【作者】 安永；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 背景与目的体外循环(CPB)对机体是一种全身性的强刺激,能引起机体产生应激反应。应激引起的过度的炎症反应造成组织损害、多器官功能障碍综合征(MODS),是心脏外科患者术后死亡的主要原因之一。因此,CPB过程中器官保护一直是心血管外科临床与基础研究的重点。一直以来,人们对CPB所致的心、脑、肺、肾等器官的研究较多,也比较深入,而对肝脏功能的影响及其机制却缺乏深入系统研究。我们知道,肝脏是应激反应的中心器官和重要靶器官之一。临床研究表明,CPB术后不仅存在较高比例肝酶谱升高,而且存在着混合性的高胆红素血症(23.2-35.1%),肝功能障碍,甚至肝功能衰竭。恶性肝损害一旦发生,治疗棘手,往往导致MODS,预后差,病死率高。而慢性肝病或肝硬变患者CPB术后死亡率更高达25-31%,并发症发生率更高达58-66%。因此肝功能障碍是CPB术后的严重并发症。我国是个肝炎大国,很多心脏病病人本身就是肝炎或肝病患者,而心脏病本身也可以导致急性或慢性肝损害,此类患者心脏手术的时机选择以及适应症也是困绕心外科医生的问题,有的患者因此而丧失治疗的机会。因此研究CPB过程中肝细胞损伤的发生机制及探讨相应的保护措施,对增强手术耐受性,促进术后肝功能恢复具有重要意义。临床研究表明,CPB术后高胆红素血症或黄疸是由结合和非结合胆红素共同升高所致。这说明并非单纯由于溶血导致,同时也存在着肝细胞转运胆红素的功能障碍。而胆红素的转运则完全依赖于位于肝细胞膜面的肝胆膜转运蛋白(HMTs),也称胆红素转运子。HMTs基因表达异常有可能导致肝细胞胆汁淤积和肝细胞受损,而这类基因表达的信号调控机制尚未完全阐明。而CPB时存在胆红素代谢异常,其分子机制尚未明确,我们推测可能与HMTs基因表达的改变有关,此类研究尚未见文献报道。综合文献报道,在大多数HMTs启动子区域含有类干扰素活化序列,相应的转录因子与该序列结合后可启动HMTs的转录。信号转导转录活化因子5(STAT5)是胞浆信号蛋白,活化后可直接转移至胞核内,作为转录因子与靶基因启动子区域的类干扰素活化序列结合,引起靶基因的活化。因此我们推测STAT5途径可能是调控HMTs基因表达的途径之一。STAT5信号途径具有广泛的生物学效应,可促进合成代谢、刺激细胞再生、抗凋亡、调节急性期反应、稳定肠粘膜屏障等功能。已知生长激素(GH)—生长激素受体(GHR)轴是引起STAT5活化的最主要的路径。GHR主要位于肝细胞,当GH分泌不足和GHR表达下降时,STAT5的活性亦随之降低。因此给予外源性GH上调STAT5活性调控HMTs表达具有理论上的可行性。本研究可丰富对肝细胞胆盐转运信号调控的分子机制的认识。由于CPB是一个可以预期的应激反应,我们完全可以提前采取措施,在非应激条件下建立机体对围手术期损伤机制的耐受性,激发机体的自然防御机制减轻炎症反应和器官损伤,是很有前景的途径。但是通过GH预处理保护CPB后肝细胞功能的作用及机制研究未见报道。深入探讨这一途径的效果和可行性对于改善CPB后全身炎症反应,减少并发症具有重要临床意义。方法1.利用显微外科器械,采用微型化CPB环路设备,经右颈静脉腔房引流、左颈动脉灌注方法建立大鼠闭胸式CPB 2h模型,对CPB的大鼠进行分组,常规CPB组和GH干预CPB组(简称GH组,术前3天开始肌注GH 2.5mg/kg,1/日,预充液中加入2.5mg/kg),同时设立假手术组(SH组)和正常对照组(N组)。2.观察CPB后肝细胞损害与全身炎症反应的关系:检测指标包括一般肝功能检测;血清肝脏结构蛋白:前白蛋白(PA)、转铁蛋白(Tf);血清急性期反应蛋白:C反应蛋白(CRP)、血清淀粉样蛋白A(SAA);血清致炎因子:TNF-α、IL-1β、IL-6等的测定;血清GH、GH结合蛋白(GHBP)改变;肝脏改变的研究(肝细胞凋亡、再生情况及病理组织学改变等)。3.观察CPB后肝细胞胆盐转运功能的改变:利用免疫荧光组化、RT-PCR、Western blot等方法从细胞和分子水平测定肝细胞HMTs (NTCP,BSEP、FXR)的表达情况;4.观察CPB对肝细胞STAT5活性的影响及GH-GHR途径上调STAT5调控HMTs的机制:利用免疫荧光组化、原位杂交、RT-PCR、Western blot等方法从细胞和分子水平测定肝细胞GHR、STAT5的表达情况;观察STAT5活性改变对肝细胞HMTs的表达及功能的影响。5.统计学方法:所有数据采用均数±标准差表示。统计学处理采用SPSS11.0软件行t检验及ANOVA。结果1.通过微型化CPB环路设备,经右颈静脉腔房引流、左颈动脉灌注方法成功建立建立大鼠CPB模型,转流过程中血流动力学、血气分析等指标均在正常范围,术后可以长期生存。2. CPB后早期存在着明显的肝细胞损害,以CPB后3h最为明显;表现为肝功能受损,ALT、TB增高;血清肝脏结构蛋白浓度降低;血清急性期反应蛋白浓度增加,肝细胞凋亡增加,炎性细胞浸润。血清GHBP降低,血清致炎因子TNF-α、IL-1β、IL-6升高。直线回归分析表明肝损害与炎症反应及GHBP水平高度相关。3.大鼠CPB后存在肝细胞胆盐转运功能障碍,肝胆膜转运蛋白HMTs对CPB高度敏感,以CPB后3h最为明显;表现为CPB早期各种HMTs的mRNA及蛋白表达水平呈现不同程度的下降和不均一;核胆酸受体FXR mRNA及蛋白表达水平亦明显受抑。4. CPB术后早期存在着肝细胞GHR mRNA及蛋白表达水平不同程度的下降;STAT5 mRNA、蛋白表达水平及活性水平也下降;以CPB后3h最为明显;STAT5活性下降与GHR抵抗有关。而且,肝损害与STAT5活性下降及GHR抵抗均相关。5. STAT5上调途径对CPB后肝细胞的影响。GH预处理可上调STAT5表达活性,直接调控HMTs的表达,提高核胆酸受体FXR的活性,促进胆盐转运,降低高胆红素血症对肝细胞的损害;同时STAT5的上调可降低肝脏炎症反应,增加肝脏结构蛋白,降低肝脏急性期反应,抑制凋亡,促进再生,改善肝脏功能。结论1.我们成功建立了大鼠闭胸式CPB模型。利用显微外科器械,采用微型化CPB环路设备,经右颈静脉腔房引流、左颈动脉灌注方法建立大鼠闭胸式CPB模型比较符合生理,具有较好的稳定性和可靠性,是进行CPB术后肝脏病理生理改变研究的理想动物模型。2. CPB可导致明显的肝细胞损害。表现为(1)肝功能受损,ALT、TB增高;血清肝脏结构蛋白浓度降低;血清急性期反应蛋白浓度增加,肝细胞凋亡增加,炎性细胞浸润等。(2) CPB导致肝细胞胆盐转运存在障碍。表现为CPB早期各种HMTs的mRNA及蛋白表达水平呈现不同程度的下降和不均一;核胆酸受体FXR mRNA及蛋白表达水平明显受抑。肝细胞NTCP,BSEP等HMTs和核胆酸受体FXR的表达程度的下降和不均一是CPB术后肝细胞损害的重要原因之一。肝细胞内胆红素蓄积、过度炎性反应和急性期反应(APR)、FXR表达受抑和GH抵抗导致的STAT5活性下调共同介导了HMTs的表达的不均一;3. GH预处理有望成为预防和减轻CPB过程中肝细胞损害及SIRS的新策略。GH预处理上调STAT5途径对CPB后肝细胞具有明显的保护作用。其可能的机制为(1)直接调控HMTs的表达,促进胆盐转运,降低高胆红素血症对肝细胞的损害;(2)提高核胆酸受体FXR的活性,从而调控HMTs的表达;(3)降低肝脏炎症反应和APR,直接减轻对肝细胞的炎性损害,间接解除炎性反应对HMTs的抑制;(4)增加肝脏结构蛋白合成,抑制肝细胞凋亡,促进肝细胞再生。更多还原

【Abstract】 Background and objective:Cardiopulmonary bypass is a severe stress which can provoke a systemic inflammatory response syndrome (SIRS). Contact of the blood components with artificial surface of the bypass circuit, ischemia-reperfusion injury, operative trauma are mainly possible causes of SIRS. The excess inflammatory reaction may contribute to the development of postoperative complications and result in multiple organ dysfunction syndromes (MODS), which is one of the leading causes of death in cardiac surgery. Therefore, protection of organs during CPB is all along the emphasis in cardiovascular surgery. Over the past years, studies were mainly focused on the important organs such as heart, lung, brain and kidney. However, we know little about the liver. As we all know, liver is a central and important target organ responsing to the surgical stress and it is also one of the vulnerable organs for the attack of proinflammatory cytokines. It has long been recognized that early jaundice and liver damage could appear followed CPB surgery. Furthermore, critical hepatic lesion is still troublesome in clinical treatments and often can lead to MODS or mortality. For patients with preoperative chronic liver disease (CLD) or liver cirrhosis, mortality of CPB surgery can achieve 25-31%. So hepatosis is serious complication after CPB surgery. It is well known that liver disease is still a major health problem in China. As techniques of open-heart surgery and postoperative patient care improve, the number of patients with preoperative comorbidities who undergo CPB surgery is increasing. However, clinical outcome after CPB surgery in patients with CLD are still unsatisfactory. Due to the systemic and hepatic effects of CPB, the risk of further hepatic damage during CPB to an already compromised liver must also be a particular concern. But the research of CPB on the hepatocellular damage is absent and its accurate mechanism is still unknown.Clinical investigations showed that hyperbilirubinemia or jaundice followed CPB came about from an increase in both conjugated and unconjugated bilirubin. It indicates that both the impaired liver function for bilirubin transport and the increasing production of bilirubin from haemolysis lead to them. As we all know, hepatobiliary membrane transporters (HMTs) in liver cell are in main charge of bilirubin transporting. The expression anormaly of HMTs genes could reuslt in hepatocellular cholestasis and damage. However, the mechanisms of signal regulation of these genes expression are still unclear. Since CPB can provoke anormaly of bilirubin metabolism, the molecular mechanism is still unknown. We presume that it is concerned with the changes of HMTs expression.Combination of literature reports, promoter regions of major HMTs genes contain interferoid (IFN)-activated sequence. The transcription of HMTs can be driven by corresponding transcription factors binding to the sequence. Signal transducer and activator of transcription (STAT)-5 is the signal protein in endochylema. STAT5 activation is attributable to an increase in nuclear translocation of phosphorylated liver STAT5, which binds to IFN-activated sequence located in the target gene promoter region and thereby stimulates gene transcription. So we make a hypothesis that STAT5 pathway can regulate the expression of HMTs genes.STAT5 signal pathway have extensive biological effects, such as promote anabolic metabolism, stimulate cell regeneration, resist apoptosis, regulate acute phase reaction, stabilize mucosal barrier function, and etc. The axis of growth hormone (GH) and growth hormone receptor (GHR) is one of the main pathways to evoke the activation of STAT5. GHR locates mainly in liver cell. The activity of STAT5 will be depressed when GH secretes insufficiently or the expression GHR decreases. Therefore, it is feasible theoreticallythat up-regulation STAT5 by exogenous GH can regulate the expression of HMTs. This study will strengthen the recognition of molecular mechanisms of signal regulation of the hepatocellular bilirubin transporting.CPB is an expectable stress response. So building tolerance to perioperative damage during the unstressed condition may result in a more advanced method to assuage CPB-induced liver damage, and exploiting a natural defense mechanism called stress response may be a potential approach to overcome this problem. But the effect and mechanism of preconditioning with GH on the protection of liver function after CPB are still unknown. Consequently, this study based on the experimental model of CPB in rats is aimed to investigate the mechanisms of hepatocellular damage induced by CPB and the protective effect and mechanism of GH at cellular and molecular level, and to build a new strategy and provide a new target for the protection of liver function during and after CPB.Methods:1) Non-transthoracic CPB-2h model in rats was established via left carotid artery and right jugular vein cannulation for arterial perfusion and venous return respectively by using microsurgical instruments and miniature circuit devices. Adult male Sprague-Dawley rats, weighing 480±20g, were housed in wire-bottomed cages in a temperature-controlled room with a 12-hour light/dark cycle. Rats were acclimatized to the environment for 7 days. All animals received humane care. Rats were randomly divided into two groups according to the administration of GH before the initiation of CPB. Group G intramuscularly received 2.5mg/kg body weight of recombinant human GH (rhGH, Serono inc., Switzerland) at 8 AM every 24h for three days and just before the initiation of CPB. Group C (n=15) served as control and only saline was added in the same way. Sham-operated and normal control groups were assigned at the same time.2) To observe relationship between hepatocellular damage and SIRS, we detect indexes inferior: liver function; serum constitutive hepatic proteins and acute-phase proteins (APPs); serum cytokines such as tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, and IL-10 by using a rat-specific enzyme linked immunosorbent assay (ELISA); serum GH, GH binding protein (GHBP), insulin-like growth factor (IGF)-I, and IGF binding protein (GHBP)-3 (radioimmunoassay, RIA); and liver changes: liver protein concentration, liver cell proliferation (immunohistochemical staining for proliferative cell nuclear antigen (PCNA)), liver cell apoptosis (TUNEL immunohistochemical method), and pathological and morphological changes.3) To observe the changes of liver function for bilirubin transport induced by CPB, we determine the expression of hepatic HMTs (NTCP,BSEP, and FXR) at the level of cells and molecules (mRNA and protein) by using immunofluorescence, RT-PCR and Western blot analysis.4) To study the influence of CPB on hepatocyte STAT5 and the mechanisms of protective effects of up-regulation STAT5 activity (by GH-GHR axis pathway) regulating HMTs, We determine the expression of hepatic GHR and STAT5 at the level of cells and molecules (mRNA and protein) by using immunofluorescence, hybridization in-situ, RT-PCR and Western blot analysis; furthermore, We also observe the influence of the changes of liver STAT5 activity on hepatocyte function.5) Statistical analysis: Values of continuous variables were expressed as mean±standard deviation. Comparisons between groups were analyzed by 2-way repeated-measures analysis of variance (ANOVA) and the unpaired Student t test. Correlation between data was analyzed with linear regression. Statistical analysises was performed with computerized statistical packages (SPSS 11.0 software, SPSS, Chicago, IL, USA). Significance was accepted at p<0.05.Results:The main results in this study were as follows:1) Non-transthoracic CPB model in rats, via left carotid artery and right jugular vein cannulation for arterial perfusion and venous return respectively by using miniature circuit devices, was successfully established and was associated with good recovery. The results of hemodynamics and blood gas analysis were within normal range during bypass.2) There were significant liver injuries at CPB termination and markedly at 3h after CPB termination in both groups. Administration of rhGH markedly increased serum IGF-I and IGFBP-3 compared with group C. Group G showed significantly lower serum concentrations of alanine aminotransferase (ALT) and total bilirubin (TB) after CPB termination. Those receiving rhGH demonstrated a significant increase in serum prealbumin and transferrin and a marked decrease in serum amyloid A and C-reactive protein. rhGH significantly decreased serum tumor necrosis factor (TNF)-αand interleukin (IL)-1β, whereas no changes were found for serum IL-6 and IL-10. rhGH significantly increased total liver protein content, hepatocyte proliferation and decreased hepatocyte apoptosis verus group C. Liver injury was positive correlation with the proinflammatory cytokine levels and serum GHBP level.3) There was significant hepatocellular bilirubin tansport dysfuntion after CPB. Hepatic HMTs were very sensitive to CPB and markedly depressed at 3h after CPB termination. In earlier period of CPB termination, the expression of HMTs at the levels of mRNA and protein was low and nonuniform to some extent, the expression of farnesoid X receptor (FXR) was also depressed obviously at the levels of mRNA and protein.4) Hepatic STAT5 and GHR were lower at the levels of mRNA and protein at CPB termination and markedly depressed at 3h after CPB termination. Administration of rhGH markedly increased the activity of STAT5 detected by EMSA compared with group C. There was marked positive correlation between the expression of STAT5 and GHR, they were all correlated with hepatocyte dysfunction negatively, which indicated resistance of GHR might contribute to the inhibition of STAT5 after CPB and they both influence the hepatocyte function.5) The effect of up-regulation of STAT5 activity on hepatocyte functions after CPB. Pretreatment with GH can up-regulate the expression of STAT5, which can control the expression of hepatic HMTs and elevate the expression of FXR. These results contribute to hepatic transportation of bilirubin and relieve the hepatocelluar damage directly.Conclusions:1) The nontransthoracic CPB model in rats is established successfully. The nontransthoracic CPB model in rats is easy to establish and is associated with good recovery, which can in principle simulate the clinical setting of human CPB. This reproducible model is fit to study on the pathophysiological process of CPB in vivo.2) CPB results in obvious hepatocellular damage. Possible patterns of manifestations are shown inferior: (1) CPB induces acute liver injury in a rat model via increases in serum ALT,serum TB, serum acute phase proteins, proinflammatory cytokines TNF-α,IL-1β, and hepatocyte apoptosis, which is associated with decreases in serum concentrations of constitutive hepatic proteins; (2)There exists significant hepatocellular bilirubin tansport dysfuntion followed CPB. Depressive and nonuniform expression of HMTs and FXR is one of the key points for hepatocellular damage induced by CPB. Bilirubin accumulation in hepatocyte, excessive inflammatory respose and APR, and down-regulation of STAT5 induced by GH resistance contribute to the Depressive and nonuniform expression of HMTs.3) GH can prevent CPB-induced acute liver injury in a rat model. This strategy of pretreatment with GH might be a prospective management for preventing acute liver injury and lessening SIRS when CPB is performed. Up-regulation of STAT5 pathway has obviously protective effects on hepatocyte after CPB. Possible mechanisms are shown inferior: (1) directly control the expression of hepatic HMTs, which can promote hepatic transportation of bilirubin and relieve the hepatocelluar damage directly; (2) elevate the expression of FXR, which can regulate the expression of HMTs; (3) relieve APR and proinflammatory cytokines TNF-αand IL-1β, which can lessen the inflammatory hepatocellular injury directly and remove the inflammatory inhibition of HMTs expression indirectly; (4) increase the synthesis of hepatic constitutive hepatic proteins decrease, decrease hepatocyte apoptosis, and stimulate hepatocyte proliferation.更多还原