The Effect And Mechanism Of ABCG2 Gene On Goat Sertoli Cells Under The Oxidative Stress

Sertoli cells provide a scaffold for the differentiation and maturation of spermatogenic cells,and provide the nutrients and energy needed for the process of spermatogenesis.Additional,Sertoli cells still can secrete a variety of proteins and growth factors.With natural immune immunity function,it is an important component of the blood testosterone barrier.Because of its unique biological characteristics,it is widely used in tissue engineering,cell therapy,and it has opened up a broad prospect for cell transplantation and the treatment of diabetes and Parkinson's disease.But when used for cell transplantation,it is necessary to support Sertoli cells in vitro.The process of cell culture in vitro is a static system,in which a variety of reactive oxygen species?ROS?can be produced,so that leads to cell death.Widely located in a variety of cytoplasmic membranes,ATP binding cassette transporter G2?ATP-binding cassette transporters G2,ABCG2?is able to transport and efflux of harmful substances in recent studies.In particular,it plays an important role in promoting and maintaining the proliferation of cells under oxidativestress.Nrf2 signaling pathway can regulate the transcription of ABCG2 gene,which is one of the key ways of cells against oxidative damage.Therefore,it is of practical significance to study the effect and mechanism of ABCG2 gene on the proliferation of Sertoli cells in vitro under oxidative stress.In this study,firstly,built themodel of oxidative stress in the goat Sertoli cell?GSC?,then study the expression and localization of ABCG2 gene in GSC under oxidative stress,to elucidate the effect of ABCG2 gene on the proliferation of GSC,last,investigate the effect of ABCG2 in Nrf2 signaling pathways on the proliferation of GSC under the condition of oxidative stress after adding the inhibitors of ABCG2 gene,to elucidate the effect and mechanism of ABCG2 gene on the proliferation of GSC in vitro under oxidative stress.Firstly,taken GSC as the research object,added different concentrations of H₂O₂respectively,4 h later,detected of each cell survival rate,the content of lipid peroxidation product,the activity of superoxide dismutase,performed Karyotype analysis on the cells and detected aneuploidy by flow cytometry.The results showed thatthe most appropriate condition of establishing the oxidative stress model for GSC is treatment with200??H₂O₂ for 4 h.Secondly,extracted RNA of cells in control group and oxidative stressgroup respectively,reverse transcribed into DNA as template,synthetized the ABCG2 gene primers according to the Genbank of ABCG2 gene sequences,amplificated,detected the expression and localization of ABCG2 gene by Real time PCR and immunohistochemistry,then carried on the significance analysis.The results showed that ABCG2 gene was expressed in both the control group and the oxidative stress group,and there was significant difference in expression.The expression levelof ABCG2 gene in oxidative stress group was significantly higher than that in control group?P<0.05?.Third,treated the cells of the oxidative stress group with ABCG2 inhibitors,then extracted RNA,reverse transcribed into DNA as template,synthetized the Nrf2?HO-1?NQO1 gene primers according to the Genbank of Nrf2?HO-1?NQO1 gene sequences,amplificated,detected the expression of Nrf2?HO-1?NQO1 gene by Real time PCR,then carried on the significance analysis.The results showed that the expression levels of Nrf2,HO-1 and NQO1 in the inhibitor group were higher than those in the control group and lower than those in the oxidative stress group.From what has been discussed above,in this study,constructed the oxidative stress model of GSC successfully,and investigate the expression and localization of ABCG2gene of Sertoli cells under the oxidative stress.After the addition of ABCG2 inhibitor,the expression of key genes in Nrf2 signaling pathway was changedaccordingly,which is speculated that ABCG2 gene may be located in the downstream of HO-1,and NQO1 gene in Nrf2 signaling pathway.This study provides a theoretical basis for maintaining proliferation of GSC in vitro,lays the foundation for the further study of Nrf2 signaling pathway,and it also opens up a new way to find a suitable target for the production in the late stage to regulate the oxidative stress state of the cultured cells in vitro.