CAJ | 학술논문

目的观察MAGE-A3基因过量表达对人肝癌细胞株增殖及侵袭能力的影响.方法采用逆转录-聚合酶链反应(RT-PCR)从肝癌组织中制备出含NheI和KpnI酶切位点的MAGE-A3目的基因,克隆到pcDNA3.1(-)真核表达载体,并通过酶切鉴定、测序验证.经脂质体法正义瞬时转染肝癌细胞株SMMC7721,通过RT-PCR、Western blot分别检测MAGE-A3 mRNA和蛋白表达产量.以噻唑蓝(MTT)比色法、Boy-den小室实验观察MAGE-A3基因过量表达对人肝癌细胞增殖及侵袭的影响.结果成功扩增出目的基因;得到重组质粒pcDNA3.1-MAGE-A3,经过酶切鉴定与测序,结果正确;RT-PCR和Western blot检测显示,MAGE-A3基因mRNA及蛋白产物的表达水平明显高于未转染组和空载体组(3组MAGE-A3 mRNA量分别为7838、2847、1557,蛋白量分别为30938、24 088、29 654);转染组细胞数量增多,穿透Matrigel膜的细胞数显著增加,与未转染组和空载体组比较差异有统计学意义(P<0.05).结论成功构建pcDNA3.1-MAGE-A3真核表达载体,转染人肝癌细胞株SMMC7721后可促进细胞增殖和侵袭能力,提示MAGE-A3基因在肝癌复发和转移中起到重要作用.
목적 관찰MAGE-A3기인과량표체대인간암세포주증식급침습능력적영향.방법 채용역전록-취합매련반응(RT-PCR)종간암조직중제비출함NheI화KpnI매절위점적MAGE-A3목적 기인,극륭도pcDNA3.1(-)진핵표체재체,병통과매절감정、측서험증.경지질체법정의순시전염간암세포주SMMC7721,통과RT-PCR、Western blot분별검측MAGE-A3 mRNA화단백표체산량.이새서람(MTT)비색법、Boy-den소실실험관찰MAGE-A3기인과량표체대인간암세포증식급침습적영향.결과성공확증출목적 기인;득도중조질립pcDNA3.1-MAGE-A3,경과매절감정여측서,결과정학;RT-PCR화Western blot검측현시,MAGE-A3기인mRNA급단백산물적표체수평명현고우미전염조화공재체조(3조MAGE-A3 mRNA량분별위7838、2847、1557,단백량분별위30938、24 088、29 654);전염조세포수량증다,천투Matrigel막적세포수현저증가,여미전염조화공재체조비교차이유통계학의의(P<0.05).결론 성공구건pcDNA3.1-MAGE-A3진핵표체재체,전염인간암세포주SMMC7721후가촉진세포증식화침습능력,제시MAGE-A3기인재간암복발화전이중기도중요작용.
Objective To observe the effects of over-expression of target gene MAGE-A3 by transient transfection on proliferation and metastasis of human hepatocellular carcinoma (HCC) cell lines.Methods MAGE-A3 target gene that containing restriction enzyme cutting site of NheI and KpnI was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from HCC tissues.The target gene was cloned into the expression vector pcDNA3.1 ( - ) to construct MAGE-A3 eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3,which was checked by restrictive enzyme digestion and DNA sequencing,and then transiently transfected into human HCC cell line SMMC7721 with Lipofectamine 2000.The mRNA and protein expression of MAGE-A3 was detected by semi-quantitive RT-PCR and Western blotting assay respectively.MTT assay and Boyden chamber assay were performed to detect the effects of MAGE-A3 on colony formation and metastasis.Results MAGE-A3 target gene was amplified and the eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3 was correctly constructed,and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western blotting revealed a strongly increased level of MAGE-A3 mRNA and protein expression in transfection group as compared with untransfected group and empty vector-transfected group (for MAGE-A3 mRNA expression level: 7838,2847 and 1557 in three groups,and for MAGE-A3 protein levels: 30 938,24 088 and 29 654,respectively).Both the number of HCC cells transfected by peDNA3.1-MAGE-A3 in MTT assay and the number of cells penetrating matrigel were increased as compared with untransfected group and empty vector-transfected group (P ＜0.05 ).Conclusion The eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-A3 was successfully constructed.The number of cells and invasiveness of the HCC cells transfected by pcDNA3.1-MAGE-A3 were significantly increased,suggesting MAGE-A3 gene may play a significant role in proliferation and metastasis of human HCC.