ABSTRACT
To develop a GFP transgenic cell model under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted. Synthetic oligonucleotide ARE motif was annealed and purified then inserted into pTK-GFP to construct the vector of pARE-TK-GFP. The TK and ARE-TK fragments were amplified by PCR and cloned into pEGFP-N1 to reconstruct eukaryotic expression vectors of pTK-GFP/Neo and pARE-TK-GFP/Neo. They were transfected into HepG2 cells and clones resistant G418 were isolated. PDTC and Lentinan were used to induce the cell levels of GFP and the fluorescence was measured using a fluorescence plate reader. The results showed that the induced level of GFP is significantly increased and have dose-dependeny in a certain range. This findings indicated that such a cell model offered a potential platform for preliminary screening of all kinds of natural or synthetic chemopreventive agents.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Base Sequence , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Methods , Enhancer Elements, Genetic , Genetics , Gene Expression , Gentamicins , Pharmacology , Green Fluorescent Proteins , Genetics , Metabolism , Hep G2 Cells , Lentinan , Pharmacology , Microscopy, Fluorescence , Molecular Sequence Data , Oligonucleotides , Genetics , Proline , Pharmacology , Recombinant Fusion Proteins , Genetics , Metabolism , Thiocarbamates , Pharmacology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the content of phytoestrogen in dissimilarity herbs.</p><p><b>METHOD</b>The activity of phytoestrogen in heat-clearing drugs, drugs for relieving exterior syndrome, diuretic, anastaltics, tonics and astringents were detected based on the recombinant yeast cell (W303-1A/hER-ERE-Lac Z). The estrogenic activity in traditional Chinese materia medica were assayed quantitatively by determining the expression of beta-galactosidase.</p><p><b>RESULT</b>The phytoestrogen concentration (6.35 x 10(-3) nmol x g(-1) E2 equivalent) in heat-clearing drugs was the highest while that in anastaltic and tonic drugs was the lowest, which was less than the detected limit.</p><p><b>CONCLUSION</b>Compared with the other traditional Chinese materia medica, the content of phytoestrogen, which can bind to estrogen receptor, in giant knotweed rhizome, forsythia suspense, ash bark, baical skullcap root and ophiopogonis tuber were higher.</p>