NKG2D-IL-21融合基因修饰的结肠癌细胞体外刺激NK细胞活化的研究

Study of NK cell activation by colon cancer cells genetically modified with NKG2D-IL-21 fusion gene ex vivo

目的观察含NKG2D-IL-21融合基因的重组表达载体转染结肠癌细胞后对NK细胞活化的影响。方法首先利用DNA限制性内切酶鉴定插入真核表达载体(pcDNA3.1-NKG2D-IL-21)的NKG2D和IL-21基因序列,并通过脂质体介导质粒转染结肠癌细胞株CT-26。流式细胞术胞内染色法检测CT-26内NKG2D-IL-21融合蛋白的表达,酶联免疫吸附实验鉴定细胞培养上清NKG2D-IL-21蛋白的含量。最后将经基因转染的CT-26细胞与小鼠脾脏NK细胞共培养,流式细胞术检测NK细胞表面活化性受体CD69的表达。结果 NKG2D-IL-21融合基因稳定插入pc DNA3.1载体。CT-26细胞经外源性NKG2D-IL-21融合基因转染后,表达含有NKG2D和IL-21的融合蛋白。分泌NKG2D-IL-21融合蛋白的CT-26细胞具有明显促进NK细胞表达CD69的活性。结论重组pcDNA3.1-NKG2D-IL-21真核表达载体可作为一种新型DNA疫苗。

Objective To observe effect of NK cell activation by colon cancer cell genetically modified with NKG2D-IL-21 fusion gene in a recombinant expression vector. Methods Firstly,the recombinant vector,pc DNA3. 1-NKG2D-IL-21,was identified with double restrictive enzymes for insertion of NKG2 D and IL-21. After transfection by liposome and plasmid complex,expression of NKG2D-IL-21 fusion protein in CT-26 cells（ a colon cancer cell line） was detected by an intracellular staining flow cytometry. Concentrations of NKG2D-IL-21 in cell-culture supernatants were measured by an ELISA assay. Finally,transfected CT-26 cells were co-cultured with mouse splenocytes overnight,and frequencies of DX5＋CD69＋cells were checked by flow cytometry. Results The NKG2D-IL-21 fusion gene was stably inserted into pc DNA3. 1. Ectopically transfection by NKG2D-IL-21 fusion gene in CT-26 cells leaded to express the corresponding protein. The secreted protein by CT-26 cells pre-transfected by the pc DNA3. 1-NKG2D-IL-21 plasmid stimulated NK cells to express CD69. Conclusion The pc DNA3. 1-NKG2D-IL-21 plasmid can be considered as a new DNA vaccine to mediate anti-tumor activity.