C/ebp Beta Regulation Sublytic C5b - 9 Stimulation Induced Il - 6 And Gmc Tgf - Beta 1 Gene Start Experiment Research

Objective: To investigate the expression and phase of CCAAT enhancer bindingproteinβ(C/EBPβ), interleukin-6(IL-6) and transforming growth factor β1(TGF-β1) in ratglomerular mesangial cells (GMCs) induced by sublytic C5b-9stimulation. Methods: The ratGMC were cultured in vitro and stimulated with or without sublytic C5b-9. The level of C/EBPβ、IL-6and TGF-β1expression was measured by Real-time PCR and Western blot in the culturedGMC following sublytic C5b-9attack at fixed time. Results: C/EBPβ and IL-6expression in theGMC at3h upon by sublytic C5b-9attack were found to be significantly up-regulated comparedto control groups, and TGF-β1exhibited remarkably up-regulation at24h after sublytic C5b-9attack. Conclusion: The level of C/EBPβ、 IL-6and TGF-β1expression in sublyticC5b-9-treated rat GMCs were markedly elevated. Moreover, C/EBPβ and IL-6had the sameexpression changes and more early than TGF-β1expression. Objective: To construct CCAAT enhancer binding protein β (C/EBPβ) and itsshRNA expression vectors, and to assess their function on rat glomerular mesangial cells (GMCs).Methods: The CDs area of C/EBPβ and six reverse repeated motifs targeting of C/EBPβ genewere synthesized and cloned into eukaryotic expression plasmid pIRES2-EGFP andpGCsi.U6.neo.GFP. After screened and confirmed, the recombinant plasmids were transfectedinto GMC, then the levels of C/EBPβ protein in rat GMCs were measured by Western blot toprove its expression and find out the optimal shRNA. Results: It was verified by partialnucleotide sequencing and restriction endonuclease digestion that the constructed eukaryoticvectors were correct. The results by Western blot showed that the constructed pIRES2-EGFP/ C/EBPβ plasmid could correctly express, and the optimal shRNA, which could effectively silencethe target gene, was C/EBPβ shRNA-1. Conclusion: The pIRES2-EGFP/C/EBPβ plasmid and itsshRNA were constructed successfully. These data provide experimental tools for studyingbiological functions of C/EBPβ gene in the future. Objective: To explore the regulatory effects on the transcription and expression ofIL-6and TGF-β1genes in the GMC triggered by sublytic C5b-9attack via C/EBPβoverexpression or knockdown. In addition, to demonstrate that C/EBPβ can bind to IL-6andTGF-β1promoters and regulate their transcription. Methods: Firstly, the cultured rat GMC weretransfected with pIRES2-EGFP/C/EBPβ and C/EBPβ shRNA-1respectively, and the mRNA andprotein levels of C/EBPβ, IL-6and TGF-β1in different groups were measured by Real-time PCRand Western blot. Moreover, the regulatory effects of IL-6and TGF-β1promoter activity byC/EBPβ were measured with dual luciferase reporter system. Results:(1) Upregulation of IL-6and TGF-β1by C/EBPβ overexpression and downregulation of IL-6and TGF-β1expression byC/EBPβ knockdown were demonstrated respectively.(2) C/EBPβ increased the activity of IL-6promoter directly using luciferase reporter assay, and its binding site located in-618bp~-126bp ofIL-6promoter. Conclusion: C/EBPβ can only bind to specific elememt on IL-6promoter in theGMC in response to sublytic C5b-9stimulation and facilitate its expression. And C/EBPβ can alsoupregulated the transcription activity of TGF-β1inderectly in the GMC upon sublytic C5b-9attack to promote its synthesis and secretion.