靶向EGFRvⅢ的CAR-NK细胞对胶质瘤细胞杀伤作用的体外实验研究  

In vitro experimental study on the killing effect of EGFRvⅢ-targeted CAR-NK cells on glioma cells

作　　者：陈举林 依日扎提·艾力 秦虎[1,2] 吉文玉 郑伊桐[1,2] 汪永新 Chen Julin;Yirizati Aili;Qin Hu;Ji Wenyu;Zheng Yitong;Wang Yongxin(Department of Neurosurgery,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830000,China;Key Laboratory of Precision Diagnosis and Clinical Translation of Neurological Tumors,Urumqi 830000,China)

机构地区：[1]新疆医科大学第一附属医院神经外科,乌鲁木齐830000 [2]神经肿瘤精准诊断与临床转化重点实验室,乌鲁木齐830000

出　　处：《中华神经外科杂志》2024年第8期841-847,共7页Chinese Journal of Neurosurgery

基　　金：新疆维吾尔自治区自然科学基金(2022D01D70)。

摘　　要：目的探索靶向表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)的嵌合抗原受体(CAR)修饰的人外周血来源自然杀伤(NK)细胞在脑胶质瘤细胞中的杀伤作用。方法通过Ficoll密度梯度离心法获得人外周血来源的NK细胞并进行体外扩增培养,并通过慢病毒感染构建靶向EGFRvⅢ的CAR-NK细胞及U87-EGFRvⅢ稳转细胞株。根据不同效应细胞作用于不同靶细胞而将实验组分为NK+U87组、NK+U87-EGFRvⅢ组、CAR-NK+U87组、CAR-NK+U87-EGFRvⅢ组,通过实时无标记细胞分析(RTCA)及乳酸脱氢酶(LDH)释放实验评价CAR-NK细胞对胶质瘤细胞的杀伤作用,同时使用酶联免疫吸附实验(ELISA)检测肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)、颗粒酶B(GzmB)以及穿孔蛋白1(PRF1)在细胞培养上清中的分泌情况。结果流式细胞分析显示分离、培养得到的人外周血来源NK细胞的纯度为97.6%,存活率为98.2%;成功构建第二代靶向EGFRvⅢ的CAR分子,并成功构建靶向EGFRvⅢ的CAR-NK细胞,流式分析显示阳性细胞率为69.6%。RTCA结果显示靶细胞的生长曲线随时间呈上升趋势,过表达EGFRvⅢ的U87细胞增殖速率要高于U87细胞;在加入效应细胞后,细胞指数呈现下降趋势,在相同效靶比(E∶T)下,CAR-NK+U87-EGFRvⅢ组的细胞指数下降趋势快于NK+U87-EGFRvⅢ组,且快于CAR-NK+U87组。LDH释放实验进一步证明随着效靶比的增加,靶细胞的裂解率逐渐升高(F_(NK+U87)=700.61、F_(CAR-NK+U87)=2063.97、F_(NK+U87)-EGFRvⅢ=5707.00、F_(CAR-NK+U87)-EGFRvⅢ=28858.57,均P<0.05);在E∶T=2∶1及E∶T=3∶1时,NK+U87-EGFRvⅢ组、CAR-NK+U87组的细胞裂解率均低于CAR-NK+U87-EGFRvⅢ组(均P<0.05)。ELISA结果显示,TNF-α、IFN-γ、GzmB及PRF1的分泌量在相同组别内均随着效靶比的增加而增加(均P<0.05);在相同效靶比下,除了在E∶T=1∶1时,CAR-NK+U87-EGFRvⅢ组较CAR-NK+U87组GzmB分泌量的差异无统计学意义(P>0.05);CAR-NK+U87-EGFRvⅢ组分别与NK+U87-EGFRvⅢ组、CAR-NK+U8Objective To explore the specific killing effect of human peripheral blood-derived NK cells modified with Chimeric Antigen Receptor(CAR)targeting epidermal growth factor receptor typeⅢmutant(EGFRvⅢ)on glioma cells.Methods Human peripheral blood-derived NK cells were obtained by Ficoll density gradient centrifugation and cultured in vitro.CAR-NK cells targeting EGFRvⅢand U87-EGFRvⅢstable cell lines were constructed by lentivirus infection.The experiments were divided into NK+U87 group,NK+U87-EGFRvⅢgroup,CAR-NK+U87 group and CAR-NK+U87-EGFRvⅢgroup.The killing effect of CAR-NK cells on glioma cells was evaluated by real-time unlabeled cell analysis(RTCA)and lactate dehydrogenase(LDH)release assay.At the same time,enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),granzyme B(GzmB)and perforin 1(PRF1)in cell culture supernatant.Results Flow cytometry analysis showed that the purity of human peripheral blood-derived NK cells was 97.6%,and the survival rate was 98.2%.The second generation of CAR molecules targeting EGFRvⅢwas successfully constructed,and CAR-NK cells targeting EGFRvⅢwere successfully obtained.Flow cytometry analysis showed that the positive cell rate was 69.6%.RTCA results showed that the growth curve of target cells increased with time,and the proliferation rate of U87 cells overexpressing EGFRvⅢwas higher than that of U87 cells.After adding effector cells,the cell index showed a downward trend.At the same effector-target ratio(E∶T),the cell index of CAR-NK+U87-EGFRvⅢgroup decreased faster than that of NK+U87-EGFRvⅢgroup,and faster than that of CAR-NK+U87 group.LDH release assay further demonstrated that the cleavage rate of target cells gradually increased with the increase of effector-target ratio(F_(NK+U87)=700.61,F_(CAR-NK+U87)=2063.97,F_(NK+U87)-EGFRvⅢ=5707.00,F_(CAR-NK+U87)-EGFRvⅢ=28858.57,all P<0.05).At E∶T=2∶1 and E∶T=3∶1,the cell lysis rates of NK+U87-EGFRvⅢgroup and CAR-NK+U87

关 键 词：神经胶质瘤 受体 表皮生长因子 杀伤细胞 天然 体外研究 

分 类 号：R739.4[医药卫生—肿瘤]