信号调节蛋白α1（Sirpalpha1）在肝细胞癌中的负调控机制：一项实验研究

Negatively regulating mechanism of Sirpalpha1 in hepatocellular carcinoma: an experimental study.

作者信息

Qin Jian-Min, Yan He-Xin, Liu Shu-Qin, Wan Xing-Wang, Zeng Jin-Zhang, Cao Hui-Fang, Qiu Xiu-Hua, Wu Meng-Chao, Wang Hong-Yang

机构信息

International Co-operational Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, Shanghai 200438, China.

出版信息

Hepatobiliary Pancreat Dis Int. 2006 May;5(2):246-51.

PMID:16698585

Abstract

BACKGROUND

Signal regulatory protein (Sirp) is a recently isolated, cloned and identified inhibitor receptor distributed in the membrane of hematopoietic and nonhematopoietic cells. Sirp alpha1 (Sirpalpha1) is a member of Sirp families. Sirpalpha1 can bind SHP-2 in the form of tyrosine phosphorylation by SH2 effect and negatively regulate growth factor, oncogene, or insulin-induced responses as its substrate. This study aimed to preliminarily clarify the negatively regulating proliferation mechanism of Sirpalpha1 in liver cancer.

METHODS

pLXSN, Sirpalpha1 and Sirpalpha1P4Y2 plasmids were respectively transfected into Sk-Hep1 liver cancer cell line, and various stable Sk-Hep1 cell lines were obtained with screening agent of G418 (1200 microg/ml). The expressing levels of cyclin D1, CDK4, Fas, beta-catenin and gankyrin in various cell lines were determined with Western blotting. Cell cycles were determined at 0, 12 and 24 hours with flow cytometry after various synchronous cell lines were cultured without serum for 72. Cell apoptosis induced with agent of TNF-alpha (50 ng/ml) was determined with flow cytometry at 0, 0.5, 1, 3, 6 and 12 hours.

RESULTS

Sirpalpha1 could significantly decrease the expression of cyclin D1, beta-catenin and gankyrin, but it couldn't affect the expression level of CDK4 and Fas. When synchronous cells were cultured for 12 hours, S phase Sk-Hep1 cell transfected with Sirpalpha1 plasmid was the lowest [(31.92+/-0.22)% vs. other cell lines, P<0.05], and the cell line was highly sensitive to TNF-alpha agent for 1 hour. (59.31+/-0.59)% of apoptotic cells occurred (vs. the other time points, P<0.05).

CONCLUSIONS

Sirpalpha1 might block the cell cycle of liver cancer, inhibit cell proliferation, promote cell apoptosis by decreasing the expression of cyclin D1, beta-catenin and gankyrin. It is one of the important mechanisms inhibiting the occurrence and development of hepatocellular carcinoma.

摘要

背景

信号调节蛋白（Sirp）是一种最近分离、克隆和鉴定的抑制性受体，分布于造血细胞和非造血细胞膜上。Sirpα1是Sirp家族的成员之一。Sirpα1可通过SH2结构域以酪氨酸磷酸化的形式结合SHP-2，并作为其底物对生长因子、癌基因或胰岛素诱导的反应进行负调控。本研究旨在初步阐明Sirpα1在肝癌中负调控增殖的机制。

方法

将pLXSN、Sirpα1和Sirpα1P4Y2质粒分别转染至Sk-Hep1肝癌细胞系，用G418（1200μg/ml）筛选剂获得各种稳定的Sk-Hep1细胞系。采用蛋白质免疫印迹法检测各细胞系中细胞周期蛋白D1、细胞周期蛋白依赖性激酶4（CDK4）、Fas、β-连环蛋白和杆状病毒RING结构域蛋白（gankyrin）的表达水平。将各种同步化细胞系无血清培养72小时后，分别于0、12和24小时用流式细胞术检测细胞周期。用肿瘤坏死因子-α（TNF-α，50ng/ml）诱导细胞凋亡，分别于0、0.5、1、3、6和12小时用流式细胞术检测细胞凋亡情况。

结果

Sirpα1可显著降低细胞周期蛋白D1、β-连环蛋白和gankyrin的表达，但不影响CDK4和Fas的表达水平。同步化细胞培养12小时时，转染Sirpα1质粒的S期Sk-Hep1细胞比例最低[（31.92±0.22）%，与其他细胞系相比，P<0.05]，且该细胞系对TNF-α诱导1小时最为敏感，凋亡细胞比例为（59.31±0.59）%（与其他时间点相比，P<0.05）。