CAJ | 학술논문

目的考察一个548位点C→T单核苷酸突变的雌激素受体(ER)在体外条件下,对乳腺癌细胞株(MCF-7)信号通路的影响,以探讨ER单核苷酸突变引起非雌激素依赖性性早熟的可能机制.方法用重叠延伸PCR定点诱变技术对548位点的碱基进行定点突变.构建定点突变表达载体pSG5-MuER.构建含雌激素受体反应元件(ERE)的萤光素酶报告基因载体pGL3-ERE-Luc.将野生和突变ER质粒分别和pGL3-ERE-Luc共转染MCF-7细胞,观察萤光素酶的变化,以检测突变ER反应活性的变化.结果成功构建ER突变质粒psG5-MuER和ER报告质粒pGL3-ERE-Luc,psG5-MuER较pSG5-ER能够增加萤光素酶的产生.结论该突变雌激素受体在体外具有高促转录活性特征,构建的载体可用于进行进一步的相关研究.
목적 고찰일개548위점C→T단핵감산돌변적자격소수체(ER)재체외조건하,대유선암세포주(MCF-7)신호통로적영향,이탐토ER단핵감산돌변인기비자격소의뢰성성조숙적가능궤제.방법 용중첩연신PCR정점유변기술대548위점적감기진행정점돌변.구건정점돌변표체재체pSG5-MuER.구건함자격소수체반응원건(ERE)적형광소매보고기인재체pGL3-ERE-Luc.장야생화돌변ER질립분별화pGL3-ERE-Luc공전염MCF-7세포,관찰형광소매적변화,이검측돌변ER반응활성적변화.결과 성공구건ER돌변질립psG5-MuER화ER보고질립pGL3-ERE-Luc,psG5-MuER교pSG5-ER능구증가형광소매적산생.결론 해돌변자격소수체재체외구유고촉전록활성특정,구건적재체가용우진행진일보적상관연구.
Objective To observe whether a 548 point C→T single nucleotide mutation of estrogen receptor (ER) affects breast cancer cells (MCF-7) signaling pathway in vitro, and to explore possible mechanism of this ER single nucleotide mutation inducing non-estrogen-dependent precocious puberty.Methods ER gene was inserted into wild-ER pSG5 plasmid as a template using site-directed mutagenesis overlap extension PCR technology to the base 548 points for site-directed mutagenesis. Expression vector of site-directed mutagenesis pSGS-MuER and the ER response element reports luciferase gene vector pGL3-ERE-Luc were constructed. Wild and mutant plasmids were cotransfected with pGL3-ERE-Luc into MCF-7 cells.Luciferase change was observed in order to detect mutation ER reactivity. Results The ER mutants and the ER response element containing the luciferase report gene vector were successfully constructed, pSG5-MuER increased luciferase production than pSG5-ER. Conclusions The mutations of estrogen receptor in vitro lead to high reactivity characteristics. The vector can be used for further reseach.