靶向ROR1的嵌合抗原受体修饰T细胞的构建及对ROR1阳性肿瘤细胞的杀伤作用

The Construction of ROR1 Targeting Chimeric Antigen Receptor Modified T Cells and Its Killing Effect for ROR1-positive Tumor Cells

目的探讨靶向Ⅰ型受体酪氨酸激酶样孤儿受体(ROR1)的嵌合抗原受体修饰的T细胞(chimeric antigen receptor T cell, CAR-T)治疗ROR1阳性肿瘤细胞的体外杀伤作用。方法设计并合成CAR基因,通过慢病毒载体质粒pWPXLd,构建靶向ROR1的CAR-T核心质粒,采用BamHⅠ/EcoRⅠ双酶切鉴定,应用流式细胞术检测多种实体瘤癌细胞株表面ROR1的表达,并采用流式细胞术、乳酸脱氢酶(LDH)检测试验检测CAR-T对ROR1阳性肿瘤细胞的杀伤作用,ELISA检测试剂盒测定培养细胞上清中γ-干扰素(IFN-γ)含量。结果构建的CAR-T质粒双酶切鉴定有CAR基因插入,流式细胞术检测结果显示CAR-T感染效率约为47.23%。多种肿瘤细胞不同程度表达ROR1。流式细胞术、LDH检测试验结果提示CAR-T能特异性地杀伤ROR1阳性肿瘤细胞。CAR-T针对ROR1阳性靶细胞能释放更多的IFN-γ(P<0.05)。结论成功构建靶向ROR1的CAR-T,并能特异地杀伤ROR1阳性肿瘤细胞,释放大量IFN-γ,为临床应用提供了实验基础。

Objective To test the killing effect of typeⅠreceptor tyrosine kinase-like orphan receptor(ROR1)chimeric antigen receptor T cell(CAR-T)on several ROR1-expressing tumor cells in vitro.Methods The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid,and BamHⅠ/EcoRⅠwas used to identify the plasmid.The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry(FCM).The killing effect of CAR-T on positive cells was detected by FCM,the LDH assay and ELISA.Results The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid.FCM detection showed that the efficiency of CAR-T infection was about 47.23%.Multiple tumor cells expressed ROR1 in varying degrees.The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells.On positive target cells,more interferonI-γ(FN-γ)could be released during the CAR-T killing process than control T(P<0.05).Conclusion We successfully constructed ROR1 CAR-T.CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ,providing an experimental basis for clinical application.