【摘要】 目的:构建人DJ-1真核表达载体pcDNA3.1(+)/DJ-1,并转染人口腔癌细胞系Tca8113,建立含有DJ-1真核表达载体的口腔癌细胞模型。方法:以人HEK293细胞为模板,应用反转录聚合酶链反应(RT-PCR)法扩增人DJ-1基因全长,与真核表达载体pcDNA3.1(+)重组,经酶切鉴定和测序分析后,通过脂质体介导法转染人口腔癌细胞系Tca8113,并设立空质粒对照组(转染pcDNA3.1空载体)和未转染组,最后采用RT-PCR法检测Tca8113细胞中DJ-1基因的表达情况。结果:RT-PCR法获得长度为500 bp左右的阳性产物,重组质粒后经酶切鉴定和序列分析证实真核表达载体pcDNA3.1(+)/DJ-1构建成功。转染Tca8113细胞后RT-PCR法证实与空质粒组和未转染组相比,转染真核表达载体pcDNA3.1(+)/DJ-1的细胞系中DJ-1基因表达明显增高。结论:成功构建人真核表达载体pcDNA3.1(+)/DJ-1,并建立含有该载体的口腔癌细胞模型,为进一步研究DJ-1基因在口腔癌中的功能及应用DJ-1进行基因治疗奠定基础。更多还原

【Abstract】 Aim:To construct human DJ-1 eukaryotic expression vector pcDNA3.1(+)/DJ-1 and transfect into oral cancer cell line Tca8113,in order to establishment an oral cancer cell model containing DJ-1 expression vector.Methods:RT-PCR method was carried out to clone DJ-1 gene using the mRNA from human HEK293 cell line.Afterwards,DJ-1 gene was recombinated with pcDNA3.1(+)vector and identificated by restriction enzyme digestion and DNA sequencing technology.This recombinated vector was transfected into oral cancer cell line Tca8113 mediated by lipofectamine 2000,with pcDA3.1(+)vector as a blank control.The expression of DJ-1 gene in these cell lines was detected by RT-PCR method.Results:By RT-PCR method,a positve product about 500bp was obtained.The recombinated vector of pcDNA3.1(+)and DJ-1 gene was identified by restriction enzyme digestion and DNA sequencing technology,confirming that eukaryotic expression vector pcDNA3.1(+)/DJ-1 was constructed successfully.After transfected into oral cancer cell line Tca8113,the expression level of DJ-1 was significantlyincreased in Tca8113 cell line containing pcDNA3.1(+)/DJ-1 vector compared to that in blank control.Conclusion:Eukaryotic expression vector pcDNA3.1(+)/DJ-1 was successfully constructed and an oral cancer cell Tca8113 containing this vector was established,providing a model to further clarify the role of DJ-1 gene in oral cancer and supplying a potential gene therapy for oral cancer.更多还原