【作者】 董秀山；

【导师】 赵浩亮；

【作者基本信息】 山西医科大学 ， 外科学， 2010， 博士

【摘要】 背景肝再生是临床肝部分切除术和活体肝移植术后重要的病理生理过程。在肝脏手术及其术后的治疗中,采取适当的治疗手段对残肝功能的恢复、预防小肝综合症、减少术后并发症和提高肝脏的再生能力极其重要。然而,在肝脏手术及其术后的治疗过程中导致胆汁酸代谢紊乱的现象普遍存在,其中以胆汁外引流(如T管引流)和长期静脉营养最为常见。胆汁外引流可造成胆汁酸的丢失和胆汁酸池的减小；长期静脉营养导致胆汁分泌减少和胆汁酸肠肝循环的障碍,这些均造成肠道胆汁酸的缺乏和胆汁酸总循环量的减少。胆汁酸不仅在机体脂类和脂溶性物质的消化吸收以及胆固醇稳态发挥着重要作用,近年来研究发现胆汁酸还作为信号分子激活丝裂原活化蛋白激酶(mitogen-activated protein kinases. MAPKs)、法尼酯衍生物X受体(farnesoid X receptor, FXR)和G-蛋白耦联受体TGR5,分别在细胞增殖、胆汁酸代谢、糖脂代谢以及能量代谢等方面发挥着重要的调节作用,而肝脏再生必须依赖于胆汁酸、糖脂代谢以及能量代谢的正常进行。因此,在肠道胆汁酸缺乏的情况下探讨肝再生的病理生理过程,对正确认识胆汁外引流和长期静脉营养在肝脏外科中的应用价值具有重要的理论和临床意义。目的1.超负荷的胆汁酸能够导致肝细胞的凋亡和坏死,而低浓度的胆汁酸对肝细胞有何影响研究较少。本课题通过细胞学体外实验初步观察低浓度胆酸(CA)对大鼠原代肝细胞的细胞毒作用和对肝细胞DNA合成的影响,并探索CA对MAPKs途径的JNK和p38-MAPK蛋白表达的影响。2.通过建立肠道胆汁酸含量改变的大鼠肝再生动物模型,明确肠道缺少胆汁酸对肝再生的影响,并且观察该动物模型胆汁酸代谢的特点,尤其是血清胆汁酸的变化规律以及法尼酯衍生物X受体(FXR)和其靶基因胆汁酸合成限速酶胆固醇7α-羟化酶(CYP7A1)和肝细胞基膜侧胆盐摄取系统Na+依赖牛黄胆酸钠协同转运蛋白(NTCP)的变化。结合肝再生和胆汁酸代谢的变化特点,探讨胆汁酸代谢变化与肝再生的关系。3.探索肠道胆汁酸含量改变对大鼠肝再生早期即刻转录因子c-jun、c-fos以及细胞周期蛋白D1表达的影响,探讨肠道缺乏胆汁酸影响肝再生的机理。方法1.原代培养大鼠肝细胞48 h,更换含有0、10、20、40、80μmol/L CA的培养液继续培养12 h,MTT法检测肝细胞存活率；BrdU标记法计算肝细胞增殖指数；流式细胞术检测细胞周期,计算合成期(S期)肝细胞的相对百分比；免疫印迹技术检测JNK和p38-MAPK的蛋白表达。2.通过喂养大鼠0.2%胆酸(胆酸负荷组)、2%考来烯胺(胆酸缺乏组)建立改变肠道胆汁酸含量的动物模型,以喂养标准饲料作为对照组,所有大鼠均行70%肝部分切除术(partial hepatectomy, PH)。分别于PH后0、1、2、3、7 d五个时间点处死大鼠,收集残肝标本和血清进行检测。比较不同时间点三组大鼠肝再生情况：计算肝再生率、免疫组织化学检测增殖细胞核抗原PCNA、Ki-67的表达；自动生化仪检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBIL)、总胆汁酸(TBA)的血清浓度；实时荧光定量PCR检测FXR及其靶基因CYP7A1的mRNA表达；免疫印迹技术检测1 d时NTCP的蛋白表达。3.实时荧光定量PCR检测PH后0、0.5、1、3、6 h肝组织c-jun和c-fos mRNA表达；免疫印迹技术检测PH后6 h和12 h时细胞周期蛋白D1的蛋白表达。结果1. 10、20、40μmo1/L的CA对肝细胞的存活率无显著差异,80μmol/L的CA使肝细胞存活率降为66.7%,显著低于其他组(P<0.01)。随着CA浓度的增加,BrdU标记指数也随之增加,20、40μmol/L CA组肝细胞的增殖明显高于0μmol/L CA组(27.81±3.13,30.21±5.29 vs 18.35±4.88,P< 0.05,P< 0.01)。80μmol/L CA组BrdU标记指数则显著下降(23.53±6.01),与0μmol/L CA组相比无显著差异(P>0.05)。20、40μmol/L CA组S期细胞百分比显著高于0μmol/L组(40.49±7.23,47.81±9.14 vs 35.69±5.64, P<0.05, P<0.01)然而,80μmol/L CA组S期细胞百分比则下降为30.56±4.99,同0μmol/L CA组相比P<0.01。此外,在40μmol/L CA组,肝细胞DNA合成具有时间依赖性,以6～12 h上升较为显著(P<0.01)。20、40μmol/L CA组JNK蛋白相对表达量显著高于0μmol/L组(P<0.05,P<0.01),而80μmol/L CA可使JNK表达显著下降(P<0.01)。80μmol/L CA使p38-MAPK蛋白表达显著上升(P<0.01),而其他组p38-MAPK蛋白相对表达量同0μmol/L CA组无显著性差异。2.PH后1 d,三组血清ALT和AST水平无显著差异,而胆酸缺乏组的血清总胆汁酸和总胆红素显著低于其他两组(P<0.01,P<0.05)。胆酸缺乏组的肝再生率在PH后3、7 d显著低于胆酸负荷组和对照组(P<0.05)；胆酸缺乏组在PH后1 d时PCNA和Ki-67标记指数(22.21±2.31、17.25±6.50)显著低于胆酸负荷组(44.4±4.92、30.83±3.91)和对照组(38.74±6.42、27.04±7.22),且标记指数高峰延迟。PH后0 d时,肝脏CYP7A1 mRNA的表达依次为胆酸缺乏组>对照组>胆酸负荷组(P<0.05),而FXR mRNA的表达恰恰相反。在PH后,胆酸缺乏组FXR mRNA表达显著低于其他两组,而FXR的靶基因CYP7A1mRNA则随时间升高,明显高于其他两组。在PH后1 d,胆酸缺乏组的NTCP蛋白相对表达量最高,依次为对照组和胆酸负荷组(P<0.05)。3.在PH后1h, c-jun mRN(?)表达依次为胆酸负荷组>对照组>胆酸缺乏组(P<0.01),在3和6 h时胆酸负荷组c-jun mRNA仍保持较高水平,显著高于其他两组,而胆酸缺乏组和对照组则下降明显。就c-fos mRNA表达而言,仅在PH后3 h时三组间有显著性差异,以胆酸负荷组表达最高,其次为对照组,胆酸缺乏组表达最低。PH后6 h,细胞周期蛋白D1蛋白相对表达量依次为胆酸缺乏组<对照组<胆酸负荷组(P<0.05)；PH后12h,胆酸缺乏组和对照组无显著性差异,但显著低于胆酸负荷组(P<0.05)。结论1.浓度低于40μmol/L的胆酸对肝细胞的存活率和肝细胞p38-MAPK蛋白表达无显著影响,但使肝细胞S期细胞比例和肝细胞JNK蛋白表达量显著升高；80μmol/L的胆酸使肝细胞存活率、肝细胞S期细胞比例、JNK表达量则显著下降,而使p38-MAPK蛋白表达显著上升。因此,认为低浓度胆酸可促进肝细胞DNA的合成,可能与上调JNK蛋白表达有关。2.与标准饲料相比,0.2%胆酸可有效降低肝组织CYP7A1 mRNA的表达,而2%考来烯胺能够有效提高肝组织CYP7A1 mRNA的表达,并且对大鼠肝脏无明显损伤作用,间接反映出该剂量的胆酸和考来烯胺能够有效改变肠道胆汁酸的含量。3.肠道胆汁酸缺乏使大鼠肝再生显著延迟,并伴随血清总胆汁酸水平的下降、FXR mRNA表达的下降以及CYP7A1 mRNA表达的升高。其机制可能为机体缺乏胆汁酸使FXR的表达下降,从而使FXR失去了对CYP7A1的抑制作用,抑或为在肝再生过程中对机体缺乏胆汁酸的反应性调节,提示维持肠道正常的胆汁酸含量对FXR的表达以及保证正常的肝再生是必需的。4.早期即刻转录因子c-jun、c-fos和细胞周期蛋白D1被认为在细胞周期调节中发挥着重要作用,而肠道胆汁酸缺乏可显著降低肝部分切除术后c-jun、c-fos mRNA的表达,并且下调细胞周期蛋白D1的蛋白表达。更多还原

【Abstract】 BACKGROUNDRegeneration of the liver is an important pathophysiological process after partial hepatectomy and living donor liver transplantation. It is extremely important that appropriate surgical approaches and postoperative treatment for the recovery of residual liver function, preventing small-for-size syndrome (SFSS), reducing complications and improving the liver regenerative capacity. However, it has been widespread during the liver surgery and postoperative therapy, both of which can cause disorder in bile acid metabolism. The most common reason is external biliary drainage (such as T-tube drainage) that can lead to the loss of bile acids and the reduction of bile acid pool size, and long-term parenteral nutrition which can result in the reduction of bile secretion and the disorder of bile acid enterohepatic circulation. All of these are able to cause the lack of intestinal bile acids and the decrease of total circulation of bile acid. Besides the roles of bile acids in dietary lipid absorption and cholesterol homeostasis, it has become clear that bile acids are also signaling molecules. Three major signaling mechanisms have been identified. Bile acids activate mitogen-activated protein kinase (MAPK) pathways, are ligands for the G-protein-coupled receptor (GPCR) TGR5, and activate nuclear hormone receptors such as farnesoid X receptor a (FXR, NR1H4), all of which have been found to play an important role in the regulation in cell proliferation, bile acid metabolism, lipid metabolism and energy metabolism, and liver regeneration must rely on their normal metabolism of bile acids, glucose, lipid and energy. Therefore, under the pathophysiology of the lack of bile acids in the intestine, the study of pathological process of liver regeneration has important theoretical and clinical significance for correctly understanding of the clinical application value of the external drainage.OBJECTIVE1. The bile acids overload can lead to liver cell apoptosis and necrosis. However, studies of the effect of the low-concentration bile acids on liver cell proliferation are still few. Therefore, the first study was to investigate the effect of the low-concentration cholic acid (CA) on rat primary hepatocyte cytotoxicity and DNA synthesis, and to explore the effect of it on JNK, p38-MAPK protein expression in vitro cytologic assay. 2. To define the effect of the lack of intestinal bile acids on liver regeneration in the animal model of both changing intestinal bile acids level and liver regeneration, and to observe the characteristics of bile acid metabolism, in particular the changes of serum bile acids level as well as the farnesoid X receptor (FXR) and its target genes cholesterol 7a-hydroxylase (CYP7A1) mRNA expression and the Na+-taurocholate cotransport protein (NTCP) expression in these models. Combining the course of liver regeneration with the feature of bile acids metabolism, the relationship between bile acids metabolism and liver regeneration was explored.3. To investigate the effect of intestinal bile acid on immediate-early gene c-jun and c-fos, and cyclin D1 expression during liver regeneration in the early period in rats, the mechanism of both liver regeneration and intestinal bile acids was investigated.METHODS1. Rat hepatocytes were cultured in primary culture for 48 hour, then they were incubated in medium containing 0,10,20,40,80μmol/L CA for 12 hours. The cell survival rate was determined by MTT assay. Liver cell proliferation index was counted by BrdU-labeling method. The cell cycles were detected by flow cytometry, and the relative percentage of S phase was calculated. The expression of JNK and p38-MAPK protein was detected by western blotting.2. Interference with intestinal bile acid metabolism in rats was established through feeding rats 0.2% cholic acid (cholic acid loading group),2% cholestyramine resin (lack of bile acids group) and feeding the standard diet as the control group. All rats were performed 70% partial hepatectomy (PH). Liver regeneration was compared among three groups at 0,1,2,3,7 d after PH in rats:calculation of restoration of liver mass, immunohistochemical examination of PCNA and Ki-67, serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum total bilirubin (TBIL), and total bile acids (TBA) were analyzed with an automatic multichannel analyzer. The mRNA expression of FXR and its target genes CYP7A1 was examined by Quantitative RT-PCR at 0,1,2,3,7 d after PH. The expression of NTCP protein was detected by western blotting at 1 d after PH.3. The mRNA expression of c-jun and c-fos was examined by Quantitative RT-PCR at 0, 0.5,1,3,6 h after PH. The expression of cyclin D1 protein was detected by western blotting at 6 h,12 h after PH.RESULTS 1. No significant difference in cell viability was found in 10,20μmol/L and 40μmol/L group, however,80μmol/L CA can make the liver cell survival rate drop to 66.7%, significantly lower than other groups (P< 0.01). With the increase of CA concentration, BrdU labeling index also increased. Liver cell proliferation was significantly higher in 20μmol/L,40μmol/L CA group (27.81±3.13,30.21±5.29 vs 18.35±4.88, P< 0.05, P< 0.01). BrdU labeling index in 80μmol/L CA group was significantly decreased (23.53±6.01), there was no significant difference with 0μmol/L CA(P> 0.05) The relative percentage of S phase cell was significantly higher in 20μmol/L,40μmol/L group than 0μmol/L group (40.49±7.23,47.81±9.14 vs 35.69±5.64, P< 0.05, P< 0.01), but it was significantly decreased in 80μmol/L group (30.56±4.99, P< 0.01). In addition, liver cell DNA synthesis showed a time-dependent in 40μmol/L group, increased more significantly between 6～12 hour (P< 0.01). The relative expression of JNK protein was significantly higher in 20μmol/L and 40μmol/L group than in 0μmol/L group (P< 0.05, P< 0.01), whilst it was significantly decreased in 80μmol/L group(P< 0.01), in which, however, p38-MAPK protein expression increased significantly (P< 0.01); there was no difference in p38-MAPK protein expression among the others.2. On the day 1 after PH, there was no significant difference in the levels of serum ALT and AST among three groups, and serum TBA and TBIL in the lack of bile acids group was significantly lower than the other two groups (P< 0.01, P< 0.05). The rate of liver regeneration was significantly lower on the day 3,7 after PH in the lack of bile acids group than the other groups (P< 0.05). At day 1, the labeling indices of PCNA and Ki-67 in the lack of bile acids group(22.21±2.31%、17.25±6.50%)was lower than the cholic acid loading group (44.4±4.92%,30.83±3.91%) and control group (38.74±6.42%,27.04±7.22%) and the peaking of labeling indices was delayed. On the day 1, CYP7A1 mRNA expression was as follows:the cholic acid loading group< the normal group< the lack of bile acids group (P< 0.05), but FXR mRNA expression was just the opposite. After PH, the mRNA expression of FXR was significantly lower than the other groups, however, CYP7A1 mRNA had a trend towards increase after PH and was higher than the other groups. The protein expression of NTCP was highest in the lack of bile acids group, followed by the control group and the cholic acid loading group (P<0.05).3. At 1 hour after PH, the c-jun expression was significantly higher in the cholic acid loading group than the control group (P< 0.01), and it was significantly lower in the lack of bile acids group than that in control group (P< 0.01). At 3,6 hour, the c-jun mRNA remained at a high level in the cholic acid loading group, significantly higher than the other two groups, and it was decreased obviously in the others. In terms of c-fos expression, it was significantly different at only 3 hour after PH, and was as followed: the cholic acid loading group> the control group> in the lack of bile acids group. At 6 hour after PH, the protein expression of cyclin D1 was as follows:the cholic acid loading group> the normal group> the lack of bile acids group (P< 0.05). At 12 hour after PH, the protein expression of cyclin D1 was no significant difference, but significantly lower than the cholic acid loading group (P< 0.05)CONCLUSION1. There was no significant difference in survival rate below the concentration of 40μmol/L CA; however, the relative percentage of S phase and JNK protein expression were significantly increased by 40μmol/L CA.80μmol/L CA significantly decreased survival rate, relative percentage of S phase, and JNK expression but significantly increased p38-MAPK protein expression. Therefore, low-concentrations CA could promote liver cell DNA synthesis, which might be caused by an increase of the JNK protein expression.2. Compared with the standard feed,0.2% cholic acid feed could effectively reduce the expression of CYP7A1 mRNA, and 2% colestyramine feed could validly enhance the expression of CYP7A1 mRNA in liver tissue without substantial liver toxicity, which indirectly reflected that these doses of cholic acid and colestyramine were able to change the intestinal bile acids content effectively.3. The lack of intestinal bile results in delayed liver regeneration of normal rat liver after hepatectomy accompanied by decreased serum total bile acids levels, decreased expression of FXR mRNA and increased expression of CYP7A1 mRNA. The mechanism might be a fact that the lack of bile acids in the body made the expression of FXR reduced, and then made FXR to lost the inhibited effect on CYP7A1 expression, or reactive regulation in the absence of bile acids in the body during liver regeneration. We proposed that liver regeneration was required in order to generate an appropriate state of bile acid pool and the associated expression level of bile acid receptor FXR.4. Transcriptional activation of immediate-early genes c-fos,c-jun and cyclin D1 have been considered to play an important role in regulating cell cycle. The lack of intestinal bile acids could significantly decrease the expression of c-jun and c-fos mRNA after partial hepatectomy, and could reduce the expression of cyclin D1 protein.更多还原