| Chapter Ⅰ The detection of differences protein expression trend in brain death liver of rabbitObjective Using the proteomics technology to screening of distinguishing patterns of protein expression in liver tissue after brain death, we look for brain death related to differences liver protein expression.Methods The immunohistochemistry, PCR and western blot were used to detect the changes in protein expression levels in brain death liver.Results The immunohistochemistry, PCR and western blot were used to detect Runxl protein expression levels to study the changes after brain death in the liver, and found that as the extension of the time of brain death, the expression in the liver decreased, and the lowest level is at8hour point.Conclusion Brain death induced by slow intermittent rabbit model of intracranial pressure, we use Two-dimensional gel electrophoresis and MALDI-TOF-MS mass spectrometry to screen protein Runxl, the protein is localized in the nucleus, it is the cell proliferation and differentiation related proteins with the extension of the time of brain death, the expression in the liver decreased. Chapter Ⅱ The experimental study in vitro on Mechanism of Runxl for the hypoxia ischemia of liver cellsObjective Further explore the role of Runxl protein on hypoxic-ischemic liver cells in vitro.Methods Using the full length of the plasmid vector Runxl cDNA pEGFP-N1-RUNX1to transfected rat liver that making the Runx1gene overexpression, we transfected with pEGFP-N1as a negative control group, the liver cells of rats transfected plasmids carry ischemia hypoxia treatment.We divided the objects into four groups:control group (group C), ischemia and hypoxia group (IS), transfected with the negative control+hypoxia-ischemia group (S+IS), Runx1+overexpression of hypoxia-ischemia group (OS+IS). Using the flow cytometry to detect the rate of apoptosis in rat liver of each group, we exam the TGF-β Bcl-2、Bim、TNF-α mRNA expression levels, and explore Runx1protein on liver cells in hypoxic-ischemic protection mechanism.Results We transfected pEGFP-N1-RUNX1to cells and48hours later rat liver protein Rux1levels peaked. PCR and Western blot of the Runx1expression levels in the IS group and S+IS group was significantly lower than the control group (P<0.05), the OS+control group was significantly increased compared to the IS group (P<0.01). Flow cytometry apoptosis rate found IS group and S+IS group was significantly higher than the control group, OS+IS group had the lower rate of apoptosis than IS and S+IS, compared with a significant difference (P and the other three groups<0.05).PCR detect liver cells of rats in each group of TGF-β, Bcl-2, Bim, TNF-α mRNA expression levels. In the IS group, S+IS group, OS+IS group, TGF-(3mRNA expression levels were all higher than the control group(P<0.05), no significant difference in the three groups (P>0.05).Compared with group C the IS group and S+IS group of Bim, TNF-α mRNA expression level were significantly higher (P<0.05), but no statistically significant difference between the two groups (P>0.05), while in the OS+IS group Bim and TNF-α mRNA expression levels were significantly lower (P<0.05).Bcl-2was all opposite in IS group、S+IS group and OS+IS group.Runxl expression level caused a significant change of Bcl-2/Bim and TNF-α mRNA expression levels, and therefore Bcl-2/Bim and TNF-α is object of downstream signaling molecules of Runxl protein.Conclusion In vitro experiments confirmed that in the hypoxia-ischemia liver cells, Runxl significantly reduced, the TGF-β, Bim, TNF-α pro-apoptotic factors also significantly higher; Using pEGFP-N1-RUNX1eukaryotic expression vector transfection methods can effectively make Runxl over-expressed at the transcriptional and translational levels. After overexpression of Runxl, TGF-β expression showed no significant change, while Bcl-2, Bim, TNF-α vincreased significantly;Apoptosis rate significantly decreased after Runxl overexpression, which Runx1induced Bcl-2expression decreased. In ischemic-hypoxia Cells Runxl decreasing help to elevate Bim, TNF-α and other pro-apoptotic factor to release. The experiment confirmed Runxl regulate cell apoptosis through Bcl-2/Bim by the mitochondrial apoptotic pathway, Runx1improve TNF-a express and induced apoptosis by extracellular. Chapter III In vivo study of mechanisms on Runxl protein in rat liver cells of brain deathObjective In vivo study further confirmed the effect of liver cells of brain death in rats through TGF-β-Runxl-Bcl-2/Bim and TGF-β-Runx1-TNF-α signaling pathways.Methods High-pressure injection of plasmid transfected rat tail vein by pEGFP-N1-RUNX1, and then implemented the death of brain surgery. We divided the experiment into sham group (Group C), the group of brain death (BD), transfected with the negative control group+brain death (S+BD), Runx1overexpression+brain death group (OS+BD).Detected the liver in2h,4h,6h,8h time points of ALT, AST levels and liver morphological changes in liver function damage in each group PCR to detect the2h,4h,8h time points of Runx1, TGF-β,Bcl-2, Bim, TNF-α mRNA expression levels, used to clear Runxl regulate apoptosis function of the liver.Results High-pressure injection of plasmid transfected rat tail vein by pEGFP-N1-RUNX1,the Runx1expression peak at48hours, then gradually declined. Compared with the control group, the ALT, AST levels of each group after the death of brain gradually increased at each time point in serum, BD group and S+BD group ALT, AST levels and the control group were significantly increased (P<0.05), but no statistically significant difference between the two groups (P>0.05); compared with the BD group and S+BD group, OS+BD group at each time point in serum ALT, AST levels are lower (P<0.05). Compared with BD group, the liver morphology manifest that OS+BD group damage were abatement at each time point in BD group. PCR detected the rat liver cells in the sham group (Group C), brain death group (BD), transfected with the negative control+brain death group (S+BD), Runx1overexpression+brain death group (OS+BD) at2h,4h,8h time point of Runx1, TGF-β, Bcl-2, Bim, TNF-α mRNA expression level, GAPDH is as a reference.Compared with the sham group, brain death group (BD), transfected with the negative control+brain death group (S+BD) and Runxl overexpression+brain death group (OS+BD) TGF-β mRNA expression levels increased at each time point and no significant difference (P<0.05) among the three treatment groups. Compared with the sham group, in the group of brain death (BD) and transfected with the negative control group+brain death (S+BD) Bim, TNF-α mRNA expression levels increased and between the two groups was not statistically significant at each time point (P>0.05);While in the over-expression of Runxl+brain death group (OS+BD), Bcl-2mRNA expression levels were significantly increased (P<0.05), whill the Bim and TNF-α level were lower. Corresponding to the liver morphology, a large area of necrosis of liver cells apoptosis in the BD and S+BD group, indicating that Bim and TNF-α are activated accordingly after the Runxl reduced, resulting in massive necrosis of hepatocytes accompanied inflammatory cell infiltration.Conclusion In vivo study of Runx1experiments in the rat brain confirmed, Runx1expression decreased levels of brain death in the liver is the reason of liver apoptosis; Runx1regulate apoptosis through TGF-β/Smad-Runxl-Bcl-2/Bim and TGF-β/Smad-Runxl-TNF-α pathway. Bim and TNF-a is a downstream target genes of Runx1. |
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