关键词: Itch, 超声介导微泡破裂技术, 基因沉默, 肺肿瘤, 免疫治疗

Abstract: Objective To silence the expression of Itch gene of T-Lymphocytes by ultrasound-targeted microbubble destruction (UTMD) and investigate the cytotoxicity activity of transfected T lymphocytes against LA795 Lung Neoplasms cells in vitro. Methods T lymphocyte were isolated by magnetic bead, and an targeted shRNA to silence Itch gene of T lymphocytes was established. 48 hours after transfection by UTMD, the transfection efficiency was detected and analyzed by fluorescence microscope and flow cytometry; the expression of Itch protein was measured with Western Blot; 72 hours after transfection, the secretion of IL-2 and IFN-γ in the cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA) and the cytotoxicity activity changes against LA795 Lung Neoplasms cells were compared between transfected T lymphocytes and negative control or simplex T lymphocytes in vitro. Results The transfection rate to silence Itch gene of T lymphocytes by UTMD was (52.3±3.8)%. At 48 h after transfection, the Itch gene expression can be effectively suppressed by UTMD. 72 hours after transfection, the secretion level of cytokines, including IL-2 and IFN-γ, were significantly rise in the group of targeted shRNA to silence Itch gene of T lymphocytes by UTMD (p<0.05) and transfected T lymphocyte also showed more efficient killing ability against LA795 Lung Neoplasms cells than negative control or blank group at E:T of 10:1、20:1 and 40:1 (p<0.05). Conclusion Silencing the expression of Itch by UTMD can significantly promoted immune activity of T lymphocyte, enhanced the cytotoxicity activity of T lymphocyte against LA795 Lung Neoplasms cells in vitro.

Key words: Itch, ultrasound-targeted microbubble destruction, gene silencing, lung Neoplasms, immunotherapy