超声微泡介导CTLA-4基因靶向抑制对T淋巴细胞免疫活性及肿瘤免疫杀伤率影响的实验研究  

Experimental study on the effect of ultrasound-targeted microbubble destruction mediated CTLA-4 gene target inhibition on T lymphocyte immune activity and tumor immune killing efficiency

作　　者：韩志芬[1] 饶程义 康佳[1] 陈玉[2] HAN Zhi-fen;RAO Cheng-yi;KANG Jia;CHEN Yu(Affiliated Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610072,China)

机构地区：[1]成都中医药大学附属医院超声医学科,四川成都610072 [2]成都中医药大学附属医院肿瘤科,四川成都610072

出　　处：《中华肿瘤防治杂志》2023年第1期14-19,共6页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的 利用超声介导微泡破裂技术(UTMD)介导T细胞转染,下调细胞毒性T细胞抗原-4(CTLA-4)基因表达,观察转染T细胞免疫活性变化,检测转染T细胞对MGC-803胃癌细胞的体外免疫杀伤率。方法 分离纯化C57小鼠脾脏T淋巴细胞,构建靶向CTLA-4基因的短发夹RNA表达质粒,超声微泡介导CTLA-4质粒转染,设置转染组、阴性对照组和空白组,流式细胞仪分析转染率,实时荧光定量聚合酶链反应(qRT-PCR)检测CTLA-4基因表达,蛋白质印迹法检测各分组CTLA-4蛋白表达,流式细胞仪检测各分组T淋巴细胞活性标志CD25表达情况。按照30∶1靶效比将转染T淋巴细胞与MGC-803胃癌细胞共培养72 h,观察各分组体外肿瘤细胞杀伤率。结果 超声微泡介导shRNA转染率达71.6%,转染组CTLA-4基因和蛋白表达降低。转染组、阴性对照组和空白组CTLA-4 mRNA的表达分别为0.994±0.091、3.571±0.342和3.924±0.422,差异有统计学意义,F=86.374,P<0.001。转染组、阴性对照组和空白组CTLA-4蛋白相对表达量分别为0.153±0.031、0.517±0.102和0.499±0.094,差异有统计学意义,F=20.394,P=0.002。转染48 h后,转染组T细胞活化标志CD25表达升高,转染组、阴性对照组和空白组CD25表达分别为(29.13±3.73)%、(9.01±1.24)%和(9.33±1.07)%,差异有统计学意义,F=72.034,P<0.001。转染72 h后,转染T淋巴细胞肿瘤杀伤率增强,MGC-803胃癌细胞杀伤率达到(49.6±5.73)%,而阴性对照组和空白组MGC-803胃癌细胞杀伤率分别为(23.2±1.91)%和(21.6±1.57)%,差异有统计学意义,F=57.138,P<0.001。结论 通过UTMD介导转染能有效沉默CTLA-4基因和蛋白表达,上调T细胞免疫活性,增强T细胞对MGC-803胃癌细胞体外免疫杀伤作用。Objective Ultrasound-targeted microbubble destruction(UTMD) was applied to mediate cell transfection, silence the expression of cytotoxic T lymphocyte antigen 4(CTLA-4) gene of T-Lymphocytes, investigate the cytotoxicity activity of transfected T lymphocytes, and observe the immune killing efficiency of transfected T lymphocytes on MGC-803 gastric cancer cells in vitro.Methods C57 mouse spleen T lymphocytes were separated and established an targeted shRNA to silence CTLA-4 gene of T lymphocytes.There were three groups in this study, including transfection group, negative control group and blank group.Flow cytometry and fluorescence microscope were used to evaluate transfection efficiency of each group;western blot was used to detect the CTLA-4 protein expression;quantitative real-time polymerase chain reaction(qRT-PCR) was used to assay the gene expression of CTLA-4;flow cytometry was used to measure T lymphocyte activation markers CD25 expression.Transfected T lymphocytes were co-cultured with MGC-803 gastric cancer cells for 72 h and the effect/target ratio was 30∶1,and the killing efficiency of tumor cells in vitro was detected and analyzed by CCK8 assay.Results The transfection rate of T lymphocytes by UTMD was 71.6% and the CTLA-4 gene expression of transfection group significantly reduced.The mRNA expression of CTLA-4 in transfection group, negative control group and blank group was 0.994±0.091,3.571±0.342 and 3.924±0.422,respectively(F=86.374,P<0.001),the difference was statistically significant.The relative expression levels of CTLA-4protein in transfected group,negative control group and blank group were 0.153±0.031,0.517±0.102and 0.499±0.094,respectively(F=20.394,P=0.002),the difference was statistically significant.Forty-eight hours after transfection,the expression level of CD25significantly increased in the transfection group,CD25expressions in transfection group,negative control group and blank group were(29.13±3.73)%,(9.01±1.24)% and(9.33±1.07)%,respectively(F=72.034,P<0.001),the differe

关 键 词：细胞毒性T细胞抗原-4 超声介导微泡破裂技术 基因沉默 T淋巴细胞 肿瘤免疫 

分 类 号：R730.3[医药卫生—肿瘤]