摘要
目的卵巢叶酸受体α(αFR)在上皮性卵巢癌中高表达。文章拟构建靶向αFR的嵌合抗原受体(CAR)修饰NK-92细胞(NK-92-αFR-CAR),并验证其对αFR阳性靶细胞的杀伤效能,为卵巢癌的临床治疗提供新策略。方法选择偏爱密码子技术设计人源化抗αFR抗原的CAR分子基因;构建慢病毒表达载体pLenti-αFR-CAR;包装慢病毒并感染NK-92细胞;Western blot、qPCR及流式细胞术检测αFR-CAR分子在NK-92细胞中的表达效率。LDH释放实验和ELISA检测NK-92-αFR-CAR细胞对αFR阳性卵巢癌细胞的杀伤活性。结果双酶切pLenti-αFR-CAR重组质粒可见分子质量和αFR-CAR分子一致的基因片段。Western blot和qPCR证明αFR-CAR分子在NK-92细胞中确有表达,并且经嘌呤霉素筛选后,αFR-CAR的表达效率超过90%。NK-92-αFR-CAR细胞对卵巢癌细胞的杀伤效应及细胞因子分泌水平呈抗原依赖性,即肿瘤细胞表面αFR表达水平越高,NK-92-αFR-CAR细胞对其杀伤效应越强。NK-92-αFR-CAR细胞与SK-OV-3细胞或A2780细胞共培养后,其IFN-γ分泌水平(5007.81±180.37、2668.30±180.37)较NK-92细胞(360.80±8.44、285.86±12.39)和NK-92-EV细胞(378.75±21.76、292.10±21.76)表达更高(P<0.01);TNF-α分泌水平(964.27±74.95、585.73±83.53)较NK-92细胞(247.92±5.96、231.03±10.47)和NK-92-EV细胞(230.04±13.66、215.13±7.89)表达更高(P<0.01)。但与A-431细胞共培养,3种细胞中IFN-γ和TNF-α表达差异无统计学意义(P<0.05)。结论NK-92-αFR-CAR细胞具有分泌大量IFN-γ和TNF-α细胞因子并特异杀伤αFR阳性卵巢癌细胞的能力,这为NK-92-αFR-CAR细胞进入后续的临床前和临床研究奠定了基础。
Objective High mortality and lack of effective treatment are characteristics of ovarian cancer.In recent years,chimeric antigen receptor modified T/NK(CAR-T/NK)cells show extraordinary effect in hematological malignant tumor and is believed to be hopeful to cure cancer.So we aimed to design and construct the third generation chimeric antigen receptor(CAR)that targets folate receptorα(αFR)in ovarian carcinoma,identify its expression on NK-92 cells,evaluate the cytotoxicity of NK-92-αFR-CAR cells onαFR positive cancer cells and eventually to provide a new strategy to treat ovarian cancer.Methods In order to reinforce the expression efficiency,we chose the biased codons technology to build the third generation CAR targetingαFR.The lentiviral expressing vector which carriesαFR-CAR gene,pLenti-αFR-CAR,was constructed and then we packaged the lentiviral particles and transfected NK-92 cells.Western Blot,qPCR and flow cytometry were used to detect the expression ofαFR-CAR molecule on NK-92 cells.And lastly,we selected lactate dehydrogenase(LDH)releasing test and ELISA assays to assess the killing ability and specificity of our NK-92-αFR-CAR cells onαFR positive ovarian cancer cells.Results After the double enzyme digestion of pLenti-αFR-CAR recombinant lentiviral plasmid,we gained a gene segment which is the same as theαFR-CAR molecule weight.Western Blot and qPCR confirmed the expression ofαFR-CAR on NK-92 cells and up to 90%NK-92 cells were transfected withαFR-CAR molecule when detected by flow cytometry after puromycin selection.The cytotoxicity and cytokine releasing level of NK-92-αFR-CAR cells is antigen-dependent,namely the higher antigen the tumor cells express,the stronger cytotoxicity the NK-92-αFR-CAR cells show and the more cytokine the NK-92-αFR-CAR cells release.Conclusion We successfully obtained high purity of NK-92-αFR-CAR cells which display specific killing capability toαFR positive ovarian carcinoma cells and secret massive IFN-γand TNF-α.And the basic study of NK-92-αFR-CAR lay a foundation for its preclinical and clinical research.
作者
杨宇
敖翔
李伟强
郭韡
谭燕
敖罗权
何敏
王证程
徐祥
郭建新
YANG Yu;AO Xiang;LI Wei-qiang;GUO Wei;TAN Yan;AO Luo-quan;HE Min;WANG Zhengcheng;XU Xiang;GUO Jian-xin(Department of Gynaecology and Obstetrics,Duping Hospital and Research Institute of Surgery,Army Medical University,Chongqing 400010,China;First Department,State Key Laboratory of Trauma,Burn and Combined Injury,Duping Hospital and Research Institute of Surgery,Army Medical University,Chongqing 400010,China)
出处
《医学研究生学报》
CAS
北大核心
2021年第1期14-19,共6页
Journal of Medical Postgraduates
基金
国家重点研发计划(2018YFC1313400)。
