摘要
目的构建高表达外源性Dickkopf1(DKK1)基因的重组腺病毒载体(Ad-DKK1);感染黑色素瘤细胞株后研究Wnt信号通路中主要信号分子β-catenin的表达变化。方法利用PCR技术扩增目的基因DKK1;应用分子克隆技术和AdEasy系统重组腺病毒技术,把目的基因亚克隆到穿梭质粒;经酶切和测序后,在BJAdeasy中同源重组;用PacⅠ酶切过的重组腺病毒DNA在HEK293中包装并扩增;实验分为5组:Ad-DKK1感染组、Ad-SimDKK1感染组、Ad-GFP感染组、Ad-RFP感染组和空白细胞对照组。按照MOI值为80感染恶性黑色素瘤细胞株A375,观察细胞形态;RT-PCR法检测DKK1、β-catenin基因的表达,MTT法检测细胞活力。结果重组腺病毒Ad-DKK1经酶切鉴定与测序证实构建成功。感染A375细胞,感染率均大于80%,感染后细胞形态无明显改变。Ad-DKK1感染组DKK1的表达水平(1.638±0.067)与其他4组(0.718±0.086、1.424±0.125、1.414±0.089、1.423±0.088)比较差异具有统计学意义(P<0.05),其β-catenin的表达水平(0.590±0.011)与其他4组(0.895±0.012,0.749±0.024,0.754±0.012,0.759±0.014)比较差异具有显著统计学意义(P<0.01)。MTT法检测细胞活力:Ad-DKK1感染组(0.370±0.014)与其他4组(0.412±0.017、0.398±0.006、0.395±0.007、0.426±0.016)比较差异有统计学意义(P<0.05)。结论成功构建重组腺病毒Ad-DKK1,经其感染的恶性黑色素瘤细胞中DKK1的表达明显增强,β-catenin的表达显著降低,A375的细胞活力受到抑制。
Objective To construct a recombinant adenovirus vector expressing Dickkopf1 （DKK1） （Ad-DKK1） and study the expression of β-catenin in melanoma cells A375 after Ad-DKK1 infection. Methods DKK1 gene was amplified by PCR and subcloned to shuttle plasmid Adeasy by molecular cloning and adenovirus recombination technique,then converted in BJAdeasy cells. The recombinant adenovirus plasmid was generated. After cut by PacⅠ enzyme,the recombinant adenovirus plasmid Ad-DKK1 was packaged and amplified in HEK293 cells. Ad-DKK1 effect group,Ad-SimDKK1 effect group,Ad-GFP effect group,Ad-RFP effect group and blank group were used to infect the A375 cells in multiplicity of infection （MOI） 80,and cell morphology was observed. The expression of DKK1 and β-catenin in A375 cells was detected by RT-PCR. Cell vitality was detected by MTT assay. Results Ad-DKK1 was proved to be recombined successfully by identification with restriction enzyme and sequencing. The infection was efficient （80%） in A375 cells. The expression of DKK1 had significant difference between Ad-DKK1 effect group （1.638±0.067） and other 4 groups （0.718±0.086,1.424±0.125,1.414±0.089 and 1.423±0.088） （P0.05）. The expression of β-catenin had significant difference between Ad-DKK1 effect group （0.590±0.011） and the other 4 groups （0.895±0.012,0.749±0.024,0.754±0.012 and 0.759±0.014） （P0.01）. MTT assay indicated that there was significance in cell vitality between Ad-DKK1 effect group （0.370±0.014） and the other 4 groups （0.412±0.017,0.398±0.006,0.395±0.007 and 0.426±0.016）（P0.05）. Conclusion DKK1-expressing recombinant adenovirus vector is established. Over-expression of DKK1 suppresses the expression of DKK1 and inhibits the expression of β-catenin and cell vitality in melanoma cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2010年第12期1286-1289,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30901711)
重庆市自然科学基金(CSTC2009BB5402)~~
