摘要
一、研究背景
胆道并发症仍然是严重影响肝移植受者生活质量及长期生存率的并发症之一。随着外科技术的进步,吻合口狭窄等并发症呈逐渐下降的趋势,而非吻合口并发症,如缺血型胆道病变,则成为肝移植术后胆道并发症的主要临床病理表现。
肝脏移植术后缺血型胆道病变的发生机制尚不明确,目前,学者普遍认为与胆道缺血、免疫等因素有关。近年来,越来越多的研究表明胆汁中胆盐/磷脂比升高在肝移植后胆管损伤中起着重要作用。然而,肝移植后胆盐/磷脂比升高引起胆管及其上皮细胞损伤的具体机制尚无研究报道。研究表明,胆盐除了有“去污作用”外,还可在细胞内发挥信号分子的作用,参与细胞的增生和凋亡;而且由于其“去污作用”需要在高浓度下才能发挥,所以胆盐以信号分子的角色参与胆管细胞的病理生理改变可能更为普遍。在生理情况下,胆管细胞可经顶膜钠依赖性胆汁酸转运体(apical sodium-dependent bile acid transporter, ASBT)进行胆汁酸的重吸收,由细胞内胆汁酸结合蛋白(ileal bile acid binding protein, IBABP)转运至细胞基底侧,并经有机溶质转运体α/β(organic solute transporter alpha/beta, Ostα/β)等转出至门脉系统,从而启动了胆汁酸的“胆肝循环”。平衡的“胆肝循环”不会引起胆盐在细胞内潴留,这也是胆管细胞对胆盐毒性的一种自我保护机制。研究表明在多种炎症性及代谢性疾病中,胆汁酸转运蛋白表达发生改变,引起胆汁酸转运的紊乱,从而参与了疾病的发生发展。据以上观点和现象,我们推测,在移植肝冷保存再灌注损伤时,由于炎症因子的作用及核受体(如类法尼醇受体)表达/功能的改变,胆管细胞的胆汁酸转运平衡可能受到破坏,导致胆管细胞对胆盐毒性的自我保护功能紊乱,胆盐在胆管细胞内聚集,从而引起胆管细胞的损伤。
二、研究目的
基于以上假设,本研究以同系大鼠肝移植模型为研究对象,探讨移植肝胆管细胞胆盐转运蛋白在胆管损伤中的作用,并利用体外培养的胆管研究类法尼醇受体(Farnesoid X receptor, FXR)对胆管细胞胆汁酸转运蛋白表达的影响,探讨肝移植后胆管细胞胆盐转运蛋白参与胆管细胞损伤的机制。
三、研究方法及结果
1.利用免疫组织化学检测肝组织中ASBT、IBABP、Ostα及Ostβ的表达分布,结果显示,ASBT表达于大胆管细胞顶侧胞膜,IBABP表达于大胆管细胞浆,Ostα/Ostβ异二聚体表达于大、小胆管细胞基底侧胞膜及肝细胞血窦面胞膜。同时,我们用Real time PCR及Western blot方法检测分离的大小胆管组织中ASBT、IBABP及Ostα-Ostβ的表达,结果也显示,大胆管组织中ASBT和IBABP的表达显著高于小胆管,而Ostα-Ostβ在大胆管和小胆管组织中的表达量无显著差异。
2.我们从mRNA水平和蛋白水平检测了移植肝胆管细胞胆盐转运蛋白的表达变化,并通过组织形态学观察和细胞凋亡检测评估移植肝胆管损伤的严重程度,结果显示,大鼠肝移植术后早期(术后1-3天)胆管细胞胆盐转运蛋白的表达均降低,降低幅度与供肝冷保存时间相关。随后,位于细胞顶侧的ASBT与位于胞浆内的IBABP及基底侧Ostα-Ostβ呈现不同的恢复速度及变化趋势,即ASBT很快恢复到正常水平,且在术后第7天其表达水平显著高于正常,而IBABP和Ostα-Osβ表达长时间降低,直到术后第7天(短冷却血组)甚至到第14天(长冷缺血组)才恢复到正常水平。ASBT与IBABP、Ostα和Osβ表达水平的比值与大胆管损伤严重程度显著相关。
3.在检测移植肝胆汁中胆汁酸和磷脂浓度变化情况的基础上,我们通过免疫组织化学、荧光实时定量PCR、Western blotting及电泳移动漂移实验等技术,探讨不同冷保存时间下移植肝FXR表达及功能变化,结果显示,供肝冷保存再灌注损伤并不能引起胆汁中总胆汁酸的浓度的改变,只是通过降低磷脂的浓度,使胆汁酸/磷脂比增加,从而升高了胆汁中胆汁酸的相对浓度。大鼠肝移植后胆管组织中FXR的表达及功能活性显著降低,供肝冷保存时间越长,降低越明显。
4.通过分离培养SD大鼠肝内胆管,探讨FXR对与胆汁共培养的胆管细胞之胆盐转运蛋白表达的影响,结果显示,在正常胆管上皮细胞中,毒性胆汁可促进ASBT、IBABP、Ostα及Ostβ的表达均增加;而在转染了FXR shRNA的胆管细胞中,毒性胆汁可同样升高ASBT的表达,而IBABP、Ostα和Ostβ的表达并未增加,反而下降。
四、结论
1.胆管细胞的胆汁酸转运主要发生在大胆管细胞。
2.肝移植后胆管细胞胆汁酸转运蛋白表达出现不平行的变化,这种不平行改变程度与大胆管损伤严重程度显著相关。
3.肝移植后毒性胆汁促进ASBT表达快速恢复甚至高于正常水平,而由于FXR表达及功能受到抑制,阻断了胆汁酸对IBABP、Ostα和Ostβ的促进作用,使IBABP、Ostα和Ostβ表达恢复缓慢,从而引起了胆管细胞胆盐转运蛋白的不平行改变。
Background
Biliary complications remain the major source of morbidity for liver transplant recipients. Owing to the improvement of surgical techniques, the incidence of anastomotic strictures has continued to decrease, with the result that the non-anastomotic form is now the main type of biliary complication. Non-anastomotic strictures in the presence of a patent hepatic artery have been described as ischemic-type biliary lesions (ITBL), recently called ischemic cholangiopathy.
The molecular mechanisms involved in the pathogenesis of ITBL remain unclear. Recent year, increasing number of studies have indicated that an altered bile composition is associated with bile duct injury and with the subsequent development of non-anastomotic biliary strictures following human liver transplantation. However, the molecular mechanism by which the altered bile induces injury to the bile duct epithelial cells remains to be elucidated. It is well-known that bile salts solubilize membrane-bound lipids, leading to damage to cell membranes. However, at physiologically relevant concentrations, a simple detergent-like action involving disruption of the membrane is probably not relevant. The intracellular toxicity of bile salts, including death receptor-mediated and/or mitochondria-mediated apoptosis, may play a more important role in inducing the injury of bile duct epithelial cells following liver transplantation. Physiologically, the balance of bile acid influx and efflux in cholangiocytes is maintained by bile acid transporters to avoid the accumulation of toxic bile acids, which is a crucial step of the“cholehepatic shunt,”a process of bile acid absorption by cholangiocytes and subsequent excretion into the peribiliary capillary plexus, followed by resorption by hepatocytes and re-excretion in the form of bile. Several bile acid transporters have been identified in the cholangiocytes in humans and rodents. These transporters are widely expressed on enterocytes, and especially on the terminal ileocytes, where enterohepatic circulation is initiated. Studies show that these transporters will often undergo expressional and functional alterations and may be involved in intestinal metabolic and inflammatory diseases. Based on the above conception and evidence, we hypothesize that due to the expressional and functional alteration of inflammatory factors and nuclear receptor, the cholangiocyte bile acid transport may be disturbed and lost the self-protection from toxic bile, and subsequently inducing the accumulation of toxic bile and bile duct injury.
Objective
The current study focuses on the role of cholangiocyte bile acid transporters in bile duct injury after liver transplantation, and the possible mechanism.
Methods and results:
1. The immunohistochemistry assay on liver tissue section suggested that ASBT and IBABP were expressed exclusively on the large bile duct epithelial cells, while Ostαand Ostβwere expressed on both large and small bile duct. Quantitative PCR and Western blotting analysis showed that the expression levels of Asbt and Ilbp in large bile ducts were significantly higher than those in small bile ducts, while the expression levels of Ostαand Ostβin large bile ducts were similar with those in small bile ducts.
2. Using rat liver transplantation model, we investigated the expression levels of cholangiocyte bile acid transporters ASBT, IBABP and Ostα/Ostβ, and evaluated the evaluation of bile duct injury histopathologically. The immunohistochemical assay showed that the expressions of these transporters on large bile ducts were significantly reduced after transplantation compared with the Sham group. Three days after transplantation, ASBT recovered but IBABP, Ostαand Ostβwere still suppressed. Western blot and quantitative PCR analysis showed that the expression levels of these transporters dramatically decreased after transplantation. It took seven to fourteen days for IBABP, Ostαand Ostβto recover, while ASBT recovered within 3 days and even reached a peak above the normal level seven days post operation. In the CP-12h group, the ratios of the ASBT/ILBP, ASBT/Ostαand ASBT/Ostβexpression levels were correlated with the injury severity scores of large but not small bile ducts.
3. In the same animal model, we observed the changes of total bile acid (TBA) and phospholipids (PL) concentration in bile, and investigated the expression of FXR by quantitative PCR and Western blottingand, and analyzed its binding ability to promoter of Ostαand Osβgenes by EMSA. Result showed that cold preservation/reperfusion injury is associated with the elevated TBA/PL ratio which could induce the elevation of relative bile acid concentration in bile. It was also showed that the expression of FXR and its binding ability to promoter of target genes were inhibited after liver transplantation, and especially in the transplant group with long donor liver cold preservation time.
4. In the in vitro study with cultured rat bile duct units, we observed that the bile with higher TBA/PL ratio, so called toxic bile, could induce the expression of ASBT、IBABP、Ostαand Ostβin normal bile duct. However, after the transfection with FXR shRNA, the expression of ASBT was still induced, while the expression of IBABP、Ostαand Ostβwere inhibited to various extent.
Conclusions:
1. The bile acid re-absorption may exclusively take places in large bile duct, as the initiator of‘cholehepatic shunt’ASBT is just expressed in large bile ducts.
2. The unparallel alteration of cholangiocyte bile acid transporters is significantly correlated with large bile duct injury after liver transplantation with prolonged donor liver preservation.
3. The‘toxic bile’could promote the rapid recover and subsequent elevation of ASBT expression, while the IBABP、Ostαand Ostβexpression were not promoted accordingly because of the inhibition of FXR, by which bile acid could induce the expression of IBABP、Ostαand Ostβ.
引文
1. Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and consequences of ischemic-type biliary lesions after liver transplantation. Journal of hepato biliary pancreatic surgery 2006; 13 (6): 517.
2. Sharma S, Gurakar A, Jabbour N. Biliary strictures following liver transplantation: past, present and preventive strategies. Liver transplantation official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society 2008; 14 (6): 759.
3. Alpini G, McGill JM, Larusso NF. The pathobiology of biliary epithelia. Hepatology Baltimore, Md 2002; 35 (5): 1256.
4. Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. Journal of clinical gastroenterology 2005; 39 (4 Suppl 2): S90.
5. Alvaro D, Mancino MG. New insights on the molecular and cell biology of human cholangiopathies. Molecular aspects of medicine 2008; 29 (1-2): 50.
6. Xia X, Francis H, Glaser S, Alpini G, LeSage G. Bile acid interactions with cholangiocytes. World J Gastroenterol 2006; 12 (22): 3553.
7. Geuken E, Visser D, Kuipers F, et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. Journal of hepatology 2004; 41 (6): 1017.
8. Buis CI, Geuken E, Visser DS, et al. Altered bile composition after liver transplantation is associated with the development of nonanastomotic biliary strictures. Journal of hepatology 2009; 50 (1): 69.
9. Hoekstra H, Porte RJ, Tian Y, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology Baltimore, Md 2006; 43 (5): 1022.
10. Chen G, Wang S, Bie P, Li X, Dong J. Endogenous bile salts are associated with bile duct injury in the rat liver transplantation model. Transplantation 2009; 87 (3): 330.
11. Billington D, Evans CE, Godfrey PP, Coleman R. Effects of bile salts on the plasma membranes of isolated rat hepatocytes. Biochemical journal, The 1980; 188 (2): 321.
12. Palmeira CM, Rolo AP. Mitochondrially-mediated toxicity of bile acids. Toxicology2004; 203 (1-3): 1.
13. Faubion WA, Guicciardi ME, Miyoshi H, et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. Journal of clinical investigation, The 1999; 103 (1): 137.
14. Xia X, Francis H, Glaser S, Alpini G, LeSage G. Bile acid interactions with cholangiocytes. World journal of gastroenterology WJG 2006; 12 (22): 3553.
15. Cai SY, Boyer JL. FXR: a target for cholestatic syndromes? Expert opinion on therapeutic targets 2006; 10 (3): 409.
16. Kim MS, Shigenaga J, Moser A, Feingold K, Grunfeld C. Repression of farnesoid X receptor during the acute phase response. Journal of biological chemistry, The 2003; 278 (11): 8988.
17. Benedetti A, Bassotti C, Rapino K, Marucci L, Jezequel AM. A morphometric study of the epithelium lining the rat intrahepatic biliary tree. Journal of hepatology 1996; 24 (3): 335.
18. Liu JJ, Glickman JN, Masyuk AI, Larusso NF. Cholangiocyte bile salt transporters in cholesterol gallstone-susceptible and resistant inbred mouse strains. Journal of gastroenterology and hepatology 2008; 23 (10): 1596.
19. Buis CI, Verdonk RC, Van der Jagt EJ, et al. Nonanastomotic biliary strictures after liver transplantation, part 1: Radiological features and risk factors for early vs. late presentation. Liver transplantation official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society 2007; 13 (5): 708.
20. Kanno N, LeSage G, Glaser S, Alvaro D, Alpini G. Functional heterogeneity of the intrahepatic biliary epithelium. Hepatology Baltimore, Md 2000; 31 (3): 555.
21. Glaser S, Francis H, Demorrow S, et al. Heterogeneity of the intrahepatic biliary epithelium. World journal of gastroenterology WJG 2006; 12 (22): 3523.
22. Ballatori N, Li N, Fang F, Boyer JL, Christian WV, Hammond CL. OST alpha-OST beta: a key membrane transporter of bile acids and conjugated steroids. Front Biosci 2009; 14: 2829.
23. Alpini G, Glaser SS, Rodgers R, et al. Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes.Gastroenterology 1997; 113 (5): 1734.
24. Bogert PT, LaRusso NF. Cholangiocyte biology. Current opinion in gastroenterology 2007; 23 (3): 299.
25. Bergheim I, Harsch S, Mueller O, Schimmel S, Fritz P, Stange EF. Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers. Journal of lipid research 2006; 47 (1): 42.
26. Renner O, Harsch S, Strohmeyer A, Schimmel S, Stange EF. Reduced ileal expression of OSTalpha-OSTbeta in non-obese gallstone disease. Journal of lipid research 2008; 49 (9): 2045.
27. Halpern MD, Holubec H, Saunders TA, et al. Bile acids induce ileal damage during experimental necrotizing enterocolitis. Gastroenterology 2006; 130 (2): 359.
28. Tanaka Y, Chen C, Maher JM, Klaassen CD. Kupffer cell-mediated downregulation of hepatic transporter expression in rat hepatic ischemia-reperfusion. Transplantation 2006; 82 (2): 258.
29. Fouassier L, Beaussier M, Schiffer E, et al. Hypoxia-induced changes in the expression of rat hepatobiliary transporter genes. American journal of physiology Gastrointestinal and liver physiology 2007; 293 (1): G25.
30. Huls M, van den Heuvel JJ, Dijkman HB, Russel FG, Masereeuw R. ABC transporter expression profiling after ischemic reperfusion injury in mouse kidney. Kidney Int 2006; 69 (12): 2186.
31. Pellicciari R, Costantino G, Fiorucci S. Farnesoid X receptor: from structure to potential clinical applications. Journal of medicinal chemistry 2005; 48 (17): 5383.
32. Chignard N, Poupon R. Targeting farnesoid x receptor in hepatic and biliary inflammatory diseases. Gastroenterology 2009; 137 (2): 734.
33. Fujino T, Murakami K, Ozawa I, et al. Hypoxia downregulates farnesoid X receptor via a hypoxia-inducible factor-independent but p38 mitogen-activated protein kinase-dependent pathway. FEBS journal, The 2009; 276 (5): 1319.
34. Chen F, Ma L, Dawson PA, et al. Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter. J Biol Chem 2003; 278 (22): 19909.
35. Stravitz RT, Sanyal AJ, Pandak WM, Vlahcevic ZR, Beets JW, Dawson PA. Inductionof sodium-dependent bile acid transporter messenger RNA, protein, and activity in rat ileum by cholic acid. Gastroenterology 1997; 113 (5): 1599.
36. Alpini G, Ueno Y, Glaser SS, et al. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology 2001; 34 (5): 868.
37. Hwang ST, Urizar NL, Moore DD, Henning SJ. Bile acids regulate the ontogenic expression of ileal bile acid binding protein in the rat via the farnesoid X receptor. Gastroenterology 2002; 122 (5): 1483.
38. Frankenberg T, Rao A, Chen F, Haywood J, Shneider BL, Dawson PA. Regulation of the mouse organic solute transporter alpha-beta, Ostalpha-Ostbeta, by bile acids. Am J Physiol Gastrointest Liver Physiol 2006; 290 (5): G912.
39. Boyer JL, Trauner M, Mennone A, et al. Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents. Am J Physiol Gastrointest Liver Physiol 2006; 290 (6): G1124.
40. Khan AA, Chow EC, Porte RJ, Pang KS, Groothuis GM. Expression and regulation of the bile acid transporter, OSTalpha-OSTbeta in rat and human intestine and liver. Biopharm Drug Dispos 2009; 30 (5): 241.
41. Balesaria S, Pell RJ, Abbott LJ, et al. Exploring possible mechanisms for primary bile acid malabsorption: evidence for different regulation of ileal bile acid transporter transcripts in chronic diarrhoea. European journal of gastroenterology and hepatology 2008; 20 (5): 413.
42. Roberts SK, Kuntz SM, Gores GJ, LaRusso NF. Regulation of bicarbonate-dependent ductular bile secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts. Proceedings of the National Academy of Sciences of the United States of America 1993; 90 (19): 9080.
43. Campion JP, Porchet N, Aubert JP, L'Helgoualc'h A, Clement B. UW-preservation of cultured human gallbladder epithelial cells: phenotypic alterations and differential mucin gene expression in the presence of bile. Hepatology Baltimore, Md 1995; 21 (1): 223.
44. Landrier JF, Eloranta JJ, Vavricka SR, Kullak-Ublick GA. The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes. Am J Physiol Gastrointest Liver Physiol 2006; 290 (3): G476.
1. Strazzabosco M, Fabris L. Functional anatomy of normal bile ducts. Anatomical record Hoboken, N J 2007 2008; 291 (6): 653.
2. Alpini G, Roberts S, Kuntz SM, et al. Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver. Gastroenterology 1996; 110 (5): 1636.
3. Alpini G, Ulrich C, Roberts S, et al. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. American journal of physiology, The 1997; 272 (2 Pt 1): G289.
4. Benedetti A, Bassotti C, Rapino K, Marucci L, Jezequel AM. A morphometric study of the epithelium lining the rat intrahepatic biliary tree. Journal of hepatology 1996; 24 (3): 335.
5. Marzioni M, Glaser SS, Francis H, Phinizy JL, LeSage G, Alpini G. Functional heterogeneity of cholangiocytes. Seminars in liver disease 2002; 22 (3): 227.
6. Lemaigre FP. Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies. Gastroenterology 2009; 137 (1): 62.
7. Ludwig J. New concepts in biliary cirrhosis. Seminars in liver disease 1987; 7 (4): 293.
8. Ludwig J, Ritman EL, LaRusso NF, Sheedy PF, Zumpe G. Anatomy of the human biliary system studied by quantitative computer-aided three-dimensional imaging techniques. Hepatology Baltimore, Md 1998; 27 (4): 893.
9. Masyuk TV, Ritman EL, LaRusso NF. Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction. American journal of pathology 2001; 158 (6): 2079.
10. Ishii M, Vroman B, LaRusso NF. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 1989; 97 (5): 1236.
11. Kanno N, LeSage G, Glaser S, Alvaro D, Alpini G. Functional heterogeneity of the intrahepatic biliary epithelium. Hepatology Baltimore, Md 2000; 31 (3): 555.
12. Glaser S, Francis H, Demorrow S, et al. Heterogeneity of the intrahepatic biliary epithelium. World journal of gastroenterology WJG 2006; 12 (22): 3523.
13. Alpini G, Glaser S, Robertson W, et al. Large but not small intrahepatic bile ducts are involved in secretin-regulated ductal bile secretion. American journal of physiology, The 1997; 272 (5 Pt 1): G1064.
14. Carrella M, Roda E. Evolving concepts in the pathophysiology of biliary lipid secretion. Ital J Gastroenterol Hepatol 1999; 31 (7): 643.
15. Kubitz R; Haussinger D. Osmoregulation of bile formation. Methods-Enzymol. 2007; 428: 313-24.
16.姚光弼.胆管细胞对胆汁分泌和形成的作用.肝脏2004年9月第9卷第3期:176-178.
17. Kanno N, LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte bicarbonate secretion. American journal of physiology Gastrointestinal and liver physiology 2001; 281 (3): G612.
18. Glaser SS, Rodgers RE, Phinizy JL, et al. Gastrin inhibits secretin-induced ductal secretion by interaction with specific receptors on rat cholangiocytes. American journal of physiology, The 1997; 273 (5 Pt 1): G1061.
19. Costa M, Furness JB. Somatostatin is present in a subpopulation of noradrenergic nerve fibres supplying the intestine. Neuroscience 1984; 13 (3): 911.
20. Cho WK. Role of the neuropeptide, bombesin, in bile secretion. Yale journal of biology and medicine, The 1997; 70 (4): 409.
21. Cho WK, Boyer JL. Vasoactive intestinal polypeptide is a potent regulator of bile secretion from rat cholangiocytes. Gastroenterology 1999; 117 (2): 420.
22. Cho WK, Mennone A, Rydberg SA, Boyer JL. Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion. Gastroenterology 1997; 113 (1): 311.
23. Baiocchi L, LeSage G, Glaser S, Alpini G. Regulation of cholangiocyte bile secretion. Journal of hepatology 1999; 31 (1): 179.
24. Esteller A. Physiology of bile secretion. World J Gastroenterol 2008; 14 (37): 5641.
25. Gradilone SA, Carreras FI, Calamita G, Lehmann GL. Liver aquaporins: significance in canalicular and ductal bile formation. Ann-Hepatol. 2004, 3(4): 130-6
26. Portincasa P, Moschetta A, Mazzone A. Water handling and aquaporins in bile formation: recent advances and research trends. J-Hepatol. 2003,39(5): 864-74]
27. Calamita G, Ferri D, Gena P, et al. Water transport into bile and role in bile formation. Curr-Drug-Targets-Immune-Endocr-Metabol-Disord. 2005, 5(2): 137-42
28. Lazaridis KN, Pham L, Tietz P, et al. Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter. Journal of clinical investigation, The 1997; 100 (11): 2714.
29. Alpini G, Glaser SS, Rodgers R, et al. Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes. Gastroenterology 1997; 113 (5): 1734.
30. Liu JJ, Glickman JN, Masyuk AI, Larusso NF. Cholangiocyte bile salt transporters in cholesterol gallstone-susceptible and resistant inbred mouse strains. J Gastroenterol Hepatol 2008; 23 (10): 1596.
31. Xia X, Francis H, Glaser S, Alpini G, LeSage G. Bile acid interactions with cholangiocytes. World journal of gastroenterology WJG 2006; 12 (22): 3553.
32. Soroka -C-JL, -J-M; Azzaroli,-F; Boyer,-J-L Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver. Hepatology 2001; 33 (4): 783.
33. Ballatori N, Christian WV, Lee JY, et al. OSTalpha-OSTbeta: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia. Hepatology 2005; 42 (6): 1270.
34. Rao A, Haywood J, Craddock AL, Belinsky MG, Kruh GD, Dawson PA. The organic solute transporter alpha-beta, Ostalpha-Ostbeta, is essential for intestinal bile acid transport and homeostasis. Proc Natl Acad Sci U S A 2008; 105 (10): 3891.
35. Ballatori N, Fang F, Christian WV, Li N, Hammond CL. Ostalpha-Ostbeta is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver. Am J Physiol Gastrointest Liver Physiol 2008; 295 (1): G179.
36. Alpini G, Glaser S, Baiocchi L, et al. Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats. Hepatology. 2005, 41(5): 1037-45.]
37. Reymann A, Braun W, Drobik C, Woermann C. Stimulation of bile acid active transport related to increased mucosal cyclic AMP content in rat ileum in vitro. Biochim Biophys Acta 1989;1011: 158-164.
38. [Alpini G, Glaser S, Chowdhury U, Francis H, Kanno N, Phinizy JL, Eisel W, LeSage G. cAMP-dependent translocation of the apical bile acid transporter (ABAT) to the cholangiocyte apical membrane regulates ductal absorption of conjugated bile acids. Hepatology 1999; 30: A1029.
39. Halpern MD, Holubec H, Saunders TA, et al. Bile acids induce ileal damage during experimental necrotizing enterocolitis. Gastroenterology 2006; 130 (2): 359.
40. Stravitz RT, Sanyal AJ, Pandak WM, Vlahcevic ZR, Beets JW, Dawson PA. Induction of sodium-dependent bile acid transporter messenger RNA, protein, and activity in rat ileum by cholic acid. Gastroenterology 1997; 113 (5): 1599.
41. Alpini G, Ueno Y, Glaser SS, et al. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology 2001; 34 (5): 868.
42. Chen F, Ma L, Dawson PA, et al. Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter. J Biol Chem 2003; 278 (22): 19909.
43. Brady LM, Beno DW, Davis BH. Bile acid stimulation of early growth response gene and mitogen-activated protein kinase is protein kinase C-dependent. Biochem J 1996; 316 (Pt 3): 765-769
44. Podevin P, Rosmorduc O, Conti F, Calmus Y, Meier PJ, Poupon R. Bile acids modulate the interferon signalling pathway. Hepatology 1999; 29: 1840-1847.
45. Di Toro R, Campana G, Murari G, Spampinato S. Effects of specific bile acids on c-fos messenger RNA levels in human colon carcinoma Caco-2 cells. Eur J Pharm Sci 2000; 11: 291-298.
46. Nathanson MH, Burgstahler AD, Masyuk A, Larusso NF. Stimulation of ATP secretion in the liver by therapeutic bile acids. Biochem J 2001; 358: 1-5.
47. Voronina S, Longbottom R, Sutton R, Petersen OH, Tepikin A. Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology. J Physiol 2002; 540: 49-55
48. Marrero I, Sanchez-Bueno A, Cobbold PH, Dixon CJ. Taurolithocholate and taurolithocholate 3-sulphate exert different effects on cytosolic free Ca2+ concentration in rat hepatocytes. Biochem J 1994; 300 (Pt 2): 383-386
49. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, Lehmann JM. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999; 284: 1365-1368
50. Kurz AK, Block C, Graf D, Dahl SV, Schliess F, Haussinger D. Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver. Biochem J 2000; 350 (Pt 1): 207-213
51. Rao YP, Studer EJ, Stravitz RT, Gupta S, Qiao L, Dent P, Hylemon PB. Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes. Hepatology 2002; 35: 307-314
52. Kurz AK, Graf D, Schmitt M, Vom Dahl S, Haussinger D. Tauroursodesoxycholate- induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats. Gastroenterology 2001; 121: 407-419.
53. Elsing C, Kassner A, Hubner C, Buhli H, Stremmel W. Absorptive and secretory mechanisms in biliary epithelial cells. Journal of hepatology 1996; 24: 121.
54. Grattagliano I, Portincasa P, Palmieri VO, Palasciano G. Contribution of canalicular glutathione efflux to bile formation. From cholestasis associated alterations to pharmacological intervention to modify bile flow. Curr-Drug-Targets-Immune- Endocr- Metabol-Disord. 2005, 5(2): 153-61.
55. Alpini G, Glaser SS, Ueno Y, Pham L, Podila PV, Caligiuri A, LeSage G, LaRusso NF. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. Am J Physiol Gastrointest Liver Physiol 1998; 274: G767-775
56. Alpini G, Ueno Y, Glaser S, Marzioni M, Phinizy JL, Francis H, LeSage G. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology 2001; 34: 868-876
57. LeSage GD, Glaser S, Marucci L, Benedetti A, Phinizy JL, Rodgers R, Caligiuri A, Papa E, Tretjak Z, Jezequel AM, Holcomb LA, Alpini G. Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver. Am J Physiol 1999; 276: G1289-1301.
58. LeSage GD, Benedetti A, Glaser S, Marucci L, Tretjak Z, Caligiuri A, Rodgers R, Phinizy JL, Baiocchi L, Francis H, Lasater J, Ugili L, Alpini G. Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes fromnormal rat liver. Hepatology 1999; 29: 307-319.
59. LeSage G, Glaser S, Robertson W, Phinizy JL, Rodgers R, Alpini G. Partial hepatectomy induces proliferative and secretory events in small cholangiocytes. Gastroenterology 1996; 110: A1250.
60. Xu WH, Ye QF, Xia SS. Apoptosis and proliferation of intrahepatic bile duct after ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int 2004; 3: 428-432.
61.李子禹,邹声泉.胆汁和胆管上皮的免疫生物学.国外医学. 2000年第27卷第6期:333-336.
62. LeSage G, Alvaro D, Benedetti A, Glaser S, Marucci L, Baiocchi L, Eisel W, Caligiuri A, Phinizy JL, Rodgers R, Francis H, Alpini G. Cholinergic system modulates growth, apoptosis, and secretion of cholangiocytes from bile duct-ligated rats. Gastroenterology 1999; 117: 191-199.
63. Alpini G, Ueno Y, Glaser SS, et al. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology Baltimore, Md 2001; 34 (5): 868.
64. Glaser SS, Gaudio E, Miller T, Alvaro D, Alpini G. Cholangiocyte proliferation and liver fibrosis. Expert reviews in molecular medicine 2009: e7.
65. Lesage G, Glaser S, Ueno Y, et al. Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis. American journal of physiology Gastrointestinal and liver physiology 2001; 281 (1): G182.
66. Alpini G, Glaser SS, Ueno Y, et al. Heterogeneity of the proliferative capacity of rat cholangiocytes after bile duct ligation. American journal of physiology, The 1998; 274 (4 Pt 1): G767.
67. LeSage GD, Benedetti A, Glaser S, et al. Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver. Hepatology Baltimore, Md 1999; 29 (2): 307.
68. Alvaro D, Mancino MG. New insights on the molecular and cell biology of human cholangiopathies. Molecular aspects of medicine 2008; 29 (1-2): 50.
69.杨洋,杨可桢,黄志强,等.胆结石与慢性胆系炎症的免疫学初步研究—胆汁、血液Ig及胆道Ig产生细胞的检测.中华内科杂志,1985 ;24 :536.
70. Khan SA, Thomas HC, Davidson BR, Taylor-Robinson SD. Cholangiocarcinoma.Lancet 2005; 366: 1303-1314.
71. Curry MP, Hegarty JE. The gallbladder and biliary tract in cystic fi brosis. Curr Gastroenterol Rep 2005; 7: 147-153.
72. Kinnman N, Lindblad A, Housset C, Buentke E, Scheynius A, Strandvik B, Hultcrantz R. Expression of cystic fi brosis transmembrane conductance regulator in liver tissue from patients with cystic fi brosis. Hepatology 2000; 32: 334-340.
73. Schlenker T, Romac JM, Sharara AI, Roman RM, Kim SJ, LaRusso N, Liddle RA, Fitz JG. Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am J Physiol 1997; 273: G1108-1117.
74. Roman RM, Feranchak AP, Salter KD, Wang Y, Fitz JG. Endogenous ATP release regulates Cl- secretion in cultured human and rat biliary epithelial cells. Am J Physiol 1999; 276:G1391-1400.
75. Wu CT, Davis PA, Luketic VA, Gershwin ME. A review of the physiological and immunological functions of biliary epithelial cells: targets for primary biliary cirrhosis, primary sclerosing cholangitis and drug-induced ductopenias. Clinical and developmental immunology 2004; 11 (3-4): 205.
76. Masyuk T, Masyuk A, LaRusso N. Cholangiociliopathies: genetics, molecular mechanisms and potential therapies. Current opinion in gastroenterology 2009; 25 (3): 265.
77. Chen XM, O'Hara SP, LaRusso NF. The immunobiology of cholangiocytes. Immunology and cell biology 2008; 86 (6): 497.
78. Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005; 39: S90-102.
79. Adams DH, Afford SC. Effector mechanisms of nonsuppurative destructive cholangitis in graft-versus-host disease and allograft rejection. Semin Liver Dis 2005; 25: 281-297.