Silencing itch by small speciifc interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cells in vitro
10.3969/j.issn.1007-3969.2014.10.011
- VernacularTitle:特异性小干扰RNA沉默Itch基因增强小鼠T细胞对MFC胃癌细胞的免疫杀伤作用
- Author:
Ying SU
;
Yaming LAN
;
Yiqiong LU
;
Guohong TIAN
;
Liexian HU
- Publication Type:Journal Article
- Keywords:
Itch;
Gene silencing;
T-lymphocytes;
Stomach neoplasms;
Adoptive immunotherapy
- From:China Oncology
2014;(10):777-782
- CountryChina
- Language:Chinese
-
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 immu-nosuppression. 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-βsignaling. Therefore, manipulation of Itch activities may provide the opportunities to develop future therapies for immune disorders such as autoimmunity and cancer. speciifc 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 speciifc 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-γwere measured by enzyme-linked immunosorbent assay (ELISA). At the end, the cytotoxicity activity changes against MFC stomach neoplasms cells was compared between transfected T lym-phocytes, 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-γin 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 efifcient 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 signiifcantly promoted the secretion level of IL-2, INF-γof mice T lymphocyte, enhanced the cytotoxicity activity of T lymphocyte against MFC stomach neoplasms cells in vitro.
