【摘要】 背景与目的:Itch蛋白是一种具有调节T细胞免疫应答起始的关键分子,属于E3泛素转移酶家族,广泛参与细胞内多种信号蛋白如ZAP70、P85、VAV、PLC-γ和PKC-θ等的泛素化修饰过程,在肿瘤诱导机体免疫耐受中起着重要作用。Itch通过调节T细胞表面受体活性及转移生长因子-β信号通路来介导T细胞免疫无反应性,诱导外周组织Treg细胞增殖。因此,通过改变Itch蛋白表达活性有望成为一种治疗自体免疫性疾病和肿瘤的有效途径。我们利用特异性小干扰RNA(small interfering RNA,siRNA)沉默T细胞Itch基因的表达,观察转染T细胞对小鼠MFC胃癌细胞的体外免疫杀伤作用。方法:分离615小鼠脾脏T细胞,筛选高效特异性沉默Itch基因的siRNA序列转染T细胞,蛋白质印迹法检测各分组Itch蛋白的表达水平;转染72 h后,利用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测细胞因子IL-2、INF-γ分泌情况,比较空白组、空转组及转染组T细胞与小鼠MFC胃癌细胞混合培养肿瘤杀伤率。结果:转染48 h后,与对照组相比,转染T细胞Itch蛋白表达率降低至16%;转染72 h后,检测转染组、空转组、空白组细胞因子IL-2分泌水平分别为(1 891.96±141.91)pg/mL,(1 241.69±91.67)pg/mL,(1 175.03±89.14)pg/mL(P<0.001),转染组、空转组、空白组细胞因子INF-γ分泌水平分别为(958.33±75.46)pg/mL,(683.33±66.67)pg/mL,(691.72±68.72)pg/mL(P<0.05)。在体外实验中,与空白组及空转组T细胞相比,转染组T细胞能更高效地杀伤小鼠MFC胃癌细胞,最高杀瘤率达到(54.18±2.96)%。结论:利用特异性siRNA技术沉默Itch基因能够促进小鼠T细胞因子IL-2、INF-γ分泌,增强T细胞对小鼠MFC胃癌细胞的体外免疫杀伤作用。更多还原

【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. Itc h 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 ofItch activities may provide the opportunities to develop future therapies for immune disorders such as autoimmuni ty and cancer. specifi c 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 neopl asms cells in vitro.Methods: T lymphocytes were isolated from the spleen of 615 mice and transfected by specifi c 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 Itchprotein 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 effi cient 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 signifi cantly 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.更多还原