Silencing itch by small specific interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cells<em>in vitro</em>

粟英; 兰亚明; 卢义琼; 田国红; 胡烈献

doi:10.3969/j.issn.1007-3969.2014.10.011

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Silencing itch by small specific interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cellsin vitro

更新时间：2025-12-31

- Silencing itch by small specific interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cellsin vitro
- China Oncology Vol. 24, Issue 10, Pages: 777-782(2014)
- 作者机构：
  
  湖北民族学院附属民大医院急诊科,湖北,恩施,445000
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.1007-3969.2014.10.011
  CLC：
- Published Online：12 November 2014，
  
  Published：12 November 2014
- 稿件说明：
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粟英,兰亚明,卢义琼,田国红,胡烈献. 特异性小干扰RNA沉默

粟英, 兰亚明, 卢义琼, et al. Silencing itch by small specific interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cellsin vitro[J]. China Oncology, 2014, 24(10): 777-782.
粟英,兰亚明,卢义琼,田国红,胡烈献. 特异性小干扰RNA沉默 DOI： 10.3969/j.issn.1007-3969.2014.10.011.

粟英, 兰亚明, 卢义琼, et al. Silencing itch by small specific interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cellsin vitro[J]. China Oncology, 2014, 24(10): 777-782. DOI： 10.3969/j.issn.1007-3969.2014.10.011.

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(P0.001)，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(P0.05)。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。

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 (P0.001)

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 (P0.05). 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.

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Silencing itch by small specific interfering RNA enhance immune activity of mouse T lymphocyte to kill MFC stomach neoplasms cellsin vitro

DOI：10.3969/j.issn.1007-3969.2014.10.011

摘要

Abstract

关键词

Keywords

references