried out with CCK-8 kit. IFN-γ and IL-4 were measured in the culture medium by ELISA and cytotoxicity assay was performed using a cytotox 96 non-radioactive cytotoxicity assay kit. Results The Ii expression of DCs was significantly down-regulated after Ii-siRNA transfection. Ii-siRNA-treated DCs effected increased allostimulatory capacity by lymphocyte proliferation assay and polarized allogeneic lymphocyte toward a Thl response [ IFN-γ：（5107 ± 351 ） pg/ml, IL-4 ： （65 ± 13 ） pg/ml, P 〈 0. 05 ]. Stronger and more specific cytotoxic activity was observed when DCs were co-transfected with Ii-siRNA and MFC RNA in vitro （ cytotoxicity% ： 66. 94% ±2.75% ,P 〈 0. 05）. Conclusions Transfection of DCs with Ii-siRNA enhances DC-based anti-tumor immunotherapy.

关 键 词：RNA干扰 免疫 细胞 树突状细胞 小鼠 

分 类 号：R686[医药卫生—骨科学]