纳米载体介导的PiggyBac转座子制备CAR-NK细胞  

Nanocarrier-mediated PiggyBac transposon system for preparation of CAR-NK cells

作　　者：岳冉 刘子洋 郑岩 陆小丹 胡珊珊 张炳勇 李修岭 李景果 韩双印 YUE Ran;LIU Ziyang;ZHENG Yan;LU Xiaodan;HU Shanshan;ZHANG Bingyong;LI Xiuling;LI Jingguo;HAN Shuangyin(Postgraduate Division,Xinxiang Medical College,Xinxiang 453003,Henan,China;Department of Gastroenterology,Zhengzhou University People’s Hospital,Zhengzhou 450003,Henan,China;Henan Provincial Eye Hospital&Eye Center of Henan Provincial People’s Hospital,Zhengzhou 450003,Henan,China)

机构地区：[1]新乡医学院研究生院,河南新乡453003 [2]郑州大学人民医院消化内科,河南郑州450003 [3]河南省立眼科医院暨河南省人民医院眼科中心,河南郑州450003

出　　处：《中国肿瘤生物治疗杂志》2020年第2期109-114,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81772670)。

摘　　要：目的:探索新型阳离子聚合物纳米载体mPEG-P(Asp-AED-g-HFB)(PAEF)和PiggyBac转座子介导嵌合抗原受体(chimeric antigen receptor,CAR)修饰自然杀伤(natural killer,NK)细胞的基因转染方法,为肿瘤细胞免疫治疗制剂研发提供新策略。方法:制备PAEF/DNA(转座酶+转座子)复合物,通过Nano-ZSE动态光散射系统(Malvern Instruments)测量PAEF/DNA复合物的粒径分布和表面电位;DNA凝胶电泳验证PAEF的DNA包封率、释放性和稳定性,结合粒径电位选择进入细胞合适的N/P值;CCK-8细胞毒性实验分析不同N/P值条件下PAEF/DNA复合物的细胞毒性;荧光显微镜及流式细胞术检测细胞转染效率,评估PAEF基因转染载体的可行性。结果:PAEF可包裹DNA形成粒径100~150 nm的纳米复合物,后者易于介导DNA进入细胞;当N/P值为20时,PAEF即可实现DNA的完全包裹;在还原剂二硫苏糖醇(dithiothreotol,DTT)存在下,PAEF对DNA有良好的释放能力;N/P值为80时,PAEF/DNA复合物转染组的NK-92细胞存活率显著高于脂质体转染组[(72.50±3.9)%vs(64.03±1.8)%,P<0.05];荧光显微镜下观察发现,PAEF/DNA组荧光多、荧光强度大;流式细胞术显示最高转染效率为83.4%。结论:纳米载体PAEF通过静电吸附作用能够很好地包裹DNA,且生物相容性好,基因转导效率较高,成功制备的CAR-NK细胞为过继免疫治疗提供良好的实验基础。Objective: To explore the gene transduction method of chimeric antigen receptor(CAR) mediated by novel cationic polymer nanocarrier mPEG-P(Asp-AED-g-HFB)(PAEF) and PigyBac transposon system to modify natural killer(NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA(transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System(Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis,and then by combining with particle size and surface potential to determine the preferential N/P ratio to enter NK cells. The cell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed.Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol(DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs(64.03±1.8)%,P<0.05];Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group;Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.

关 键 词：纳米载体 PIGGYBAC转座子 嵌合抗原受体 NK细胞 肿瘤免疫治疗 

分 类 号：R730.51[医药卫生—肿瘤] Q782[医药卫生—临床医学]