摘要
目的构建真核表达质粒EX-Y2069-M29,检测其在人子宫内膜癌HEC-1-B细胞中的表达,并观察LRP16对细胞增殖的影响。方法从HEC-1-B细胞中提取总RNA,应用PCR技术,扩增获得LRP16基因编码序列片段,克隆入真核表达载体EX-Y2069-M29,对重组质粒进行酶切和测序鉴定后,以脂质体介导法转染至HEC-1-B细胞,采用Westernblot法检测LRP16蛋白的表达,用WST-1法检测细胞增殖情况。结果酶切和测序结果证明真核表达质粒EX-Y2069-M29的DNA序列完全正确,将其转染HEC-1-B细胞后,LRP16蛋白表达明显增加;转染后HEC-1-B细胞在体外能继续增殖,但增殖能力明显下降。结论 LRP16基因重组真核表达质粒EX-Y2069-M29构建成功,并能在HEC-1-B细胞中表达,为进一步研究LRP16基因奠定了基础;转染LRP16后并没有促进HEC-1-B细胞体外增殖,LRP16基因用于子宫内膜癌基因治疗可能具有潜在价值。
Objective To construct the eukaryotic expression plasmid EX-Y2069-M29,investigate the expression of LRP16 in human endometrial carcinoma HEC-1-B cells and its role in regulating proliferation of HEC-1-B cells.Methods The coding sequence of LRP16 was generated by PCR using total RNA extracted from the HEC-1-B cells.LRP16 gene was cloned into the eukaryotic expression vector EX-Y2069-M29.After restriction enzyme analysis and sequence identification,the recombinant plasmid was transfected into HEC-1-B cells with liposome mediated method.The expression of LRP16 was detected by Western blot.WES-T was used to measure the proliferation of HEC-1-B cells when transfected with LRP16.Results The results of enzyme analysis and sequencing both identified DNA sequence of LRP16 gene eukaryotic expression plasmid EX-Y2069-M29 correctly.The expression of LRP16 increased obviously after transfection into HEC-1-B cells.HEC-1-B cells continued to proliferate in vitro after transfected with LRP16,but cell proliferation is decreased.Conclusion LRP16 gene eukaryotic expression plasmid EX-Y2069-M29 was constructed successfully and it can be sustainly expressed in HEC-1-B cells.This provides experimental basis for further study on the function of LRP16.
出处
《四川医学》
CAS
2013年第5期587-589,共3页
Sichuan Medical Journal
