关键词: Itch, 基因沉默, T淋巴细胞, 胃肿瘤, 免疫疗法

Abstract:

Background and purpose: Itch protein is an established regulator of T cell immune response thresholds, belong to a class of E3 ubiquitin-transferring enzymes, widely involve in the ubiquitination of several key signaling molecules, such as ZAP70, P85, VAV, PLC-γ, PKC-θ, etc, plays a critical role in tumor induced immunosuppression.Itch ligase activity regulate T-cell anergy and development of regulatory T cells in the periphery by modulating key components of T-cell receptor and transforming growth factor-β signaling. Therefore, manipulation of Itch activities may provide the opportunities to develop future therapies for immune disorders such as autoimmunity and cancer. specific small interfering RNA(siRNA) was utilized to silence the expression of Itch gene of T-lymphocytes and investigate the cytotoxicity activity of transfected T lymphocytes against MFC stomach neoplasms cells in vitro. Methods: T lymphocytes were isolated from the spleen of 615 mice and transfected by specific siRNA to silence the expression of Itch gene, The expression of Itch protein were examined by Western bolt in each group; 72 hours after transfection, The secretion level of IL-2, INF-γ were measured by enzyme-linked immunosorbent assay (ELISA). At the end, the cytotoxicity activity changes against MFC stomach neoplasms cells was compared between transfected T lymphocytes, negative control and blank control in vitro. Results: Compared with control group, the expression rate of Itch protein of transfected T-lymphocytes was decreased to 16% after transfection 48 hours; 72 hours after transfection, the secretion level of IL-2 in transfection group, negative control and blank control respectively were (1 891.96±141.91)pg/mL, (1 241.69±91.67)pg/mL and (1 175.03±89.14)pg/mL (P<0.001), the secretion level of INF-γ in transfection group, negative control and blank control respectively were (958.33±75.46)pg/mL, (683.33±66.67)pg/mL and (691.72±68.72) pg/mL (P<0.05). Transfected T lymphocyte also showed more efficient killing ability against MFC stomach neoplasms cells than negative control and blank control in vitro, the highest killing rate has reached (54.18±2.96)%. Conclusion: Silencing Itch gene can significantly promoted the secretion level of IL-2, INF-γ of mice T lymphocyte, enhanced the cytotoxicity activity of T lymphocyte against MFC stomach neoplasms cells in vitro.

Key words: Itch, Gene silencing, T-lymphocytes, Stomach neoplasms, Adoptive immunotherapy