CAJ | 학술논문

目的探讨联合应用靶向血管内皮生长因子(VEGF)小干扰RNA(siRNA)与双自杀基因yCDglyTK对人胃癌细胞的体外杀伤作用。方法以磷酸钙纳米颗粒为载体介导空白质粒pcDNA3.1（-）null(空白质粒组)、靶向VEGF的干扰质粒pGenesil-shVEGF(干扰质粒组)、双自杀基因质粒pcDNA3.1（-）CV-yCDglyTK(双自杀基因组)及联合基因质粒pcDNA3.1（-）shVEGF-yCDglyTK（联合基因组）转染胃癌SGC7901细胞，未转染的胃癌细胞为空白对照组，经G418筛选稳定转染的胃癌细胞株，采用RT-PCR和免疫印迹法验证目的基因表达。给予前体药物5-氟胞嘧啶(5-FC)后，通过四甲基偶氮唑盐(MTT)生长曲线、旁观者效应实验、Hoechst 33258染色及流式细胞术，观察各组细胞的生物学特性变化、凋亡细胞形态及凋亡率。采用SPSS 13.0统计学软件进行数据处理分析，组间多重比较采用LSD检验。结果成功建立4种转染不同质粒的胃癌细胞株，联合基因组及双自杀基因组均可检测到双自杀基因yCDglyTK的表达。MTT生长曲线显示5-FC作用24 h后，与空白对照组和空白质粒组相比，干扰质粒组、双自杀基因组及联合基因组吸光度值明显降低(P＜0.01)。当稳定转染联合基因的SGC7901细胞占60％、80％和100％时，细胞相对存活率分别为13.09％±2.40％、9.74％±2.83％及5.68％±1.03％。荧光显微镜下见双自杀基因组及联合基因组大量细胞呈现凋亡形态改变。流式细胞检测结果示干扰质粒组、双自杀基因组以及联合基因组的凋亡率分别为16.40％±4.68％、57.63％±4.96％及69.07％±4.69％，与空白对照组相比，差异有统计学意义(P＜0.01)。结论采用靶向VEGF siRNA与双自杀基因联合治疗可有效杀伤胃癌SGC7901细胞，诱导凋亡是其杀伤瘤细胞的重要机制之一。
목적 탐토연합응용파향혈관내피생장인자(VEGF)소간우RNA(siRNA)여쌍자살기인yCDglyTK대인위암세포적체외살상작용。방법 이린산개납미과립위재체개도공백질립pcDNA3.1（-）null(공백질립조)、파향VEGF적간우질립pGenesil-shVEGF(간우질립조)、쌍자살기인질립pcDNA3.1（-）CV-yCDglyTK(쌍자살기인조)급연합기인질립pcDNA3.1（-）shVEGF-yCDglyTK（연합기인조）전염위암SGC7901세포，미전염적위암세포위공백대조조，경G418사선은정전염적위암세포주，채용RT-PCR화면역인적법험증목적기인표체。급여전체약물5-불포밀정(5-FC)후，통과사갑기우담서염(MTT)생장곡선、방관자효응실험、Hoechst 33258염색급류식세포술，관찰각조세포적생물학특성변화、조망세포형태급조망솔。채용SPSS 13.0통계학연건진행수거처리분석，조간다중비교채용LSD검험。결과성공건립4충전염불동질립적위암세포주，연합기인조급쌍자살기인조균가검측도쌍자살기인yCDglyTK적표체。MTT생장곡선현시5-FC작용24 h후，여공백대조조화공백질립조상비，간우질립조、쌍자살기인조급연합기인조흡광도치명현강저(P＜0.01)。당은정전염연합기인적SGC7901세포점60％、80％화100％시，세포상대존활솔분별위13.09％±2.40％、9.74％±2.83％급5.68％±1.03％。형광현미경하견쌍자살기인조급연합기인조대량세포정현조망형태개변。류식세포검측결과시간우질립조、쌍자살기인조이급연합기인조적조망솔분별위16.40％±4.68％、57.63％±4.96％급69.07％±4.69％，여공백대조조상비，차이유통계학의의(P＜0.01)。결론채용파향VEGF siRNA여쌍자살기인연합치료가유효살상위암SGC7901세포，유도조망시기살상류세포적중요궤제지일。
Objective To investigate the cytotoxicity of vascular endothelial growth factor (VEGF) siRNA combined with fusion suicide gene yCDglyTK on human gastric cancer cell line in vitro.Methods The gastric cancer cell line SGC7901 was transfeeted with blank plasmid pcDNA3.1 (-) null [pcDNA3.1 (-) group], or VEGF-siRNA expression plasmid pGenesil-shVEGF (SGC7901/shVEGF group), or fusion suicide gene plasmid pcDNA3.1 (-)CV-yCDglyTK (SGC7901/CDTK group), or combined gene plasmid pcDNA3.1 (-)shVEGF-yCDglyTK (SGC7901//shVEGF-CDTK group) with calcium phosphate nanoparticles (CPNPs).Un-transfected gastric cells were set as control group.The stable transfected cells were selected by G418.The target gene expression was verified by RT-PCR and Western-blot.After given prodrug 5-fluorocytosine (5-FC), the biologic characters variation, apoptotic morphology and apoptotic rate of cells in each group were observed through cell growth curve by MTT assays, by-stander effect, Hoechst 33258 staining and flow cytometry.The data was analyzed with SPSS 13.0 software and multiple groups’ comparison was analyzed with LSD test.Results Four gastric cancer cells lines transfected with different plasmids were successfully established.The expression of gene yCDglyTK was detected both in SGC7901/CDTK cells and SGC7901/shVEGF-CDTK cells.By MTT assays, the cell growth curve indicated that the A570 value of SGC7901/shVEGF cells, SGC7901/CDTK cells and SGC7901/shVEGF-CDTK cells decreased significantly compared with that of SGC7901 and SGC7901/null cells after a 24-hour 5-FC treatment (P＜0.01).When the percentage of stable gene trasfected SGC7901 cells was 60％, 80％and 100％, the cell relative viability was 13.09％±2.40％, 9.74％±2.83％ and 5.68％±1.03％,respectively. A large number of cells in SGC7901/CDTK and SGC7901/shVEGF-CDTK group appeared typical apoptotic morphology under fluorescence microscope.The result of flow cytometry showed that the apoptosis rates in SGC7901/shVEG group、 SGC7901/CDTK group and SGC7901/shVEGF-CDTK group were 16.40％ ±4.68％, 57.63％ ± 4.96％ and 69.07％ ± 4.69％,respectively, and there were significant differences compared with control (P＜0.01).Conclusion VEGF siRNA combined with suicide gene can effectively kill gastric cancer cell line SGC7901.Apoptosis induction may be one of the important mechanisms of killing tumor cells.