【作者】 陈晓光；

【导师】 徐存拴； 张富春；

【作者基本信息】 新疆大学 ， 动物学， 2011， 博士

【摘要】 哺乳类动物肝脏在物理的(如部分肝切除、缺血/再灌注)或化学的(如CCL4)刺激后,便显示出强大的再生能力。肝脏是一种由十多种细胞构成的复杂器官,肝脏结构及其功能的恢复要依赖各种肝脏细胞体积和数量的增加,以及不同肝脏细胞或同种细胞间的相互作用。作为组成肝脏的主要非实质细胞,肝窦内皮细胞(Liver sinusoidial endithlial cells, LSEC)在肝再生启动中起重要作用。但目前在分子水平上有关肝窦内皮细胞在肝再生中作用的研究很少。因此,本论文试图通过检测窦内皮细胞的基因动态表达谱以揭示它与肝脏再生的关系。首先,我们按Higgins等描述的方法建立大鼠70%部分肝切除(Partial hepatectomy, PH)动物模型,用两步灌流和胶原酶消化法分散肝脏细胞,用Percoll密度离心与抗CD31-免疫磁珠分选相结合的方法分离纯化PH后10个不同恢复时点的再生肝窦内皮细胞。利用窦内皮细胞标志蛋白CD14和ET-1进行免疫细胞化学检测窦内皮细胞的纯度,分析显示分离的窦内皮细胞纯度在95%以上。最后用含11789个已知基因和13231个未知基因,占大鼠基因组基因85%的Rat Genome 230 2.0芯片检测了PH后10个恢复时间点大鼠再生肝窦内皮细胞的基因转录谱。为验证芯片结果的可靠性,本研究用荧光定量RT-PCR检测了再生肝窦内皮细胞中GAPDH、UBC、JUN、TTR、MYC、PCNA、APOE、TRIM24、CD14和ET-1等10个基因的表达变化情况,对肝窦内皮细胞的芯片检测结果进行分析,发现共1629个基因与肝再生相关,包括833个已知基因及796个功能未知的未知基因。K-means方法分析显示,833个已知基因大致呈现8种表达模式:即(1)肝再生启动期快速上调,PH后6 h达到表达高峰,然后快速降低,72 h时再次达到高峰;(2)肝再生增殖期显著上调,然后逐渐减弱;(3)肝再生启动期快速上调,并迂回减弱,终止期再次上调;(4)PH后逐渐上调;(5)PH后快速下调并快速恢复;(6)PH后快速下调并逐渐恢复;(7)PH后整体下调,30 h达到最低;(8)肝再生早期稍微上调后逐渐下调,终止期达到最低。基因功能分析显示,这些基因参与细胞信号转导、凝血反应、血管形成、血管收缩、细胞代谢、细胞发生、生长、增殖、分化、免疫炎症、解毒、活性物质分泌等多种生物活动。用Fisher精确检验分析这些基因功能群在8种表达模式中的富集情况,并结合通过普函数计算的基因协同效应发现,PH后短时间内,参与窦内皮细胞重要生理功能(凝血、细胞吞噬和物质运输)的基因被激活,表明肝再生早期窦内皮细胞功能有恢复的趋势;解毒相关基因在再生早期表达增强,利于肝再生中废物的清除;活性物质分泌调控活动在PH后6 h开始增强,与下游细胞因子基因表达在时间上相偶联;免疫炎症、防御应答及免疫炎症信号途径相关基因虽在再生早期减弱,但协同效应结果显示它们肝再生几乎无显著变化,很显然基因的协同效应并非是单个基因作用的简单叠加;氨基酸代谢在肝再生早期增强,在终止期减弱;糖合成相关基因在再生早期表达上调,而糖代谢基因在再生后期表达减弱,根据协同效应结果发现,肝再生早期窦内皮细胞的糖合成活动增强,终止阶段无论糖合成还是糖酵解活动均减弱。另外,细胞增殖相关基因的表达在部分肝切除术30 h后恢复,这有利于肝再生中肝窦内皮细胞增殖。总之,肝再生中基因功能群富集和基因协同作用相结合的分析方法将助于揭示窦内皮细胞与大鼠肝再生的相关性。为探讨796个未知基因在肝再生中作用,本研究首先通过电子克隆预测它们编码的氨基酸序列,发现其中486个未知基因具有相应的氨基酸序列。根据本实验室建立的大鼠蛋白质亚细胞定位方法进行预测,发现大部分基因编码产物定位在细胞外基质、细胞质膜和细胞核中,表明它们可能参与组织结构重建、细胞膜形成和基因转录调控等活动。总之,上述预测结果为下一步实验研究其功能提供了有价值的参考。更多还原

【Abstract】 The mammalian adult liver exerts a strong regenerative capacity in response to mechanical stimulus (i.e., surgical hepatectomy, ischemia/reperfusion) or chemical stimulus (i.e., CCL4 administration). Because the liver is composed of multiple cell populations, rebuilding of the liver after 2/3 partial hepatectomy (PH) requires the synergistical actions of various liver cell types. It is well documented that liver sinusoidal endothelial cells (LSECs) are the largest number of liver non-parenchymal cells and the second largest number among all resident liver cells next to hepatocytes, and play a pivotal role in liver regeneration (LR), especially in the initiation of regeneration. However, details about the relevance of LSECs with liver regeneration remains yet to be delineated. It is likely that the measurement of dynamic expression profiles of LSECs would reveal the underlying mechanism of the action of this cell on liver regeneration (LR). In this study, the animal model of 70% hepatectomy in rats was established according to the method described by Higgins and Anderson; the methods of two-step perfusion combined with collagenase digestion were used to disperse liver cells; Percoll centrifugation and immunomagentic bead sorting methods were applied to isolate and purify LSECs from regenerating liver at 10 different recovery time points; CD14 and endothelin-1 (ET-1) immunostaining were employed for identifying the isolated LSECs. The immunochemical results showed that the proportion of ET1- and CD14-positive cells among the isolated LSECs were both at least 95%. The comprehensive analysis of gene expression profiles of LSECs during rat LR was carried out using Affymetrix rat genome 230 2.0 microarray, which is composed of 11789 known genes and 13231 EST sequences (totally 25020 genes), accounting for about 85% of rat genome. To evaluate the reliability of microarray results, this study used quantitative real-time PCR technology to detect the dynamic transcriptional profiles of several selected genes in LSECs from rat regenerating liver, including UBC, GAPDH, JUN, TTR, MYC, PCNA, APOE, TRIM24, CD14 and ET-1. Analysis on microarray data demonstrated that 833 known genes and 796 unknown genes with unknown structure (totaling 1629 genes) were identified as LR-related.Of them, 833 known genes were classified into eight clusters based on their expression patterns by means of K-means clustering method. The features of eight expression patterns are as follows: (1) rapid increase at priming phase and then quick return; (2) significant increase at proliferation phase; (3) quick increase at priming phase and then gradual decrease, again increase at terminal phase; (4) gradual enhancement and persistence; (5) transient downregulation and rapid recovery to baseline; (6) quick downregulation and slow recovery; (7) gradual downregulation and quick recovery with the lowest level at 30 h; (8) slight increase at priming phase and then downregulation. Gene function analysis indicated that these 833 known genes participate in many biological activities, such as signal transduction, blood coagulation, blood vessel morphogenesis, cell metabolism, extraculluar matrix and organelle organization and biogenesis, cell growth, proliferation, differentiation, immunity and inflammation, detoxification, secretion of active substances, and others. Frequencies of the functionally categorized genes in each expression cluster were represented with their statistical significance evaluated by Fisher’s exact test. Analyses of functional group enrichement combined with gene synergy showed that LSEC’s major functions, i.e. blood coagulation, phagocytosis and transportation, significantly enriched in the cluster characterized by rapid activation and gradual reduction, exhibited the quick recovery of LSEC function after partial hepatectomy. The catergories“immunity&inflammation”,“defense response”and“the related signaling pathways”were enriched in clusters exhibiting the features of transient gene down-regulation and quick recovery, but they were not drastically changed in response to partial hepatectomy during almost the whole LR based on gene synergy. In addition, the“glycogen synthesis”and“glycolysis”groups were significantly distributed in those clusters marked by“significant increase at proliferation phase and gradual decrease”and“up-regulation at early phase and down-regulation at late phase”, and as does“amino acid metabolism”functional group; while the“detoxification”functional group highly enriched in“up-regulation in early phase”became active at early phase, which is helpful in the elimination of waste substance released during LR. The recovery of expression levels of cell proliferation-involved genes after 30 h could favor the proliferation of LSECs during LR. Taken together, analyses of expression patterns of functionally classified genes and gene synergy gave insights into the potential mechanism of roles of LSECs in liver regeneration.To investigate the role of above 796 unknown genes in LR, we firstly obtained the putative full-length cDNA of unknown genes using in silico cloning method, then searched amino acid sequences encoded by cDNA sequences by open reading frame analysis based on ORF finder program. Following above procedure, we finally obtained 486 predicted protein products of unknown genes. Then we predicted the subcellular localization of 486 predicted amino acid sequence by API-SVM method using the dataset of rat protein subcellular localization made by our lab. The results showed that a majority of unknown genes were predictively localized in extracellular space, plasma membrane and nucleus, which inferred that the products encoded by these unknown genes possibly are dedicated to the composition of extracellular matrix, cell membrane formation and gene transcriptional regulation. Briefly, the above results may provide useful clues to confirm their biological functions by conducting the further experiments.更多还原