The Preliminary Relationship Of Secreted Protein ZBED3with Type2Diabetes

PART ONE Construction and identification of the eukaryotic ex-pression vector of human ZBED3geneObjective: TO construct and authenticate the eukaryotic expressionvector of human ZBED3gene.Methods: The cDNA fragment of human ZBED3containing tworestriction sites(ECORI,SalI) was obtained from human muscle tissues byusing reversal transcript-polymerase chain reaction(RT-PCR).And then,itcloned into the pIRES2-EGFP vector by ECORI/SalI double digestion andligation.The recombinant plasmid was authenticated by enzyme digest andDNA sequence analysis.Results: The recombinant plasmid pIRES2-EGFP-ZBED3digestedwith ECORI/SalI or without were by agarose gel electrophoresis andemerged two bands(687bp:ZBED3cDNA product,5.3kb: pIRES2-EGFPplasmid)or one band(5.98kb:recombinant plasmid pIRES2-EGFP-ZBED3)severally.This was a preliminary marker of successful construction.Thesequencing results were ananlysised by pairs using DNAssistsoftware,confirmed the cloned gene was human ZBED3cDNA. Conclusion: The eukaryotic expression plasmid pIRES2-EGFP-ZBED3has been successfully constructed.In order to prove the ZBED3protein is a kind of secreted proteins to lay the solid foundation. PART TWO Proving ZBED3is a kind of secreted proteinsObjective: ZBED3zinc finger protein is a kind of secreted proteins,tobe prepared for the elisa test.Methods: Developed HEK293T cells, collected HEK293T cells andextracted cell proteins when cells grew well. Used of low-temperaturefreeze-drying suction technique to remove moisture from the culture toachieve the purpose of moderate concentration cell cultures. Divided intonegative control group, blank vectoe group and transfection group toexperiment.ZBED3levels were measured by western blot in HEK293Tcells and the culture medium.Results: ZBED3levels were detected by western blot in HEK293Tcells and the culture medium in negative control group, blank vectoe groupand transfection group.In addition, ZBED3levels of transfection groupwere higher compared with blank plasmid group and negative controlgroup. Conclusion: ZBED3is a kind of secreted proteins. PART THREE The preliminary relationship of ZBED3with type2diabetesObjective:Discussing the preliminary relationship of ZBED3withtype2diabetes.Methods: ZBED3mRNA levels of several kinds of tissues ofC57BL/6J mice were measured by RT-qPCR; Developing liver tissues ofC57BL/6J mice and db/db mice,after using different concentrations ofglucose insulin and liraglutide to stimulate liver tissues, ZBED3levelswere measured by western blot; ZBED3levels were measured by RT-qPCRand western blot in in all the people include normal controls and T2DM.Results: ZBED3mRNA levels were detected by RT-qPCR in severalkinds of tissues of C57BL/6J mice, the relative expressions of ZBED3mRNA in C57BL/6J mice muscle spleen and kidney were the highest; Theliver ZBED3levels were higher in db/db mice compared with C57BL/6Jmice (P<0.05),the results of using insulin and liraglutide to stimulate livertissues were in accordance with the firegoing (P<0.01and P<0.05); Usedfour kinds of concentrations of glucose to intervent, db/db mice liver tissueZBED3protein expression increased most obviously(P<0.01)after used 10nmol/L glucose to stimulate and db/db mice liver tissue ZBED3proteinexpression increased minimalitily after used5nmol/L glucose to stimulate.The muscle and fat ZBED3levels were higher in type2diabetes groupcompared with NGT group (P<0.01).Conclusion: Insulin and liraglutide can stimulate the secretion ofZBED3, its expression showed the concentration dependence of insulin.ZBED3may be an important factor of the downstream of insulin signalpathway. ZBED3release reached the highest under a certain concentrationof glucose stimulation.Afterwards,with the increase of concentration,thesecretion of ZBED3did not increased no longer.ZBED3levels were higherin type2diabetes compared with normal controls revealed that it could be akind of protection and compensation reaction.