This study investigates the estrogenic activity of phytoestrogens using engineered reporter cells. The methodology involves assessing luminescence as an indicator of estrogen receptor activation.
Estrogens are steroid hormones that act as endocrine signaling molecules and bind to nuclear receptors — estrogen receptors beta, forming estrogen-beta receptor complexes.
These ligand-receptor complexes dimerize and form homodimers in the cell cytoplasm. The resultant homodimer enters the nucleus, binds to the estrogen response element, ERE — a specific DNA sequence within the target gene's promoter — and initiates transcription.
To determine the estrogenic activity of phytoestrogens — plant-derived secondary metabolites that mimic estrogen function — begin with a multi-well plate containing reporter cells suspended in an appropriate medium.
The reporter cells are engineered to overexpress the estrogen receptors beta and transfected with a luciferase gene fused to ERE promoter. Add the sample solution containing phytoestrogens and incubate.
During incubation, the phytoestrogen binds to the estrogen receptors, beta — expressed in the reporter cells. Once bound, the phytoestrogen-receptor complexes dimerize and translocate into the nucleus.
Inside the nucleus, the dimerized complex binds to the ERE. The binding initiates luciferase gene transcription, producing the luciferase enzyme. Next, remove the medium and add a reagent containing a substrate for luciferase. Incubate to allow the expressed luciferase enzyme to oxidize its substrate and produce luminescence.
Measure the luminescence, which confirms the estrogenic activity of the compound in the sample.
Vortex the samples. Then, add 4 microliters of each sample to 496 microliters of compound-screening medium, to yield a 0.8% DMSO solution.
Disinfect the outside surface of a pre-warmed cell recovery medium tube with 70% ethanol, and transfer 10 milliliters of the medium into a tube of frozen reporter cells to thaw them. Close the tube of reporter cells, and place it in a 37-degree Celsius water bath for 5 to 10 minutes.
After retrieving the cells from the water bath, gently invert the tube several times to break up aggregates of cells, and produce a homogeneous suspension. Then, clean the surface of the tube with 70% ethanol.
Use a multi-channel pipette to dispense 100 microliters of the reporter cell suspension into each well of a 96-well plate. Then, dispense 100 microliters of samples in triplicate into the appropriate wells. Incubate the plate at 37 degrees Celsius and 5% carbon dioxide for 22 to 24 hours.
Just prior to the end of the plate incubation, remove detection substrate and detection buffer from the refrigerator, and place them in a low-light area until equilibrated to room temperature. Then, gently invert each tube to mix the solutions.
Immediately before the plate incubation is complete, pour the entire contents of the detection buffer into the tube of detection substrate, to create luciferase detection reagent. Mix the tube gently, so as not to produce foam.
Discard the contents of the sample plate into an appropriate waste container, and gently tap it on a clean absorbent paper towel to remove the last droplets from the wells. Add 100 microliters of the luciferase detection reagent to each well, and allow the assay plate to rest at room temperature for 15 minutes. Then, quantify the luminescence using a 96-well-plate-reading luminometer.
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