【作者】 赵杰；

【导师】 叶啟发；

【作者基本信息】 中南大学 ， 临床医学， 2014， 博士

【摘要】 第一章兔脑死亡肝脏差异蛋白表达趋势检测目的根据前期蛋白质组学技术筛选出的显著性差异蛋白Runx1,研究其在脑死亡肝脏中表达趋势变化。方法使用免疫组化、PCR、western blot在转录翻译水平验证Runx1蛋白在脑死亡肝脏中表达水平变化。结果免疫组化、RT-PCR、Western blot检测脑死亡后Runx1蛋白在肝脏中表达水平变化,发现随着脑死亡持续时间延长,Runx1蛋白呈下降趋势,在8小时时间点表达水平最低(P<0.05)。结论缓慢间断颅内加压法脑死亡后肝脏采用双向凝胶电泳和质谱分析,鉴定出Runx1蛋白表达水平在脑死亡前后肝脏细胞中有显著变化,该蛋白定位于细胞核内,是细胞增殖与分化相关蛋白质,随着脑死亡时间的延长,在肝脏中的表达逐渐减少,可能参与致肝脏损伤过程。第二章Runx1蛋白对缺血缺氧肝脏细胞作用的体外实验研究目的体外细胞实验探索Runx1蛋白对缺血缺氧肝脏细胞的作用。方法使用质粒为载体的全长度Runx1cDNA pEGFP-N1-RUNX1转染大鼠肝脏,使Runx1基因过表达；转染pEGFP-N1为阴性对照组,对质粒转染后的大鼠肝脏细胞进行缺血缺氧处理；实验分为四组：对照组(C组)、缺血缺氧组(IS)、转染阴性对照+缺血缺氧组(S+IS)、Runxl过表达+缺血缺氧组(OS+IS)。流式细胞仪检测各组大鼠肝脏的细胞凋亡率,检测TGF-β、Bcl-2、Bim、 TNF-α mRNA表达水平,研究Runxl蛋白对缺血缺氧细胞肝脏作用机制。结果pEGFP-N1-RUNX1转染大鼠肝脏细胞48小时后Rux1蛋白表达水平达到峰值。PCR和Western blot检测Runx1表达水平在IS组和S+IS组较对照组明显降低(P<0.05),在OS+IS组较对照组表达明显升高(P<0.01)。流式细胞仪检测各组细胞凋亡率,IS组和S+IS组较对照组细胞凋亡明显升高(P<0.01), OS+IS组细胞凋亡率较IS组和S+IS组明显降低(P<0.05)。PCR检测大鼠肝脏细胞各组TGF-β、Bcl-2、Bim、TNF-α mRNA表达水平。在IS组、S+IS组、OS+IS组中,TGF-13mRNA表达水平全部升高(P<0.05),三组之间没有明显差异(P>0.05)。在IS组和S+IS组中,Bcl-2mRNA与C组相比表达水平显著降低(P<0.05),但两组间没有统计学差异(P>0.05), Bim、TNF-α表达水平显著升高(P<0.05)；在OS+IS组中Bcl-2mRNA表达水平明显升高(P<0.05), Bim、TNF-α mRNA表达水平明显降低(P<0.05)。Runx1表达水平的改变引起了Bcl-2/Bim、TNF-α mRNA表达水平的显著改变,因此Bcl-2/Bim、TNF-α是受到Runx1蛋白调控的下游信号分子。结论体外细胞实验证实,肝脏细胞在缺血缺氧后,Runx1明显降低,TGF-β、Bim、TNF-α等细胞凋亡相关因子明显升高；采用pEGFP-N1-RUNX1真核载体转染方法可有效的使Runx1在转录和翻译水平表达增加。过表达Runx1后,TGF-β表达含量无明显变化,而Bcl-2、Bim、TNF-α等凋亡相关因子表达显著变化；过表达Runx1后细胞凋亡率显著降低,这与Runx1诱导凋亡抑制因子Bcl-2升高并使得促凋亡因子Bim、TNF-α表达降低有关。Runx1是通过调控Bcl-2/Bim家族表达,经线粒体凋亡途径诱导细胞凋亡；同时,Runx1促进TNF-α表达,通过细胞外凋亡途径引起细胞凋亡；第三章Runx1蛋白对脑死亡大鼠肝脏细胞作用机制的在体实验研究目的通过脑死亡大鼠在体实验研究进一步证实TGF-β-Runx1-Bcl-2/Bim和TGF-β-Runx1-TNF-α信号通路对脑死亡肝脏细胞作用机制。方法高压注射质粒转染大鼠尾静脉pEGFP-N1-RUNX1,并实行脑死亡手术。将实验分为假手术组(C组)、脑死亡组(BD)、转染阴性对照+脑死亡组(S+BD)、Runxl过表达+脑死亡组(OS+BD).检测各组肝脏在2h、4h、6h、8h时间点ALT、AST水平和肝脏形态学变化,以观察各组肝脏功能损伤；PCR检测各组2h、4h、8h时间点Runx1、TGF-β、Bcl-2、Bim、TNF-α mRNA表达水平,验证Runx1对肝脏作用机制。结果高压注射质粒转染大鼠尾静脉pEGFP-N1-RUNX1在48小时后Runx1表达达到峰值,后逐渐下降。检测各组ALT、AST水平,与对照组相比,BD组与S+BD组ALT、AST的水平明显升高(P<0.05),但两组问相差无统计学意义(P>0.05); OS+BD组在各个时间点血清中ALT、AST水平较BD组与S+BD组降低,差异均具有统计学意义(P<0.01)。肝脏形态学检测显示,与BD组相比,OS+BD组各时间点肝脏细胞损伤程度均减轻PCR检测大鼠肝脏细胞假手术组(C组)、脑死亡组(BD)、转染阴性对照+脑死亡组(S+BD)、Runx1过表达+脑死亡组(OS+BD)中2h、4h、8h时间点Runxl、TGF-β、Bcl-2、Bim、TNF-α mRNA表达水平,GAPDH为参照。在各个时间点,脑死亡组(BD)、转染阴性对照+脑死亡组(S+BD)、Runx1过表达+脑死亡组(OS+BD)TGF-β mRNA表达水平升高,三组处理方式间无统计学差异(P<0.05)。在各个时间点与假手术组相比,脑死亡组(BD)、转染阴性对照+脑死亡组(S+BD) Bcl-2mRNA表达水平降低,Bim、TNF-α表达水平升高(P<0.05)；而Runx1过表达+脑死亡组(OS+BD),Bcl-2mRNA表达水平明显升高,而Bim、TNF-α表达水平降低(P<0.05),相对应肝脏形态学,肝脏细胞坏死凋亡面积减轻,炎症细胞浸润减少。结论Runx1在体大鼠脑死亡实验证实,Runx1在脑死亡肝脏中表达含量的降低,引起肝脏细胞凋亡增加；Runx1对肝脏细胞的作用是通过TGF-β-Runx1-Bcl-2/Bim和TGF-β-Runx1-TNF-α信号通路来发挥对缺血缺氧肝脏细胞作用的。Bcl-2/Bim、TNF-α受到Runx1的表达调控,是Runxl的下游靶基因。更多还原

【Abstract】 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.更多还原