肝癌干细胞标志物及细小病毒H-1非结构蛋白NS-1对肝癌干细胞生长的影响  

Preliminary study of hepatocarcinoma stem cell markers and the impact of parvovirus nonstructural protein gene on hepatocarcinoma stem cells

作　　者：张洁[1] 俞翀曌[1] 汪铮 葛海良[3] 

机构地区：[1]上海交通大学医学院附属仁济医院检验科,上海200001 [2]上海市消化病研究所,上海200001 [3]上海交通大学医学院免疫学教研室,上海200025

出　　处：《诊断学理论与实践》2010年第2期146-151,共6页Journal of Diagnostics Concepts & Practice

基　　金：上海市科委基金(06ZR14063)

摘　　要：目的:研究肝癌细胞系SMMC7721肿瘤干细胞表面标志物,探讨细小病毒H-1非结构蛋白NS-1对肝癌细胞的生长抑制作用,以及其对肝癌细胞中具有干细胞特性群体的影响。方法:用流式细胞仪将肝癌细胞按细胞表面标志CD133、CD44进行分离,分为CD133+CD44+、CD133+CD44-、CD133-CD44+、CD133-CD44-4个群体,采用脂质体法将细小病毒H-1非结构蛋白NS-1质粒转染入人肝癌细胞SMMC7721,采用G418筛选稳定转染的细胞克隆,反转录-聚合酶链反应(RT-PCR)检测转染细胞NS-1基因的表达。流式细胞仪分析SMMC7721转染前后肿瘤干细胞表面标志CD133、CD90、CD44的表达和细胞生长周期,检测细胞群体的变化。以裸鼠成瘤实验和软琼脂克隆形成实验评价具肿瘤干细胞性质的肝癌细胞表面标志物以及细小病毒H-1非结构蛋白NS-1转染对肝癌细胞生长的影响。结果:肝癌SMMC7721细胞分成CD133+CD44+、CD133+CD44-、CD133-CD44+、CD133-CD44-4组亚群的细胞存在着异质性,4组亚群细胞中均有少数肿瘤干细胞样细胞能在软琼脂上形成克隆,其中CD133+CD44+、CD133+CD44-亚群克隆形成率分别为3.5%和1.8%,CD133-CD44+、CD133-CD44-亚群克隆形成率为0.7%和0.5%。转染细小病毒H-1非结构蛋白NS-1后,4组亚群细胞克隆的形成率都显著下降。裸鼠成瘤实验提示,CD133+表型的细胞成瘤能力强于CD133-表型细胞,CD133-CD44+亚群细胞成瘤能力最弱。流式细胞仪分析显示转染NS-1后,CD133+群体细胞被明显抑制,由31.9%下降至6.3%,而CD90+和CD44+的表达未出现改变;转染NS-1后细胞S期百分率明显下降。结论:肝癌细胞表型为CD133+CD44+和CD133+CD44-亚群中富含肝癌干细胞,转染细小病毒H-1非结构蛋白基因NS-1的部分亚群SMMC7721细胞生长被抑制,提示其对肝癌细胞株中具有干细胞特性的CD133+群体有一定的抑制效应。Objective To study tumor stem cell surface markers of human hepatocarcinoma cell line SMMC7721. Cells with tumor stem cell characteristics were isolated,and parvovirus nonstructural protein gene （NS-1） was transfected to study the inhibitory effect of parvovirus NS-1 on proliferation of human hepatocarcinoma cell and the effect on hepatocarcinoma cell with tumor stem cell characteristics. Methods Hepatocarcinoma cells were isolated according to cell surface markers CD133 and CD44 by flow cytometry into four subpopulations：CD133＋CD44＋,CD133＋CD44-,CD133-CD44＋ and CD133-CD44-. Parvovirus NS-1 plasmid was transfected into hepatocarcinoma cell line SMMC7721 cells by lipofectamine 2000. Stable tranfected cell clones were screened by G418. RT-PCR was used to detect the expression of NS-1 in transfected cells. Flow cytometry was used to assess the expression of tumor stem cell surface markers CD133,CD90 and CD44 in SMMC7721 cells before and after transfection with NS-1,and to assess the cell growth cycles and the changes of cell populations. Tumorigenecity in nude mice and colony formation assay in soft agar media were used to evaluate the effect of tumor stem cell surface markers and transfection of NS-1 on hepatocarcinoma cell proliferation. Results There were heterogeneities among the four subpopulations——CD133＋CD44＋,CD133＋CD44-,CD133-CD44＋,CD133-CD44-. Of these four subpopulations,some tumor stem cell like cells could form spherical clones in soft agar media. The clone formation rates of CD133＋CD44＋ and CD133＋CD44- subpopulations were 3.5% and 1.8%,respectively; that of CD133-CD44＋ and CD133-CD44- subpopulations were 0.7% and 0.5%,respectively. The clone formation rates of all these four subpopulations decreased markedly after transfection with NS-1. The tumorigenicity in nude mice experiments showed that tumorigenic potential of CD133＋ cells was stronger than that of CD133-cells. The tumorigenic potential of CD133-CD44＋ cells was the weakest. In nude mice inoculated with NS-1 tr

关 键 词：肝肿瘤 干细胞标志物 细小病毒 

分 类 号：R735.7[医药卫生—肿瘤]