细小病毒H-1非结构蛋白NS1基因对人胃癌细胞的抑制作用研究

Inhibitory Effect of Parvovirus H-1 Nonstructural Protein NS1 Gene on Human Gastric Cancer Cells

背景:前期研究表明细小病毒H-1(H-1 PV)对胃癌细胞生长具有一定抑制作用,非结构蛋白NS1可能是其细胞毒作用的效应蛋白。胃癌细胞CD44+群体中可能含有肿瘤干细胞。目的:探讨H-1 PV非结构蛋白NS1基因对不同分化程度的人胃癌细胞的影响。方法:以双酶切和质粒测序鉴定真核表达质粒pcDNA3.1-NS1,采用脂质体法将重组质粒转染高分化胃癌MKN28细胞、中分化胃癌SGC7901细胞和低分化胃癌MKN45细胞,并设置空质粒转染对照。G418筛选稳定转染的细胞克隆,以RT-PCR法检测NS1基因表达,裸鼠体内成瘤实验检测NS1基因对不同分化程度胃癌细胞的作用,流式细胞术分析CD44表达。结果:重组质粒pcDNA3.1-NS1转染的三种胃癌细胞均稳定表达NS1基因。以106/300μl浓度的肿瘤细胞皮下接种裸鼠3周后,MKN28细胞空质粒转染组和重组质粒pcDNA3.1-NS1转染组均观察到肿瘤生长;SGC7901、MKN45细胞空质粒转染组形成瘤体,而重组质粒pcDNA3.1-NS1转染组未见瘤体。转染重组质粒pcDNA3.1-NS1后,MKN28细胞不表达CD44,SGC7901和MKN45细胞CD44表达明显降低。结论:H-1 PV非结构蛋白NS1基因表达对中低分化的胃癌细胞有较好的抗肿瘤活性,可能与其降低胃癌干细胞表型CD44的表达有关。

Previous studies demonstrated that parvovirus H-1 （H-1 PV） has an inhibitory effect on the growth ofhuman gastric cancer cells, and nonstructural protein NSI may be the effector protein of its cytotoxic effect. The gastric cancer cell CD44^＋ population contains tumor stem cells. Aims： To investigate the effect of H-1 PV nonstructural protein NS1 gene on human gastric cancer cells with different differentiation. Methods： Eukaryotic expression plasmid pcDNA3.1-NS1 was identified by double enzyme digestion and sequence analysis, and then was transfected into well differentiated gastric cancer MKN28 cells, moderately differentiated gastric cancer SGC7901 cells and poorly differentiated gastric cancer MKN45 cells by LipofectamineTM 2000, and empty plasmid-transfected cells were served as blank controls. Clones of stable transfected cells were selected by G418. NS1 gene expression was determined by RT-PCR, the effect of NS1 gene on gastric cancer cells with different differentiation was evaluated by tumorigenicity in nude mice, CD44 expression was assessed by flow cytometry. Results： It was demonstrated that all three gastric cancer cell lines transfected with recombinant plasmid pcDNA3.1-NS1 could express NS1 stably. Three weeks after subcutaneous inoculation of 106/300 p.1 tumor cells, tumor growth was observed in both empty plasmid-transfected and recombinant plasmid pcDNA3.1-NSl-transfected MKN28 cells. Tumor growth was observed in empty plasmid-transfected 5GC7901 and MKN45 cells, while no tumor was seen in these two recombinant plasmid pcDNA3.1-NSl-transfected ceils. Recombinant plasmid pcDNA3.1-NSl-transfected MKN28 cells showed no expression of CD44, and CD44 expression in recombinant plasmid pcDNA3.1-NSl-transfected SGC7901 and MKN45 cells was obviously decreased. Conclusions： H-1 PV nonstructural protein NS1 gene expression shows well antitumor effect on moderately to poorly differentiated gastric cancer cells, which may be related to the decreased expression of cancer stem cell phenotype CD44.