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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。
Objective To construct eukaryotic expression vector of ADAM10 and observe its effect on the proliferation and migration of stably transfected breast cancer cell line MCF-7. Methods The gene fragment of human ADAM10 was amplified by PCR and cloned into eukaryotic expression vector pcDNA3.1. The positive clones were identified by PCR, restriction enzyme digestion and sequencing. The recombinant plasmids were transfected into MCF-7 cells by Lipofectamine 2000, and the cells stably transfected with ADAM10 were screened by G418. The expression of ADAM10 was detected by Western blot. The proliferation and migration of cells were detected by MTT assay and Transwell assay. RESULTS: The eukaryotic expression vector of ADAM10 was successfully constructed by PCR, restriction enzyme digestion and sequencing. The results of Western blot showed that ADAM10 protein was highly expressed in stable transfected cells. MTT assay showed that the proliferation rate of transfected cells was slightly higher than that of control (P <0.05) Transwell results showed that transfected cells had stronger migration ability than control (P <0.01). Conclusion The ADAM10 eukaryotic expression vector was successfully constructed. After transfected MCF-7 cells with stable expression, the proliferation and migration of ADAM10 cells were enhanced, which laid the foundation for further study on the biological function of ADAM10.