中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2014年
11期
1517-1522
,共6页
李宁%吉晓滨%谢景华%刘启才
李寧%吉曉濱%謝景華%劉啟纔
리저%길효빈%사경화%류계재
黑色素瘤抗原A%真核载体%基因转染%绿色荧光蛋白%基因表达%肿瘤细胞模型
黑色素瘤抗原A%真覈載體%基因轉染%綠色熒光蛋白%基因錶達%腫瘤細胞模型
흑색소류항원A%진핵재체%기인전염%록색형광단백%기인표체%종류세포모형
Melanoma-associated antigen A3%Eukaryotic expression vector%Gene transfection%Green fluorescent protein%Expression%The tumor cells model
目的：建立pIRES2-EGFP/MAGE-A3真核质粒在小鼠黑色素瘤B16细胞稳定表达的肿瘤细胞模型。方法：将喉癌来源的黑色素瘤抗原A3（Melanoma-associated antigen A3，MAGE-A3）构建成真核质粒pIRES2-EGFP/MAGE-A3，脂质体法将pIRES2-EGFP/MAGE-A3转染小鼠黑色素瘤B16细胞，G418筛选阳性克隆，荧光显微镜检测阳性克隆中增强型绿色荧光蛋白（ Enhanced green fluorescent protein，EGFP）的表达，荧光定量PCR（ qRT-PCR）检查MAGE-A3在B16细胞中的转录。结果：pIRES2-EGFP/MAGE-A3真核质粒转染B16细胞后筛选得到阳性克隆，可见融合蛋白表达产生明亮的绿色荧光， qRT-PCR可检测到MAGE-A3 mRNA的转录。结论：pIRES2-EGFP/MAGE-A3真核质粒通过脂质体法能有效转染，并在B16稳定表达，成功建立了MAGE-A3肿瘤细胞模型。
目的：建立pIRES2-EGFP/MAGE-A3真覈質粒在小鼠黑色素瘤B16細胞穩定錶達的腫瘤細胞模型。方法：將喉癌來源的黑色素瘤抗原A3（Melanoma-associated antigen A3，MAGE-A3）構建成真覈質粒pIRES2-EGFP/MAGE-A3，脂質體法將pIRES2-EGFP/MAGE-A3轉染小鼠黑色素瘤B16細胞，G418篩選暘性剋隆，熒光顯微鏡檢測暘性剋隆中增彊型綠色熒光蛋白（ Enhanced green fluorescent protein，EGFP）的錶達，熒光定量PCR（ qRT-PCR）檢查MAGE-A3在B16細胞中的轉錄。結果：pIRES2-EGFP/MAGE-A3真覈質粒轉染B16細胞後篩選得到暘性剋隆，可見融閤蛋白錶達產生明亮的綠色熒光， qRT-PCR可檢測到MAGE-A3 mRNA的轉錄。結論：pIRES2-EGFP/MAGE-A3真覈質粒通過脂質體法能有效轉染，併在B16穩定錶達，成功建立瞭MAGE-A3腫瘤細胞模型。
목적：건립pIRES2-EGFP/MAGE-A3진핵질립재소서흑색소류B16세포은정표체적종류세포모형。방법：장후암래원적흑색소류항원A3（Melanoma-associated antigen A3，MAGE-A3）구건성진핵질립pIRES2-EGFP/MAGE-A3，지질체법장pIRES2-EGFP/MAGE-A3전염소서흑색소류B16세포，G418사선양성극륭，형광현미경검측양성극륭중증강형록색형광단백（ Enhanced green fluorescent protein，EGFP）적표체，형광정량PCR（ qRT-PCR）검사MAGE-A3재B16세포중적전록。결과：pIRES2-EGFP/MAGE-A3진핵질립전염B16세포후사선득도양성극륭，가견융합단백표체산생명량적록색형광， qRT-PCR가검측도MAGE-A3 mRNA적전록。결론：pIRES2-EGFP/MAGE-A3진핵질립통과지질체법능유효전염，병재B16은정표체，성공건립료MAGE-A3종류세포모형。
Objective:To construct tumor cell model by determination of the pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid expressing steadily in mouse melanoma B16 cells.Methods:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid being constructed from the melanoma-associated antigen A3 genes sourcing laryngocarcinoma in advance was translated into the mouse melanoma B16 cells under the mediation of lipofectamine,and the positive clones were detected with G418.The expression of enhanced green fluorescent protein( EGFP) and MAGE-A3 mRNA in positive clones were detected by fluorescence microscopy and fluorescence quantitative PCR ( qRT-PCR ) assay, respectively.Results:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected into B16 cells successfully, the green fluorescence of fusion protein expression was found, and MAGE-A3 mRNA transcription in B16 cells expressions were detected in positive clones.Conclusion:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected effectively and expressed stably by liposome method in the B16 cells.The expression of MAGE-A3 tumor cell model has been successfully established,which provide data for the study of laryngocarcinoma immunotherapy.
