Studies On The Effect And Mechanism Of CISH Gene On Inflammatory Factors Expression In Hepatocyte LO2

Objective The aims of the work are to regulate the expression of cytokine-inducible SH2-containing protein(CISH)gene in hepatocytes LO2 by gene intervention method and explore the potential mechanism of CISH regulation on the expression of inflammatory factors in hepatocytes LO2.Methods1.Fabricate the overexpressed plasmids of CISH recombinant and up-regulate the expression of CISH gene in hepatocytes LO2 The template of CISH plasmid was purchased from Shanghai Jikai Gene Technology Company.The special primer was designed to amplify target fragment of CISH.The PCR amplified product of target gene and vacuous plasmids GV230 were recombined using Xho I/Kpn I cleavage to fabricate overexpressed plasmids of the CISH recombinant,which was used to transfect hepatocytes LO2 through lipo2000.Then the change of m RNA and protein level of CISH in hepatocytes LO2 was examined by RT-PCR and Western Blot assay respectively.2.Synthesize CISH si RNA and down-regulate the expression of CISH in hepatocytes LO2 CISH si RNA(#1/#2/#3)was purchased from Guangzhou Ruibo Biotechnology,Hepatocytes LO2 was transfected by CISH si RNA(#1/#2/#3)through lipo2000,then,the change of m RNA and protein level of CISH in hepatocytes LO2 was examined by RT-PCR and Western blot assay respectively.3.Test the influence of the change of CISH expression to the inflammatory factors expression in hepatocytes LO2 After the hepatocyte LO2 was transfected by the overexpressed plasmids of the CISH recombinant and CISH si RNA 48 h,the content of inflammatory factors TNF-??IL-1? in hepatocyte LO2 bacterium spent culture supernatant was tested by ELISA.4.Test the change of signaling molecules STAT5 a in hepatocyte LO2 after CISH expression change After the hepatocyte LO2 was transfected by the overexpressed plasmids of the CISH recombinant and CISH si RNA 48 h,the change of signaling molecules STAT5 a in hepatocyte LO2 was tested by Western Blot.Results1.Fabricate the overexpressed plasmids of CISH recombinant successfully The overexpressed plasmids of the CISH recombinant were identified by RT-PCR and sequencing.CISH gene segment amplification was in accordance whith the sequence published in the gene bank and the hepatocytes LO2 transfected could express.The above indicated that we fabricated the overexpressed plasmids of CISH recombinant successfully.2.Fabricate the hepatocyte models up-regulated and down-regulated by CISH gene successfully Compared with the negative control,RT-PCR and Western Blot assay showed that the experimental group 1 transfected by the overexpressed plasmids of CISH recombinant could up-regulate the expression of CISH gene and those transfected by CISH si RNA fragments could down-regulate the expression of CISH gene,which indicated that we fabricated the hepatocyte models up-regulated and down-regulated by CISH gene successfully.3.The changing of CISH expression could affect the levels of inflammatory cytokines TNF-? and IL-1? secreted from hepatocytes LO2 Compared with the negative control,the results of ELISA indicated that the levels of the inflammatory factors TNF-? and IL-1? secreted from hepatocytes LO2 in experimental group 1 transfected by the overexpressed plasmids of CISH recombinant showed a decreasing trend,while it showed an increasing trend in experimental group 2transfected by CISH si RNA fragments.4.The expression of signaling molecules STAT5 a in hepatocytes LO2 could change when CISH gene was up-regulated or down-regulated Compared with the negative control,Western Blot assay showed that the expression of signaling molecules STAT5 a in experimental group 1 transfected by the overexpressed plasmids of CISH recombinant decreased,however,it increased in experimental group 2 transfected by CISH si RNA fragments.Conclusion1.CISH gene can regulate the secretion of inflammatory factors TNF-? and IL-1? in hepatocytes LO2 negatively;2.CISH gene may affect the secretion of inflammatory cytokines in hepatocytes LO2 by inhibiting the expression of its downstream signaling molecule STAT5 a.