CAJ | 학술논문

目的构建pcDNA3.1(+)-MEK1真核表达载体,体外转染人肝癌MHCC97H细胞,观察MEK1在肝癌细胞中的表达及其对ERK通路活性的影响.方法应用RT-PCR扩增出MEK1基因插入pcDNA3.1(+)中,形成重组载体pcDNA3.1(+)-MEK1.经测序确认后,利用LipofectamineTM 2000将重组载体转染MHCC97H肝癌细胞,使用含G418的培养基进行筛选;将肝癌细胞分为不转染组、转染空载体组和转染重组载体组,运用Western-blot方法检测MEK1、p-MEK1、ERK1/2、p-ERK1/2在各组细胞中的表达并绘制各组细胞的生长曲线对比其增殖能力.结果重组载体pcDNA3.1(+)-MEK1酶切鉴定电泳条带大小正确,测序结果经Blast比对与人MEK1基因开放性读码框100％吻合;重组载体载体转染肝癌细胞后MEK1表达水平较不转染及转染空载体载体组细胞显著升高,且磷酸化的MEK1及ERK1/2水平也明显升高,转染重组载体载体的肝癌细胞增殖能力得到显著增强.结论成功构建了真核表达载体pcDNA3.1(+)-MEK1,过表达MEK1蛋白可提高肝癌细胞MAPK/ERK通路活性.
목적 구건pcDNA3.1(+)-MEK1진핵표체재체,체외전염인간암MHCC97H세포,관찰MEK1재간암세포중적표체급기대ERK통로활성적영향.방법 응용RT-PCR확증출MEK1기인삽입pcDNA3.1(+)중,형성중조재체pcDNA3.1(+)-MEK1.경측서학인후,이용LipofectamineTM 2000장중조재체전염MHCC97H간암세포,사용함G418적배양기진행사선;장간암세포분위불전염조、전염공재체조화전염중조재체조,운용Western-blot방법검측MEK1、p-MEK1、ERK1/2、p-ERK1/2재각조세포중적표체병회제각조세포적생장곡선대비기증식능력.결과 중조재체pcDNA3.1(+)-MEK1매절감정전영조대대소정학,측서결과경Blast비대여인MEK1기인개방성독마광100％문합;중조재체재체전염간암세포후MEK1표체수평교불전염급전염공재체재체조세포현저승고,차린산화적MEK1급ERK1/2수평야명현승고,전염중조재체재체적간암세포증식능력득도현저증강.결론 성공구건료진핵표체재체pcDNA3.1(+)-MEK1,과표체MEK1단백가제고간암세포MAPK/ERK통로활성.