基于磷脂酰肌醇蛋白聚糖3和巨噬细胞特异性胞内区域的嵌合抗原受体的构建  

Construction of chimeric antigen receptor based on phosphatidylinositol proteoglycan 3 and macrophage specific intracellular region

作　　者：王康 刘双双 陈文魏 殷爱红 邓蕾 俞悦 Wang Kang;Liu Shuangshuang;Chen Wenwei;Yin Aihong;Deng Lei;Yu Yue(Department of Hepatobiliary Surgery,the First Affiliated Hospital of Nanjing Medical University,Key Laboratory on Living Donor Liver Transplantation,National Health Commission,Key Laboratory of Live Liver Transplantation,Chinese Academy of Medical Sciences,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院肝胆中心,国家卫健委活体肝移植重点实验室,中国医学科学院活体肝移植重点实验室,南京210029

出　　处：《中华实验外科杂志》2024年第10期2206-2210,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82270607)。

摘　　要：目的:构建基于肝癌标志物磷脂酰肌醇蛋白聚糖3(GPC3)和巨噬细胞(MΦ)特异性胞内区域结构的嵌合抗原受体(CAR)并验证其功能。方法:采用基因重组的方法,构建2种编码抗人磷脂酰肌醇蛋白聚糖3(GPC3 -ScFv)的CAR的慢病毒载体:含T细胞胞内区域结构的t-CAR和含MΦ特异性胞内区域结构的m-CAR。包装慢病毒并感染人THP-1细胞,制备成t-CAR-THP-1和m-CAR-THP-1,由THP-1、t-CAR-THP-1和m-CAR-THP-1分化的M1型MΦ分别命名为M1、t-CAR-M1和m-CAR-M1。采用共培养、实时荧光定量聚合酶链反应(RT-qPCR)和流式细胞术(FCM)检测两种CAR修饰的M1体外的抗肿瘤功能。进一步在B-NDG小鼠体内构建GPC3阳性肝癌细胞Hep-3B的皮下移植瘤模型,以不做处理作为对照组,注射3×10^(6) M1、3×10^(6) t-CAR-M1和3×10^(6) m-CAR-M1作为实验组,观察裸鼠体内肿瘤生长情况以及裸鼠存活时间。两组间差异比较采用配对样本 t检验,多组间差异比较采用方差分析(ANOVA)。 结果:成功构建靶向GPC3的t-CAR和m-CAR慢病毒载体,制备了t-CAR-THP-1和m-CAR-THP-1细胞,并获得M1、t-CAR-M1和m-CAR-M1,RT-qPCR结果显示,t-CAR-THP-1组和m-CAR-THP-1组GPC3 -ScFv和绿色荧光蛋白(GFP)表达高于THP-1组(9.43±0.21、11.97±1.01比1.00±0.00, t=68.40、18.91, P<0.05)。在体外功能实验中,t-CAR-THP-1和m-CAR-THP-1细胞能够特异性地亲和GPC3蛋白。FCM结果显示,t-CAR-M1和m-CAR-M1对Huh7细胞(GPC3 -)的吞噬差异无统计学意义(10.93±1.10比11.30±0.46, t=0.53, P>0.05),而t-CAR-M1组和m-CAR-M1组的吞噬效应高于M1组(10.93±1.10、11.30±0.46比8.29±0.63, t=3.60、6.67, P<0.05),同时,t-CAR-M1和m-CAR-M1对Hep-3B细胞的吞噬效率显著高于对Huh7细胞(21.57±0.66、27.30±1.58比10.93±1.10、11.30±0.46, t=15.27、16.19, P<0.05)。在GPC3阳性肝癌移植瘤模型小鼠体内,t-CAR-M1和m-CAR-M1治疗组肿瘤生长速度显著低于第一组和第二组[ F(5,15)=6.86, P<0.05],m-CAR-M1组的存活率高于对照组、M1组、Objective:To investigate the anti-tumor effect of macrophages(MΦ)modified by chimeric antigen receptor(CAR)based on phosphatidylinositol proteoglycan 3(GPC3)and the macrophage-specific intracellular region.Methods:Two lentiviral vectors encoding anti human GPC3(GPC3-ScFv)were constructed using gene recombination:t-CAR containing T cell intracellular region and m-CAR containing MΦ-specific intracellular region structure.THP-1 cells were transduced with CAR vectors to get t-CAR-THP-1 and m-CAR-THP-1.M1 type MΦdifferentiated from THP-1,t-CAR-THP-1,and m-CAR-THP-1 were named M1,t-CAR-M1,and m-CAR-M1,respectively.Co-culture,quantitative real-time polymerase chain reaction(RT-qPCR),and flow cytometry(FCM)were used to detect the anti-tumor function of the two types of cells in vitro.Further we constructed a subcutaneous transplant tumor model of GPC3 positive HCC cell Hep-3B in B-NDG mice.With no treatment as the control group,injecting 3×10^(6) M1,3×10^(6) t-CAR-M1,and 3×10^(6) m-CAR-M1 as the experimental groups,we observed the growth of tumors and survival in nude mice.Paired sample t test was used to compare the differences between two groups,and analysis of variance(ANOVA)was used to compare the differences between mutiple groups.Results:T-CAR and m-CAR lentiviral vectors targeting GPC3 were constructed,and t-CAR-THP-1 and m-CAR-THP-1 cells were prepared,and also the M1,t-CAR-M1 and m-CAR-M1.The RT-qPCR results showed that the expression of GPC3-ScFv and green fluorescent protein(GFP)in the t-CAR-THP-1 and m-CAR-THP-1 groups was higher than that in the THP-1 group(9.43±0.21,11.97±1.01 vs.1.00±0.00,t=68.40,18.91,P<0.05).In vitro functional experiments showed that both t-CAR-THP-1 and m-CAR-THP-1 cells can specifically bind to GPC3 protein.The FCM results showed no significant difference in the phagocytosis of Huh7 cells(GPC3-)between t-CAR-M1 and m-CAR-M1(10.93±1.10 vs.11.30±0.46,t=0.53,P<0.05),but phagocytosis of t-CAR-M1 and m-CAR-M1 groups were higher than that of M1 group(10.93±1.10,11.30±0.46 vs.8.

关 键 词：嵌合抗原受体 巨噬细胞 磷脂酰肌醇蛋白聚糖3 肝细胞肝癌 免疫治疗 

分 类 号：R735.7[医药卫生—肿瘤]