Immune activity enhancement of T lymphocyte by ultrasound microbubble mediated Itch gene silencing / 中华超声影像学杂志

ABSTRACT

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 interferon γ (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-γ were (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-γ in 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.