特异性嵌合抗原受体-T细胞对不同表皮生长因子受体表达肿瘤的杀伤效应研究

Killing efficacy of specific chimeric antigen receptor-T cells on tumors with different epidermal growth factor receptor expression

目的探讨特异性嵌合抗原受体(CAR)-T细胞对不同表皮生长因子受体(EGFR)表达肿瘤的杀伤效应。方法北部战区总医院呼吸与危重症医学科已制备所需CAR-T细胞,将其分为UTD对照组、EGFR CAR-T组和CD19 CAR-T组。选取H23非小细胞肺癌细胞系、SKOV3卵巢癌细胞系、Hela宫颈癌细胞系及人胃癌细胞系(HGC)27胃癌细胞系,采用流式细胞术检测肿瘤细胞表面EGFR表达情况,通过免疫磁珠分选技术分选SKOV3和Hela细胞,分选得到EGFR阳性率更高的肿瘤细胞,采用流式细胞术检测其表达EGFR阳性率。将UTD对照组、EGFR CAR-T组和分化抗原簇(CD)19 CAR-T组分别与不同EGFR阳性率肿瘤细胞共培养,通过实时无标记细胞功能分析仪(RTCA)检测其对多种EGFR表达率肿瘤细胞的杀伤曲线。结果流式细胞术检测H23、SKOV3、Hela及HGC27细胞系的EGFR阳性率分别为83.7%、73.8%、48.3%和6.1%。免疫磁珠法分选后SKOV3及Hela细胞系EGFR表达率为81.1%和64.2%。RTCA法证实EGFR CAR-T组能够杀伤EGFR高表达的H23细胞,对EGFR低表达的HGC27细胞无明显杀伤作用,差异有统计学意义(P<0.001)。与SKOV3及Hela细胞比较,EGFR CAR-T细胞组对分选后SKOV3及分选后Hela细胞杀伤更强,差异有统计学意义(P<0.001)。结论不同肿瘤细胞EGFR表达阳性率不同,针对EGFR的CAR-T细胞能够杀伤EGFR阳性的肿瘤细胞,且肿瘤细胞EGFR表达率越高对其杀伤作用越强。

Objective To explore the killing effect of specific chimeric antigen receptor(CAR)-T cells on tumors with different epidermal growth factor receptor(EGFR)expression.Methods The Respiratory and Critical Care Medicine department of the Northern Theater Command General Hospital has prepared CAR-T cells for the experiment and divided them into UTD control group,EGFR CAR-T group,and CD19 CAR-T group.We select H23 non-small cell lung cancer cell line,SKOV3 ovarian cancer cell line,Hela cervical cancer cell line,and human gastric cancer(HGC)27 gastric cancer cell line,and use flow cytometry to detect the expression of EGFR on the surface of tumor cells,using immunomagnetic bead sorting technology to select SKOV3 and Hela cells,and select tumor cells with higher EGFR positivity.Using flow cytometry to detect the positive rate of EGFR expression.UTD control group,EGFR CAR-T group and cluster of differentiation(CD)19 CAR-T group were co-cultured with tumor cells with different EGFR positive rates,respectively.Their killing curves against tumor cells with multiple EGFR expression rates were detected by real-time labeling free cell function analyzer(RTCA).Results The positive rates of EGFR in H23,SKOV3,Hela,and HGC27 cell lines detected by flow cytometry were 83.7%,73.8%,48.3%,and 6.1%.The expression rates of EGFR in SKOV3 and Hela cell lines were 81.1%and 64.2%after immunomagnetic bead sorting.The RTCA method confirmed that the EGFRCAR-T group was able to kill H23 cells with high EGFR expression,but had no significant killing effect on HGC27 cells with low EGFR expression,the differences were statistically significant(P<0.001).Compared with SKOV3 and Hela cells,the EGFR CAR-T cell group showed stronger cytotoxicity to sorted SKOV3 and Hela cells,the differences were statistically significant(P<0.001).Conclusions Different tumor cells have different EGFR expression.CAR-T cells targeting EGFR can kill EGFR positive tumor cells,and the higher EGFR expression of tumor cells,the stronger killing efficacy.