ADAM10真核表达载体的构建及其对MCF-7增殖迁移的影响

目的构建ADAM10真核表达载体,观察稳定转染乳腺癌细胞MCF-7后对其增殖和迁移的影响。方法利用PCR技术扩增人ADAM10的基因片段,克隆入pcDNA3.1真核表达载体,阳性克隆通过PCR、酶切和测序鉴定。脂质体法将重组质粒转染MCF-7细胞,通过G418筛选出稳定转染ADAM10的细胞株,通过Western blot检测细胞ADAM10的表达,通过MTT法和Transwell法分别检测细胞的增殖和迁移变化。结果经PCR、酶切和测序鉴定证明ADAM10真核表达载体构建成功,Western blot结果显示稳定转染细胞的ADAM10蛋白高表达,MTT实验显示转染细胞增殖速度稍快于对照(P<0.05),Transwell结果显示转染细胞迁移能力比对照强(P<0.01)。结论成功构建ADAM10真核表达载体,稳定转染MCF-7细胞后可增强细胞的增殖和迁移,为进一步研究ADAM10生物学作用奠定基础。

Construction of a eukaryotic expression vector containing ADAM10 gene and its effects on MCF-7 cell proliferation and migration

To investigate the effects of ADAM10 on MCF-7 cell proliferation and migration,an eukaryotic expression vector containing ADAM10 gene was constructed in this study.Firstly,ADAM10 gene was amplified by PCR and inserted into the eukaryotic expression plasmid pcDNA3.1,and then the positive clones were identified by PCR,restriction enzymes digestion and DNA sequencing.Secondly,MCF-7 cells were transfected by recombinant plasmids,and steady cell clones was obtained by G418 screening.Western blot indicated that ADAM10 genes were highly expressed in transfected MCF-7 cells,while MTT showed that ADAM10 could promote the proliferation of transfected cells(P < 0.05),and Transwell method demonstrated that the migration was also promoted(P<0.01).This result of our study provides a basis for the study of biological effects of ADAM10.