摘要
目的采用重组报道基因的方法开发一套可筛选环境雌激素和检测青春期前儿童体内雌激素水平的雌激素受体介导的转录激活测评系统。方法构建含有雌激素受体反应元件的报道载体pGL3-ERE-LUC和pGL3-overERE-LUC以及人类雌激素受体表达载体pSG5-ER。将两种报道载体分别转染入MCF-7细胞中,加入不同浓度雌激素受体配体,观察其对该系统的影响。将两种报道载体分别与雌激素受体表达载体共转染,加入不同浓度雌激素受体配体,观察其对该系统的影响。结果酶切结果证实pSG5-ER表达载体及报告载体构建成功。转染了pGL3-ERE-LUC或pGL3-overERE-LUC的MCF-7细胞,当加入一定浓度雌二醇(E2)及异黄酮(isoflavone)时,荧光素酶活性均增加;加雌激素受体拮抗剂他莫惜芬(TAM)后,荧光素酶活性下降。结论将pGL3-ERE-LUC或pGL3-overERE-LUC分别与pSG5-ER共转染入MCF-7细胞,可建立较敏感的环境雌激素测评系统。
Objective To construct an estrogen receptor mediated transcriptional actixation assay system to dectect environmental estrogen and the level of estrogen in prepubertal chihtren. Methods Reporter plasmids pGL3-ERE-LUC and pGL3-overERE-LUC that contained the estrogen receptor response element and human estrogen receptor expression plasmid were constructed. The reporter plasmids were transfected into MCF-7 cells then induced luciferase activity was observed when different concentration of estrogen receptor ligands were added. And the luciferase activity of cotransfection of the estrogen receptor expression plasmid pSG5-ER with the reporter plasmids was assayed. Results pSG5-ER vector and reporter vector were successfully constructed and identified by restriction enzyme cutting. When certain concentrations of E2 and isoflavone were cocuhured with MCF-7 cells transfected with above plasmids and vector, lueiferase activity increased, but when TAM was cocuhured, the luciferase activity decreased. Conclusion When pGL3-ERE-LUC or pGL3-overERE-LUC are stablely cotransfected with human estrogen receptor expression vector pSG5-ER into the MCF-7 cells, the sensitive estrogen and estrogen-like substances assay system can be constructed.
出处
《现代临床医学生物工程学杂志》
2006年第4期338-341,共4页
Journal of Modern Clinical Medical Bioengineering
关键词
雌激素
性早熟
雌激素受体
雌激素受体反应元件
Estrogen
Ecocious puberty
Estrogen receptor
Estrogen receptor response element
