Establishment Of Drug Screening Models Based On Steroid Signal Transduction

Drug screening model is one of the most important conditions in drug discovery and testing the mechanism of drugs' effects.During the long period of drug research,a lot of drug screening models come forth,which played active roles in drug discovery. Based upon the latest developments in molecular biology, cellular biology and bioinformatics, etc., new technologies or experimental methods have come into being,that made drug screening directional,minimal,quick and convenient.If these technologies and methods have been applied to drug screening,it can significantly accelerate the pace of drug development.Along with the orientation of drug screening,we established drug screening cell models based on steroid signal pathway. Then we can use these models to analyse components of traditional Chinese medicines and try to find active components of these drugs.1. Construction of reporter vectors based on estrogen and androgen signal pathway.First,we synthesized the valid response elements of estrogen and androgen,then we inserted them into the multiple cloning region of pGL3-Promoter vector. Thus we constructed reporter vectors in which the reporter gene is luciferase.In addition,we tried to make GFP to be a reporter gene,so we subcloned this gene from EGFP-N1 plasmid,replaced Luc+ with GFP in pGL3-P vector.the vector with GFP reporter gene was constructed. Then we combined this vector with estrogen response element and androgen response element,the vector with GFP reporter gene based on steroid signal pathway was constructed.The four vectors we constructed were prove correct by enzyme and sequencing.2. Establishment and application of drug screening models based on estrogen and androgen signal pathway.Human breast cancer cell MCF-7 and human prostate PC-3 were transfected with constructed plasmids,then cells were treated with estrogen and androgen for two days.By measuring the quantities of luciferase and green fluorescence protein,we can validate whether the models are right or effective.According to our experiments,wecan sure the model based on estrogen signal pathway is effective.So,we choose this model to analyse a traditional Chinese medicine(CMZYC)which plays roles in women's disease,we found that in the three distilled components.one component distilled by water could make great change in signal pathway,while the other two components distilled by ethanol and ethyl acetate could not.Thereby we think that the active component of this Chinese medicine is in the component distilled by water.Our research afford a clue for more analysis of the active component. 3. The effects of Chinese medicine CMZYC on growth of BEL-7402 ^ SMMC-7721and LO2 cells.MTT,FACS and cell counter methods were used to determine whether the three components have effects on cell morphology,growth and cell cycle.We found that the component of CMZYC dissolved by ethyl acetate could significantly affect the growth of BEL-7402 and SMMC-772l(P<0.05) at the concentration of 10~2,at the same time, cell morphology of BEL-7402 had changed significantly,cells were longer and slimer,while a lot of SMMC-7721 cells detached.whereas normal liver cell LO2 had no obvious change in growth and morphology.According to the result of FACS, the number of BEL-7402 cells in S phase decreased, while LO2 had no change. Our results suggested that component of CMZYC dissolved by ethyl acetate could be anticancer in a way,but the detailed mechanism need to be further studied.