The Construction Of MERα-labeling Platform And Related Research

Estrogen is an important endogenous substance，the metabolic disorder ofestrogen synthesis in body will lead to brain disease, osteoporosis and breast cancer.Estrogen combines with estrogen receptor to activate the intracellular signalingpathways to regulate metabolic process. Studies have found that sustained activationof estrogen-receptor pathways will lead to the occurrence of cancers. Similarly,recent studies have found that estrogen in many tissues can mediate rapid inductionsof rapid cell responses within seconds or minutes, it may directly activate amembrane-bound signaling pathway, which is called the non-genomic estrogenmechanism of reactions. Therefore, it is very critical to study the non-genomicestrogen mechanism of research for further study of estrogen-mediated cell signalingpathways. However, how to effectively distinguish the ER in the cytoplasm and ERon the membrane becomes a difficult bottlenecks to study the mechanism ofnon-genomic pathways of mER. At the same time there are no such research aboutsignal termination mechanism of mER so far. Therefore new methods with aPPTase-catelyzed system are used to distinguish mER and pER, and this subject willcontribute to a further study on the communications among estrogen receptors and afound of new mechanism. On the other hand, the screening of the regulators ofestrogen chemical molecular based on the cell model of miRNA-interference is ofgreat significance to real-time study the dynamic distribution of mER with thestimulation of different chemical factors inside and outside the cell membrane.In the study of membrane labeling methods, there are so many flaws in thetraditional methods, for example, cells needs to be fixed according to immunofluorescence method, GFP-Integrated method can’t be used to mark the proteinsinside and outside the cell membrane dynamicly, as a result, PPTase method wereused to label mERα, experimental results in vitro indicated that the enzyme and the marking system could be used, experimental results in vivo suggested that the Cterminus of ERαis exposed on the cell surface and provides the basis for living cellslabeling, the labeling results of live cells further demonstrated that this labelingmethod is fitted to distinguise the receptor proteins on the membrane and in thecytoplasm, and a critical scientific problem has been solved.In the study of signaling pathways in which chemical molecules regulate thetranscription of miRNAs, we used miRNA-based cell model to screen the naturalproduct in a compound library, as a result, we got2kinds of effective compounds,the results suggested as the concentration of the compounds increases, the decreasingeffect on the transcription of miRNA-21is enhanced, and after a right concentration,the effect increased. These compounds will be very critical for the following studys.Finally, we did some research about the interaction between Endogenousproteins and some compounds whose structure is very special, for the first time, wefound2compounds that can interact with endogenous proteins in a static quenchingprocess, they change the conformations of proteins to play a role in pharmacologicalfunctions, this part provided an important supplement for the screening of regulationof cell membrane estrogen receptors.