CAJ | 학술논문

目的构建CD133慢病毒表达载体,建立稳定过表达CD133的人胃腺癌SGC7901细胞株.方法聚合酶链反应法扩增CD133基因全长序列,将序列插入pGMLV-PB1-1载体,构建pGMLV-PB1-1-CD133慢病毒表达载体,随后转入293T细胞中对慢病毒进行包装,用获得的慢病毒毒液感染人胃腺癌细胞株SGC7901.建立稳定过表达CD133的SGC7901细胞株.荧光显微镜下观察pGMLV-PB1-1-CD133慢病毒表达载体的转染效果,逆转录-聚合酶链反应(RT-PCR)和Western blot法检测了SGC7901、pGMLV-PB1-1-SGC7901和pGMLV-PB1-1-CD133-SGC79013种细胞中CD133 mRNA及蛋白的表达水平.结果经限制性内切酶鉴定及测序分析,我们成功构建了pGMLV-PB1-1-CD133慢病毒表达载体质粒.Western blot结果显示,pGMLV-PB1-1-CD133慢病毒表达载体质粒成功转染293T工具细胞,pGMLV-PB1-1-CD133慢病毒表达载体有较好的过表达效果.pGMLV-PB1-1-SGC7901和pGMLV-PB1-1-CD133-SGC7901细胞在荧光显微镜下均发出绿色荧光,并且转染效率高.RT-PCR和Western blot检测显示,与对照组比较,转染pGMLV-PB1-1-CD133慢病毒表达载体组的细胞,CD133的表达在mRNA和蛋白两个水平均显著提高(1.160 ±0.051、0.835±0,077),差异有统计学意义(P＜0.01).结论成功构建CD133慢病毒表达载体和SGC7901-CD133细胞株.
목적 구건CD133만병독표체재체,건립은정과표체CD133적인위선암SGC7901세포주.방법 취합매련반응법확증CD133기인전장서렬,장서렬삽입pGMLV-PB1-1재체,구건pGMLV-PB1-1-CD133만병독표체재체,수후전입293T세포중대만병독진행포장,용획득적만병독독액감염인위선암세포주SGC7901.건립은정과표체CD133적SGC7901세포주.형광현미경하관찰pGMLV-PB1-1-CD133만병독표체재체적전염효과,역전록-취합매련반응(RT-PCR)화Western blot법검측료SGC7901、pGMLV-PB1-1-SGC7901화pGMLV-PB1-1-CD133-SGC79013충세포중CD133 mRNA급단백적표체수평.결과 경한제성내절매감정급측서분석,아문성공구건료pGMLV-PB1-1-CD133만병독표체재체질립.Western blot결과현시,pGMLV-PB1-1-CD133만병독표체재체질립성공전염293T공구세포,pGMLV-PB1-1-CD133만병독표체재체유교호적과표체효과.pGMLV-PB1-1-SGC7901화pGMLV-PB1-1-CD133-SGC7901세포재형광현미경하균발출록색형광,병차전염효솔고.RT-PCR화Western blot검측현시,여대조조비교,전염pGMLV-PB1-1-CD133만병독표체재체조적세포,CD133적표체재mRNA화단백량개수평균현저제고(1.160 ±0.051、0.835±0,077),차이유통계학의의(P＜0.01).결론 성공구건CD133만병독표체재체화SGC7901-CD133세포주.
Objective To construct a CD133 lentiviral expression vector and to establish its stably transfected SGC7901 cell line.Methods Full-length CD133 gene amplified by reverse transcription-poly-merase chain reaction (RT-PCR) was inserted into a pGMLV-PB1-1 vector to construct pGMLV-PB1-1-CD133 and pGMLV-PB1-1 vectors,and then transfected into 293T cells to precede the lentivirus equipment package.Subsequently,we collected the lentivirus venom to infect the SGC7901 cells and establish a stably overexpressed cell line named SGC7901-CD133.Results A CD133 lentiviral expression vector (pGMLV-PBl-1-CD133) was successfully constructed by restrictive enzyme digestion and plasmid sequencing.RT-PCR and Western blotting revealed the increased mRNA and protein expression of CD133 gene in cells transfected with pGMLV-PB1-1-CD133 (1.160 ± 0.051,0.835 ± 0.077) as compared with the control groups.Therefore the SGC7901-CD133 cell line was successfully established through the experiment.Conclusion A lentiviral CD133 expression vector and its SGC7901 cell line were successfully constructed.