Establishment Of Stable PK15 Cell Lines Overexpressed Procine Selenoprotein S And Its Cytoprotecion Against OTA

Selenoprotein S(SelS)is known to localize to endoplasmic reticulum,which is widely distributed in biological organism,such as liver,kidneys,skeletal muscles,adipose tissue,hypothalamus and so on.Research shows that selenoprotein S has been linked to antioxidative protection in a cell-type-dependent manner and is suggested to be involved in various inflammatory diseases and endoplasmic reticulum homeostasis regulation.Ochratoxins are a group of mycotoxins produced by some Aspergillus species and some Penicillium species.Ochratoixn A(OTA)is the most prevalent and relevant fungal toxin of this group,which is known to occur in many crops and some commodities.OTA can be accumulated in biological organisms when we take the food contaminated by OTA.The present study was conducted to design the primer of porcine selenoprotein S with the restriction sites and construct the eukaryotic vector,then to transfect the recombinant plasmid into porcine kidney 15(PK15)cells with liposomes encapsuled and screen the stable transfection and store the stalbe cell lines.And then,a study about doing the research on groups with different treatments through the cellular models which have undertaken the oxidative damage by OTA,so as to the object of study on the biological function of selenoprotein S.Experiment 1:The construction and indentification of pDNA3.1-SelSIn the Genbank of NCBI website,it is necessary to search for the mRNA of porcine selenoprotein S.Accroding to the porcine selenoprotein S Genbank ID,primer could be designed with restriction sites.Then pcDNA3.1 would be chose as a eukaryotic vector,which could be used to link with the target gene SelS.And then through digestion and sequencing,we identified the validity of the reorganization of the product.The result showed that the eukaryotic vetors have been tested correct.Then name the vector pcDNA3.1-SelS and store the strains.Experiment 2:The stable transfection of reorganization pcDNA3.1-SelS and its detection of expressionIn the study,the pcDNA3.1-SelS was transfected into PK15 cell,and screened by G418.After the stable cell lines had been screened,the cell lines would be grouped as the PK15+pcADNA3.1-SelS group,the PK15+pcDNA3.1 group and the PK15group.Then both RT-PCR technique and western blot would be used to check the expression of SelS.The results of both RT-PCR and Western Blot showed that expression of SelS of PK15+pcADNA3.1-SelS group is almost twice as many as the control groups.Also the cell proliferation and the intracellular redox status of PK15 cells in groups would be measured.And the results showed that no difference is between the proliferation of different groups,and the PK15+pcDNA3.1-SelS was better than the control groups according to the detection of the MDA,SOD and GSH.Experiment 3:The antagonism of stable PK15 cell lines overexpressed porcine SelS caused by OTAIn the study,the cell lines in groups would be cutured for 48 hours with OTA.Then cell activity would be measured by MTT.The result showed that,when OTA reached 2ug/ml,we could observe an antagonist effect of the PK15+pcDNA3.1-SelS group,which had significant differences as compared with control groups(P<0.01).Also,the concentration of Malondialdehyde(MDA),Superoxide dismutase activity(SOD),Reduced glutathione(GSH)in groups cultured with OTA for 48 hours would be measured.The results showed that the MDA concentration of the PK15+pcDNA3.1-SelS group is less than the control groups,which had significant differences as compared with control groups(P<0.01).And both the SOD and GSH concentration of the PK15+pcDNA3.1-SelS group is more than the control groups,which had significant differences as compared with control groups(P<0.01).The study showed that the PK15 cell lines overexpressed porcine SelS could have its cytoprotection against OTA to some degree.