多药耐药相关蛋白1（MRP1）在梗阻性黄疸肝组织中表达及意义的初步研究
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摘要
梗阻性黄疸(Obstructive Jaundice，OJ)，是临床上肝外胆管各种原因的阻塞所引起的胆汁淤积症，常见于壶腹周围癌、胰头癌、肝门部胆管癌、胆总管结石等疾病。OJ时胆汁淤积常可导致复杂而严重的全身性病理生理改变，长期重度淤胆还可影响手术方式的选择，导致术后恢复慢以及严重并发症，因此深入研究重度OJ时病理生理改变的分子机制，对临床如何增强长期重度OJ患者的手术耐受性、提高手术安全性、促进术后的恢复具有重要意义。
近年来，肝脏胆红素转运子越来越受到人们的关注。多药耐药相关蛋白1 (Multidrug Resistance-associated Protein 1 , MRP1/Mrp1分别指人和大鼠)是位于多种极性细胞膜表面介导其底物依赖ATP自细胞内转运至细胞外的一种膜糖蛋白，属ATP结合盒式(ATP Binding Cassette，ABC)基因家族，含有1531个氨基酸，对结合性胆红素及其它胆汁成分的亲合转运能力很强。MRP1/Mrp1在正常情况下表达量极少，当各种原因所致胆汁淤积时，排泄胆红素的主要转运子MRP2表达有特征性的下调，与此同时，作为一种“诱生型”胆红素转运子，Mrp1表达在内毒素诱导的肝内淤胆大鼠模型中明显升高，推测其与结合性胆红素的代偿排泄密切相关。我们已在临床上发现，部分重度OJ和淤胆性肝炎患者在用常规治疗无法使黄疸减轻时，试用重组人生长激素(recombinant human Growth Hormone, rhGH)治疗可以获得较满意的疗效，但其作用机制研究较少。
本研究从重度OJ患者以及胆道梗阻-胆道再通的肝外淤胆大鼠模型中观察肝组织中MRP1/Mrp1表达的变化及上调的可能机制，探讨MRP1/Mrp1在重度OJ病理生理改变中的意义，为临床治疗提供依据。
主要的结果和结论如下：
1. OJ患者肝组织中MRP1的表达明显强于对照组；
2. 胆道再通-生长激素注射组大鼠7d病死率明显低于胆道再通组，说明rhGH在胆道再通术前的应用增强了OJ大鼠的手术耐受性，提高了手术安全性；
3. 大鼠结扎胆总管术后ALT、AKP显著升高，ALB、PA逐渐下降，TBIL在术后7d达到峰值，此后水平有所回落，而尿中DBIL在梗阻期间一直上升；TNF-α在结扎胆总管术后上升，随梗阻时间的延长，与假手术组逐渐出现非常显著的差异(P<0.01)。
胆道再通术后上述指标逐步好转，至术后7d接近或已达到正常水平；给予rhGH的大鼠各项指标要明显优于未用rhGH者，随用药天数的增加，效果更趋明显，说明应用rhGH能明显改善OJ大鼠再通术前的肝功能，并能促进术后的恢复；
4. 动物实验证实了Mrp1的表达随着梗阻时间的延长明显升高，同时血清TBIL水平下降，尿样DBIL水平上升，在梗阻解除后Mrp1仍处于较高水平，推测Mrp1在梗阻性黄疸时以及梗阻解除早期都起到了代偿排泄胆红素的作用，是机体的一种自身调节机制；
5. 梗阻期间Mrp1的表达与TNF-α水平密切相关，说明TNF-α可能通过上调Mrp1来减轻淤胆；rhGH可能通过限制TNF-α的过度释放间接上调Mrp1，发挥TNF-α两面性作用中积极的一面；rhGH亦可直接上调Mrp1，其信号途径尚需深入研究。
Objectives: To study the potential molecular mechanism in the pathophysiological changes of obstructive jaundice, and provide a possible approach to enhance surgical tolerance and safety, promote recovery. Multidrug resistance-associated protein 1 is a membrane glycoprotein, which pumps its substrates, eg. conjugated bilirubin and bile salts, out of cytosol. Whatever causes cholestasis may derive from, there is a characteristic down-regulation of MRP2, which is considered as the main bilirubin transporter under normal circumstances. At the same time, as a novel inducible bilirubin transporter, the expression of Mrp1 protein, normally expressed at very low levels in hepatocytes, is elevated in endotoxin-induced intrahepatic cholestatic rats, suggesting that Mrp1 mediates the efflux of conjugated bilirubin and divalent bile salt conjugates into plasma followed by urinary elimination, namely a compensatory mechanism under conditions of impaired bile excretion into biliary tree. So far, its expression and clinical significance remain unclear in obstructive jaundice.
Methods:
1. The expression of MRP1 was detected by indirect immunofluorescence in the liver specimens of twenty control patients without jaundice and thirty patients with obstructive jaundice.
2. One hundred and forty six Wistar male rats were randomizedly divided into five groups, namely SHAM, CBDL, CBDL-GH, REF, REF-GH. Experimental model was established by common bile duct ligation(CBDL) and internal drainage 2 weeks later, and rhGH was injected subcutaneously by 0.5u/kg bw per day.
Such liver function indices as ALT, ALB, AKP, TBIL in rat serum, in addition to DBIL in rat urine, were assayed by ultraviolet spectrophotometer at each selected timepoint. TNF-αand PA were assayed by RIA and immune turbidity respectively. The expression of Mrp1 was detected by indirect immunofluorescence, which was quantified by average integration optic density.
Results:
1. The expression of MRP1 in OJ patients' liver specimens was stronger than in control ones.
2. One-week mortality of REF-GH group was much lower than REF group (p<0.05).
3. Serum ALT, AKP and TNF-α, in addition to urinary DBIL were ascending, while serum ALB and PA descending significantly after CBDL. The level of TBIL peaked on the seventh day postoperatively, followed by a moderate decline. After bile reflow, both serum liver function indices and urinary DBIL were taking a gradually favourable turn and drew near to normal levels. The above-mentioned indices of rhGH-treated rats were much better than that of non-rhGH-treated rats.
4. The expression of Mrp1 in SHAM group was so weak that could hardly be detected under fluorescence microscope. However, in CBDL and CBDL-GH groups, obvious fluorescence could be detected from the seventh day postoperatively, showing significant discrepancies when compared with SHAM group. As duration elongated, both the intensity and range of specific fluorescence grew, even after bile reflow, moderate level of fluorescence were maintained with a small amount of descent. Compared with non-rhGH-treated rats at the same timepoints, the expression of Mrp1 in rhGH-treated rats was much stronger, and the discrepencies showed statistical significance when quantified by average integration optic density.
5. TNF-αcorrelated positively with ALT and Mrp1, but negatively with PA. When performed significance-checkout, P value was smaller than 0.05, indicating that TNF-αbore close relations to both the deterioration of liver function and the expression of Mrp1 in obstructive jaundiced rats.
Conclusions:
1. Compared with control group, the expression of MRP1 in OJ group was much stronger, suggesting an important role of MRP1 in obstuctive jaundice.
2. The high level of Mrp1 was confirmed in experimental rat model, together with descent of serum TBIL and ascent of urine DBIL in obstructive period, even after bile reflow, moderate expression of Mrp1 was maintained, suggesting a compensatory function of Mrp1 to excrete bilirubin and other bile components
近年来，肝脏胆红素转运子越来越受到人们的关注。多药耐药相关蛋白1 (Multidrug Resistance-associated Protein 1 , MRP1/Mrp1分别指人和大鼠)是位于多种极性细胞膜表面介导其底物依赖ATP自细胞内转运至细胞外的一种膜糖蛋白，属ATP结合盒式(ATP Binding Cassette，ABC)基因家族，含有1531个氨基酸，对结合性胆红素及其它胆汁成分的亲合转运能力很强。MRP1/Mrp1在正常情况下表达量极少，当各种原因所致胆汁淤积时，排泄胆红素的主要转运子MRP2表达有特征性的下调，与此同时，作为一种“诱生型”胆红素转运子，Mrp1表达在内毒素诱导的肝内淤胆大鼠模型中明显升高，推测其与结合性胆红素的代偿排泄密切相关。我们已在临床上发现，部分重度OJ和淤胆性肝炎患者在用常规治疗无法使黄疸减轻时，试用重组人生长激素(recombinant human Growth Hormone, rhGH)治疗可以获得较满意的疗效，但其作用机制研究较少。
本研究从重度OJ患者以及胆道梗阻-胆道再通的肝外淤胆大鼠模型中观察肝组织中MRP1/Mrp1表达的变化及上调的可能机制，探讨MRP1/Mrp1在重度OJ病理生理改变中的意义，为临床治疗提供依据。
主要的结果和结论如下：
1. OJ患者肝组织中MRP1的表达明显强于对照组；
2. 胆道再通-生长激素注射组大鼠7d病死率明显低于胆道再通组，说明rhGH在胆道再通术前的应用增强了OJ大鼠的手术耐受性，提高了手术安全性；
3. 大鼠结扎胆总管术后ALT、AKP显著升高，ALB、PA逐渐下降，TBIL在术后7d达到峰值，此后水平有所回落，而尿中DBIL在梗阻期间一直上升；TNF-α在结扎胆总管术后上升，随梗阻时间的延长，与假手术组逐渐出现非常显著的差异(P<0.01)。
胆道再通术后上述指标逐步好转，至术后7d接近或已达到正常水平；给予rhGH的大鼠各项指标要明显优于未用rhGH者，随用药天数的增加，效果更趋明显，说明应用rhGH能明显改善OJ大鼠再通术前的肝功能，并能促进术后的恢复；
4. 动物实验证实了Mrp1的表达随着梗阻时间的延长明显升高，同时血清TBIL水平下降，尿样DBIL水平上升，在梗阻解除后Mrp1仍处于较高水平，推测Mrp1在梗阻性黄疸时以及梗阻解除早期都起到了代偿排泄胆红素的作用，是机体的一种自身调节机制；
5. 梗阻期间Mrp1的表达与TNF-α水平密切相关，说明TNF-α可能通过上调Mrp1来减轻淤胆；rhGH可能通过限制TNF-α的过度释放间接上调Mrp1，发挥TNF-α两面性作用中积极的一面；rhGH亦可直接上调Mrp1，其信号途径尚需深入研究。
Objectives: To study the potential molecular mechanism in the pathophysiological changes of obstructive jaundice, and provide a possible approach to enhance surgical tolerance and safety, promote recovery. Multidrug resistance-associated protein 1 is a membrane glycoprotein, which pumps its substrates, eg. conjugated bilirubin and bile salts, out of cytosol. Whatever causes cholestasis may derive from, there is a characteristic down-regulation of MRP2, which is considered as the main bilirubin transporter under normal circumstances. At the same time, as a novel inducible bilirubin transporter, the expression of Mrp1 protein, normally expressed at very low levels in hepatocytes, is elevated in endotoxin-induced intrahepatic cholestatic rats, suggesting that Mrp1 mediates the efflux of conjugated bilirubin and divalent bile salt conjugates into plasma followed by urinary elimination, namely a compensatory mechanism under conditions of impaired bile excretion into biliary tree. So far, its expression and clinical significance remain unclear in obstructive jaundice.
Methods:
1. The expression of MRP1 was detected by indirect immunofluorescence in the liver specimens of twenty control patients without jaundice and thirty patients with obstructive jaundice.
2. One hundred and forty six Wistar male rats were randomizedly divided into five groups, namely SHAM, CBDL, CBDL-GH, REF, REF-GH. Experimental model was established by common bile duct ligation(CBDL) and internal drainage 2 weeks later, and rhGH was injected subcutaneously by 0.5u/kg bw per day.
Such liver function indices as ALT, ALB, AKP, TBIL in rat serum, in addition to DBIL in rat urine, were assayed by ultraviolet spectrophotometer at each selected timepoint. TNF-αand PA were assayed by RIA and immune turbidity respectively. The expression of Mrp1 was detected by indirect immunofluorescence, which was quantified by average integration optic density.
Results:
1. The expression of MRP1 in OJ patients' liver specimens was stronger than in control ones.
2. One-week mortality of REF-GH group was much lower than REF group (p<0.05).
3. Serum ALT, AKP and TNF-α, in addition to urinary DBIL were ascending, while serum ALB and PA descending significantly after CBDL. The level of TBIL peaked on the seventh day postoperatively, followed by a moderate decline. After bile reflow, both serum liver function indices and urinary DBIL were taking a gradually favourable turn and drew near to normal levels. The above-mentioned indices of rhGH-treated rats were much better than that of non-rhGH-treated rats.
4. The expression of Mrp1 in SHAM group was so weak that could hardly be detected under fluorescence microscope. However, in CBDL and CBDL-GH groups, obvious fluorescence could be detected from the seventh day postoperatively, showing significant discrepancies when compared with SHAM group. As duration elongated, both the intensity and range of specific fluorescence grew, even after bile reflow, moderate level of fluorescence were maintained with a small amount of descent. Compared with non-rhGH-treated rats at the same timepoints, the expression of Mrp1 in rhGH-treated rats was much stronger, and the discrepencies showed statistical significance when quantified by average integration optic density.
5. TNF-αcorrelated positively with ALT and Mrp1, but negatively with PA. When performed significance-checkout, P value was smaller than 0.05, indicating that TNF-αbore close relations to both the deterioration of liver function and the expression of Mrp1 in obstructive jaundiced rats.
Conclusions:
1. Compared with control group, the expression of MRP1 in OJ group was much stronger, suggesting an important role of MRP1 in obstuctive jaundice.
2. The high level of Mrp1 was confirmed in experimental rat model, together with descent of serum TBIL and ascent of urine DBIL in obstructive period, even after bile reflow, moderate expression of Mrp1 was maintained, suggesting a compensatory function of Mrp1 to excrete bilirubin and other bile components
引文
1.
徐忠立. 实用黄疸病学. 北京: 中国医药科技出版社, 1995, 496.
2.
蒋飞照，陈哲京，张启瑜. 梗阻性黄疸并发急性肾功能损害相关因素分析. 肝胆胰外科杂志, 2002, 14(1): 40.
3.
俞毅君，王毓，徐德征. 内毒素血症和高胆红素血症对外科梗阻性黄疸患者的毒性作用. 中国综合临床, 2001, 17(5): 383.
4.
Paulusma CC, Bosma PJ, Zaman GJR, et al. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science, 1996, 271: 1126.
5.
Keppler D, Kamisako T, Leier I, et al. Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family. Advan Enzyme Regul, 2000, 40: 339.
6.
Vos TA, Hooiveld GJEJ, Koning H, et al. Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, spgp, in endotoxemic rat liver. Hepatology, 1998, 28:1637.
7.
McGrath T，Latoud C，Arnold S，et al. Mechanisms of multidrug resistance in HL60 cells: analysis of resistance associated membrane proteins and levels of mdr gene expression. Biochem Pharmacol, 1989, 38(20): 3611.
8.
Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science, 1992, 258:1650.
9.
Kullak-Ublick GA, Stieger B, Hagenbuch B, et al. Hepatic transport of bile salts. Semin Liver Disease, 2000, 20(3): 273.
10.
Kamisako T, Kobayashi Y, Takeuchi K, et al. Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance. J Gastroenterol, 2000, 35:659.
11.
Meier PJ and Stieger B. Bile salt transporters. Annu Rev Physiol, 2002, 64: 635.
12.
Trauner M,Arrese M,Soroka CJ,et al. The rat canalicular conjugate export pump (Mrp2) is down regulated in intrahepatic and obstructive cholestasis. Gastroenterology, 1997, 113:255.
Trauner M,Arrese M,Soroka CJ,et al. The rat canalicular conjugate export pump (Mrp2) is down regulated in intrahepatic and obstructive cholestasis. Gastroenterology, 1997, 113:255.
Konig J，Rose D，Cui Y and Keppler D. Characterization of the human multidrug resistance protein isoform MRP1 localized to the basolateral hepatocyte membrane. Hepatology, 1999, 29:1156.
Gerloff T，Geier A，Sueger B，et al. Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver. Gastroenterology, 1999, 117: 1408.
Kartenbeck J，Leuschner U，Mayer R and Keppler D. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome.Hepatology, 1996, 23:1061.
Kountouras J, Scheuer PJ, Billing BH. Effect of prolonged bile duct obstruction in the rat on hepatic transport of bilirubin. Clin Sci(Lond), 1985, 68: 341.
Tanaka Y, Kobayashi Y, et al. Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive jaundice. Am J Physiol:Gastrointest Liver Physiol, 2002, 282(4):G656.
Tilg H. Cytokines and liver diseases. Can J Gastroenterol, 2001, 15(10): 661.
Fox ES, Kim JC, Tracy TF. NFκB activation and modulation in hepatic macrophages during cholestatic injury. J Surg Res, 1997, 72(2): 129.
Scopa CD, Koureleas S, Tsamandas A, et al. Beneficial effects of growth hormone and insulin-like growth factor Ⅰ on intestinal translocation, endotoxemia, and apoptosis in experimentally jaundiced rats. J Am Coll Surg, 2000, 190(4): 423.
Beg AA, Baltimore D. An essential role for NF-κB in preventing TNF-α-induced cell death. Science, 1996, 274(1): 782.
Wang CY, Mayo MW, Baldwin AS. TNF- and therapy-induced apoptosis: potentiation by inhibition of NF-κB. Science, 1996, 274(1): 784.
Van Antwerp D, Martin Seamus, Kafri T, et al. Suppression of TNF-α-induced apoptosis by NF-κB. Science, 1996, 274(1): 787.
Hartmann G, Cheung AKY, Piquette-Miller M. Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. J Pharmacol Exp Ther, 2002, 303(1): 273.
24.
Hartmann G, Cheung AKY, Piquette-Miller M. Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. J Pharmacol Exp Ther, 2002, 303(1): 273.
25.
Stein U, Walther W, Laurencot CM, et al. Tumor necrosis factor-α and expression of the multidrug resistance-associated genes LRP and MRP1. J Natl Cancer Inst, 1999, 89(11): 807.
26.
Ram PA，Park SH，Choi HK，et al. Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liver. Differential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation. J Biol Chem，1996, 271(10): 5929.
27.
Comez DS. Compara tive effects of growth hormone in large and small bowel resection in the rat. J Surg Res, 1996, 62: 5.
28.
Greig JD, Krukowski ZH, Matheson NA. Surgical morbidity and mortality in one hundred and twenty nine patients with obstructive jaundice. Br J Surg, 1998,75:216.
29.
Chang TC, Lin JJ, Yu SS, et al. Absence of growth hormone receptor in hepatocellular carcinoma and cirrhotic liver. Hepatology, 1999, 11: 123.
徐忠立. 实用黄疸病学. 北京: 中国医药科技出版社, 1995, 496.
2.
蒋飞照，陈哲京，张启瑜. 梗阻性黄疸并发急性肾功能损害相关因素分析. 肝胆胰外科杂志, 2002, 14(1): 40.
3.
俞毅君，王毓，徐德征. 内毒素血症和高胆红素血症对外科梗阻性黄疸患者的毒性作用. 中国综合临床, 2001, 17(5): 383.
4.
Paulusma CC, Bosma PJ, Zaman GJR, et al. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science, 1996, 271: 1126.
5.
Keppler D, Kamisako T, Leier I, et al. Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family. Advan Enzyme Regul, 2000, 40: 339.
6.
Vos TA, Hooiveld GJEJ, Koning H, et al. Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, spgp, in endotoxemic rat liver. Hepatology, 1998, 28:1637.
7.
McGrath T，Latoud C，Arnold S，et al. Mechanisms of multidrug resistance in HL60 cells: analysis of resistance associated membrane proteins and levels of mdr gene expression. Biochem Pharmacol, 1989, 38(20): 3611.
8.
Cole SPC, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science, 1992, 258:1650.
9.
Kullak-Ublick GA, Stieger B, Hagenbuch B, et al. Hepatic transport of bile salts. Semin Liver Disease, 2000, 20(3): 273.
10.
Kamisako T, Kobayashi Y, Takeuchi K, et al. Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance. J Gastroenterol, 2000, 35:659.
11.
Meier PJ and Stieger B. Bile salt transporters. Annu Rev Physiol, 2002, 64: 635.
12.
Trauner M,Arrese M,Soroka CJ,et al. The rat canalicular conjugate export pump (Mrp2) is down regulated in intrahepatic and obstructive cholestasis. Gastroenterology, 1997, 113:255.
Trauner M,Arrese M,Soroka CJ,et al. The rat canalicular conjugate export pump (Mrp2) is down regulated in intrahepatic and obstructive cholestasis. Gastroenterology, 1997, 113:255.
Konig J，Rose D，Cui Y and Keppler D. Characterization of the human multidrug resistance protein isoform MRP1 localized to the basolateral hepatocyte membrane. Hepatology, 1999, 29:1156.
Gerloff T，Geier A，Sueger B，et al. Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver. Gastroenterology, 1999, 117: 1408.
Kartenbeck J，Leuschner U，Mayer R and Keppler D. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome.Hepatology, 1996, 23:1061.
Kountouras J, Scheuer PJ, Billing BH. Effect of prolonged bile duct obstruction in the rat on hepatic transport of bilirubin. Clin Sci(Lond), 1985, 68: 341.
Tanaka Y, Kobayashi Y, et al. Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive jaundice. Am J Physiol:Gastrointest Liver Physiol, 2002, 282(4):G656.
Tilg H. Cytokines and liver diseases. Can J Gastroenterol, 2001, 15(10): 661.
Fox ES, Kim JC, Tracy TF. NFκB activation and modulation in hepatic macrophages during cholestatic injury. J Surg Res, 1997, 72(2): 129.
Scopa CD, Koureleas S, Tsamandas A, et al. Beneficial effects of growth hormone and insulin-like growth factor Ⅰ on intestinal translocation, endotoxemia, and apoptosis in experimentally jaundiced rats. J Am Coll Surg, 2000, 190(4): 423.
Beg AA, Baltimore D. An essential role for NF-κB in preventing TNF-α-induced cell death. Science, 1996, 274(1): 782.
Wang CY, Mayo MW, Baldwin AS. TNF- and therapy-induced apoptosis: potentiation by inhibition of NF-κB. Science, 1996, 274(1): 784.
Van Antwerp D, Martin Seamus, Kafri T, et al. Suppression of TNF-α-induced apoptosis by NF-κB. Science, 1996, 274(1): 787.
Hartmann G, Cheung AKY, Piquette-Miller M. Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. J Pharmacol Exp Ther, 2002, 303(1): 273.
24.
Hartmann G, Cheung AKY, Piquette-Miller M. Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia. J Pharmacol Exp Ther, 2002, 303(1): 273.
25.
Stein U, Walther W, Laurencot CM, et al. Tumor necrosis factor-α and expression of the multidrug resistance-associated genes LRP and MRP1. J Natl Cancer Inst, 1999, 89(11): 807.
26.
Ram PA，Park SH，Choi HK，et al. Growth hormone activation of Stat 1, Stat 3, and Stat 5 in rat liver. Differential kinetics of hormone desensitization and growth hormone stimulation of both tyrosine phosphorylation and serine/threonine phosphorylation. J Biol Chem，1996, 271(10): 5929.
27.
Comez DS. Compara tive effects of growth hormone in large and small bowel resection in the rat. J Surg Res, 1996, 62: 5.
28.
Greig JD, Krukowski ZH, Matheson NA. Surgical morbidity and mortality in one hundred and twenty nine patients with obstructive jaundice. Br J Surg, 1998,75:216.
29.
Chang TC, Lin JJ, Yu SS, et al. Absence of growth hormone receptor in hepatocellular carcinoma and cirrhotic liver. Hepatology, 1999, 11: 123.