超声微泡介导T淋巴细胞Itch基因沉默增强T细胞免疫活性的研究

Immune activity enhancement of T lymphocyte by ultrasound microbubble mediated Itch gene silencing

目的利用超声靶向微泡破裂(UTMD)技术介导T细胞Itch基因沉默,观察T细胞免疫活性变化。方法磁珠分选纯化T细胞,构建靶向Itch基因的短发夹RNA(shRNA)表达质粒。实验组为超声辐照+Itch-shRNA质粒-SonoVue微泡,对照组为超声辐照+阴性对照shRNA质粒-SonoVue微泡,空白组未处理。转染48 h,荧光显微镜和流式细胞术评估细胞转染效率,免疫印迹(Western blot)法检测Itch基因蛋白表达水平。转染72 h,酶联免疫吸附测定(ELISA)法检测各组细胞上清液中白介素2(IL-2)、γ干扰素(IFN-γ)水平。采用单因素方差分析比较多组间差异,两两比较LSD-t检验。结果利用UTMD技术介导shRNA转染效率达到52.3%,转染后实验组、对照组和空白组Itch相对蛋白表达量分别为0.301±0.080、0.773±0.101和0.719±0.090,实验组Itch蛋白表达显著降低,差异有统计学意义(F=24.441,P<0.01)。转染72 h,实验组、对照组和空白组IL-2浓度分别为(417.3±37.1)ng/L、(158.7±17.3)ng/L和(147.0±10.2)ng/L,差异有统计学意义(F=118.701,P<0.001);IFN-γ浓度分别为(168.3±12.1)ng/L、(74.3±3.7)ng/L和(74.6±7.1)ng/L,差异有统计学意义(F=126.833,P<0.001);实验组IL-2和IFN-γ表达水平较对照组和空白组显著升高。结论利用UTMD技术介导Itch-shRNA转染能明显降低T细胞Itch水平,增强T细胞免疫活性。

Objective To inhibit the Itch gene expression of T-lymphocytes and investigate the immune activity of T lymphocytes using ultrasound-targeted microbubble destruction(UTMD).Methods T lymphocytes were separated by magnetic bead,and to establish an Itch gene targeted short hairpin RNA(shRNA)plasmid.There were three groups in this study:①experimental group,Itch-shRNA plasmid-SonoVue microbubbles combined with ultrasound irradiation;②control group,negative control shRNA plasmid-SonoVue microbubbles combined with ultrasound irradiation;③blank group,untreated.Forty-eight hours after UTMD transfection,transfection efficiency was detected and analyzed by fluorescence microscopy and flow cytometry,the expression of Itch protein was measured with Western blotting.Seventy-two hours after UTMD transfection,the secretion of interleukin 2(IL-2)and interferonγ(IFN-γ)in the cell supernatant were measured by enzyme-linked immunosorbent assay(ELISA).One-way ANOVA was used to compare the differences between groups,and LSD-t test was used to compare the differences between groups.Results The UTMD mediated shRNA transfection rate was 52.3%,and the relative expression levels of Itch protein in the experimental group,control group and blank group were 0.301±0.080,0.773±0.101 and 0.719±0.090,respectively,and the difference was statistically significant(F=24.441,P<0.01).The Itch gene expression can be effectively suppressed in the experimental group.Seventy-two hours after transfection,the concentrations of IL-2 in the experimental group,control group and blank group were(417.3±37.1)ng/L,(158.7±17.3)ng/L and(147.0±10.2)ng/L,respectively,and the difference was statistically significant(F=118.701,P<0.001)and the concentrations of IFN-γwere(168.3±12.1)ng/L,(74.3±3.7)ng/L and(74.6±7.1)ng/L,respectively,and the difference was statistically significant(F=126.833,P<0.001).The expression levels of IL-2 and IFN-γin the experimental group were significantly higher than those in the control group and the blank group.Conclusions UTMD mediated shRNA transfection can significantly decrease the expression of Itch and promote immune activity of T lymphocyte.