嵌合抗原受体修饰的诱导多能干细胞来源的M1极化的巨噬细胞治疗肝癌的研究  

Treatment of hepatocellular carcinoma with M1 polarized macrophages derived from induced pluripotent stem cells modified with chimeric antigen receptors

作　　者：刘双双 王康 史啸立 陈文魏 邓蕾 殷爱红 俞悦 Liu Shuangshuang;Wang Kang;Shi Xiaoli;Chen Wenwei;Deng Lei;Yin Aihong;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2188-2194,共7页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82270607)。

摘　　要：目的:探讨嵌合抗原受体(CAR)修饰的诱导多能干细胞(iPSCs)分化的M1型巨噬细胞(MΦ)治疗肝细胞肝癌的应用研究。方法:选用前期构建的抗人磷脂酰肌醇蛋白聚糖3(GPC3^(-)scFv)且含有MΦ特异性胞内区域结构的CAR元件慢病毒载体感染人iPSCs,获得CAR修饰的iPSCs即CAR-iPSCs,分化为MΦ,命名为CAR-iM(由iPSCs分化的巨噬细胞称为iM),进而极化成M1型MΦ,命名为CAR-iM1。采用实时荧光定量聚合酶链反应(RT-qPCR)、流式细胞术、酶联免疫吸附试验检测CAR-iM1的体外抗肿瘤功能。进一步在B-NDG裸鼠体内构建GPC3^(+)肝癌细胞Hep-3B的皮下移植瘤模型,分别不做处理、注射3×10^(6) iM1和3×10^(6) CAR-iM1,观察裸鼠体内肿瘤生长情况、存活时间、肿瘤内肿瘤相关巨噬细胞(TAMs)的表型。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:FCM检测CAR-iPSCs的绿色荧光蛋白(GFP)阳性表达率分别为89.7%,RT-qPCR检测CAR-iPSCs的GPC3^(-)scFv和GFP表达显著高于iPSCs[F(1,8)=266.6,P<0.05]。iPS组、EBs D4组的LIN28A显著高于iMc组(6.53±0.23、3.44±0.08比0.50±0.01,t=44.53、61.85,P<0,05),iPS组、EBs D4组的POU5F显著高于iMc组(5.35±0.36、5.55±0.15比0.50±0.01,t=23.37、58.16,P<0.05)。成功建立CAR-iPSCs细胞系,分化为CAR-iM并极化为CAR-iM1。共培养实验结果显示,CAR-iM1对Huh7细胞(GPC3^(-))的吞噬与iM1比较差异无统计学意义(16.97±0.40比14.10±1.11,t=4.19,P>0.05);于Hep-3B(GPC3^(+))细胞,CAR-iM1的吞噬效应高于iM1(34.60±0.78比14.97±2,27,t=14.15,P<0.05)。iM1与Huh7、Hep-3B细胞共培养时分泌的白细胞介素(IL)-6和肿瘤坏死因子-α(TNF-α)显著高于iM1单独培养(183.20±2.73、194.60±4.02比94.32±3.97,t=31.96、30.47,P<0.05);CAR-iM1与Huh7、Hep-3B细胞共培养时分泌的IL-6和TNF-α显著高于CAR-iM1单独培养(101.80±18.44比185.50±24.29、314.20±24.22,t=4.75、12.09,P<0.05);与Hep-3B细胞共培养时CAR-iM1分泌的IL-6和TNF-α水Objective:To study the chimeric antigen receptor(CAR)modified induced pluripotent stem cells(iPSCs)differentiated-and polarized-M1 macrophage(MΦ)in the treatment of hepatocellular carcinoma(HCC).Methods:We made CAR-iPSCs by transducing human iPSCs with the previously constructed lenti-CAR vector containing anti-human phosphatidylinositol proteoglycan 3(GPC3^(-)scFv)and MΦ-specific intracellular region structure.These CAR-iPSCs were differentiated into macrophages,named CAR-iM,and then polarized into M1-MΦ(CAR-iM1).Quantitative real time polymerase chain reaction(RT-qPCR),flow cytometry,and enzyme-linked immunosorbent assay were used to detect the in vitro anti-tumor function of CAR-iM1.Further we constructed a subcutaneous transplant tumor model with GPC3 positive HCC cell line Hep-3B in B-NDG mice,injected with 3×10^(6) iM1 and 3×10^(6) CAR-iM1 and then observed the tumor growth,survival time,and phenotype of tumor associated macrophages(TAMs).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:The positive expression rates of green fluorescent protein(GFP)in CAR-iPSCs detected by FCM were 89.7%,and the GPC3 scFv and GFP expression in CAR-iPSCs detected by RT-qPCR were significantly higher than those in iPSCs[F(1,8)=266.6,P<0.05].LIN28A in the iPS and EBs D4 groups(6.53±0.23,3.44±0.08)was significantly higher than that in the iMc group.(0.50±0.01,t=44.53,61.85,P<0.05),and POU5F in the iPS and EBs D4 groups(5.35±0.36,5.55±0.15)was significantly higher than that in the iMc group(0.50±0.01,t=23.37,58.16,P<0.05).CAR-iPSCs cell line were successfully established,differentiated into CAR-iM,and polarized into CAR-iM1.Co-culture tests showed that there was no significant difference in the phagocytosis of huh7 cells(GPC3^(-))by CAR-iM1(16.97±0.40)compared to iM1(14.10±1.11,t=4.19,P>0.05).The phagocytic effect of CAR-iM1 in Hep-3B(GPC3^(+))cells(34.60±0.78)was higher than that in iM1(14.9

关 键 词：嵌合抗原受体 巨噬细胞 诱导多能干细胞 磷脂酰肌醇蛋白聚糖3 肝细胞肝癌 

分 类 号：R735.7[医药卫生—肿瘤]