Construction The Vectors Of CREB-1and The Effect To Endogenous TGF-β3Secretion In HSC

Objective:To construct and obtain effective respectively CREB-1interference and expression plasmid carrier, for the next step of the experiment providing the related materials of the intervention.Method:On the basis of complementary matching principle, through the bioinformatics software and CREB-1mRNA sequences, design3stem-loop shRNAs that can target to inhibit the expression of CREB-1named sh-CREB1-1, sh-CREB1-2and sh-CREB1-3, respectively. The pGenesil-1.1for the carrier, through genetic engineering technology synthesis of such a shRNA plasmid. For internal control GADPH synthesis of sh-GADPH and not at any of the gene blank plasmid sh-BLANK. CREB-1expression vector pRSV-CREB-1was presented by Michael E. Greenberg, conventional extracted from scraps of paper and amplification, parallel DNA sequencing to verify its preliminary sequence.6groups of plasmid above respectively with liposome method transfection to HSC-T6. After transfection12h,24h,36h,48h,60h,72h cells were observed EGFP under the fluorescence microscope to preliminarily estimate he effect and efficiency of transfection. After transfection48h,6groups of cells were extracted total proteins, then Western Blot test CREB-1and GAPDH proteins expression changes in all groups.Result:The base sequences of pRSV-CREB-1expression vector after sequencing were compared to the CREB-1DNA sequences from GenBank and the result indicate that both sequences which coding amino acid of CREB-1protein are accordant to93.4%, and the regional amino acid sequences which contain critical phosphorylated site (S133) of CREB-1protein are completely consistent. All groups of cells containing EGFP plasmid are able to observe green fluorescent after transfection24h to72h. After transfection48h fluorescence is the strongest, and the transfection efficiency is about15%～20%. After transfection48h, Western Blot results show, compared with the sh-BLANK group, the relative proteins expression of CREB-1in group of sh-CREB1-1, sh-CREB1-2, sh-CREB1-3and pRSV-CREB-1were0.61±0.17(P<0.05),0.55±0.07(P<0.01),0.94±0.27(P>0.05) and1.60±0.11(P<0.05). On the side, compared with sh-BLANK group, the relative protein expression of GADPH in group of sh-GAPDH significantly reduced to0.20±0.08(P<0.01).Conclusion:Successful construction and obtain CREB-1interference and expression plasmid carriers, the sh-CREB1and pRSV-CREB-1. The sh-CREB1-2has best inhibition effect and can be used as the first choice of carrier interference follow-up experiment. The result of Sh-GAPDH group further confirmed that this transfection system was effective and feasible. Backgound and Purpose:There is a feedback loop of promote expression between CREB-1and TGF-β3each other in rat intestinal epithelial cell. Our previous studies showed that exogenous TGF-β3can increase CREB-1protein expression and induce the phosphorylation of CREB-1proteins in HSC. Whether CREB-1is also promote the secretion of TGF-β3in HSC is not yet clear. This part of the experiment as for discuss the expression and phosphorylated station of CREB-1to influence secretion of endogenous TGF-p3in HSCMethod:Using the transfection system be tested in part before and the best CREB-1interference carrier sh-CREB1-2and expression plasmid pRSV-CREB1and blank plasmid be transfected to HSC. After transfection46h, changed the medium with no contain BSA medium and treated with or without exogenous TGF-β3protein. After2h be detected expressions of the CREB-1protein in HSC cytoplasm and the phosphorylation-CREB-1(p-CREB-1) protein in cell nucleus by Western Blot, and be detected the secretion of endogenous TGF-β3in extracellular fluid of HSC by ELISA. In addition, Forskolin the promoting phosphorylation reagent and H89the inhibit phosphorylation reagent were exposed in HSCs, respectively. After the processing, be detected the expression of the p-CREB-1in HSC nucleoprotein and the secretion of endogenous TGF-β3in extracellular fluid of HSC.Result:After plasmids transfection and treated with(+) or without(-) TGF-β3, compared with the sh-BLANK(-) group, the relative expression of CREB-1and p-CREB-1proteins in group of sh-CREB1-2(-) were0.63±0.11(P<0.05) and0.93±0.10(P>0.05) that both down-regulation, the secretion of TGF-β3in extracellular fluid (EF) of HSC was0.81±0.18(P>0.05); and in group of pRSV-CREB1(-) the relative expression of CREB-1and p-CREB-1were1.66±0.14(P<0.05) and1.97±0.12(P<0.05) that both up-regulation, and the secretion of TGF-β3in EF of HSC was1.13±0.07(P>0.05). Compared with the sh-BLANK(+) group, the relative expression of CREB-1and p-CREB-1proteins in sh-CREB1-2(+) were0.45±0.08(P<0.05) and0.32±0.08(P<0.05), the secretion of TGF-β3in EF of HSC was0.35±0.05(P<0.05); and in group of pRSV-CREB1(+) the relative expression of CREB-1and p-CREB-1were2.19±0.08(P<0.05) and2.75±0.14(P <0.05), and the secretion of TGF-β3in EF of HSC was to3.76±0.11(P<0.05). Compared with Control group, the relative protein expression of p-CREB-1significantly decreased to0.53±0.07(P<0.05) and the secretions of TGF-β3also reduced to0.44±0.05(P<0.05) in H89treated group; and the expression of p-CREB-1elevated to2.13±0.09(P<0.05) and secretions of TGF-β3increased to3.76±0.11(P<0.05) in FSK treated group.Conclusion:Up-regulated the protein expression of CREB-1and p-CREB-1can increase the secretion of endogenous TGF-β3in HSC;Conversely, Down-regulated the protein expression of CREB-1and p-CREB-1can reduce the secretion of endogenous TGF-β3in HSC. This suggest p-CREB-1is the key factor for mediating endogenous TGF-β3secretion in HSC. The result could provide new experimental basis to consummate the effects of p-CREB-1and TGF-β3in prevent the course of liver fibrosis.