AIM： To study the association of three common ABCB11 and ABCC2 polymorphisms （ABCB11： 1331T〉C→V444A; ABCC2： 3563T〉A → V1188E and 4544G 〉A → C1515Y） with intrahepatic cholestasis of pregnancy （ICP） and con...AIM： To study the association of three common ABCB11 and ABCC2 polymorphisms （ABCB11： 1331T〉C→V444A; ABCC2： 3563T〉A → V1188E and 4544G 〉A → C1515Y） with intrahepatic cholestasis of pregnancy （ICP） and contraceptive-induced cholestasis （CIC）. METHODS： ABCB11 and ABCC2 genotyping data were available from four CIC patients and from 42 and 33 ICP patients, respectively. Allele-frequencies of the studied polymorphisms were compared with those in healthy pregnant controls and Caucasian individuals. Furthermore, serum bile acid levels were correlated with the presence or absence of the 1331 C allele. RESULTS： The ABCB11 1331T〉C polymorphism was significantly more frequent in cholestatic patients than in pregnant controls： C allele 76.2% （CI, 58.0-94.4） vs 51.3% （CI 35.8-66.7）, respectively （P = 0.0007）; and CC allele 57.1% （CI 36.0-78.3） vs 20% （CI 7.6-32.4）, respectively （P = 0.0065）. All four CIC patients were homozygous carriers of the C allele. In contrast, none of the studied ABCC2 polymorphism was overrepresented in ICP or CIC patients. Higher serum bile acid levels were found in carriers of the 1331CC genotype compared to carriers of the TT genotype. CONCLUSION： Our data support a role for the ABCB11 1331T〉C polymorphism as a susceptibility factor for the development of estrogen-induced cholestasis, whereas no such association was found for ABCC2. Serum bile acid and 7-glutamyl transferase levels might help to distinguish ABCB4- and ABCB11-related forms of ICP and CIC.展开更多
To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency. METHODSMutations in the gene encoding BSEP leading ...To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency. METHODSMutations in the gene encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney (HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatography-tandem mass spectrometry. Wild-type and mutant BSEP transport of [<sup>3</sup>H]-labeled taurocholate (TC) and taurochenodeoxycholate (TCDC) was assessed by vesicular transport assays. RESULTSA girl (at 2 mo) presented with pruritus, jaundice and elevated serum bile salts (BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919del and the nonsense mutation p.R1235X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy (age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives (< 5%). The patients’ native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919del and p.G1032R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively. CONCLUSIONIn summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2 (PFIC-2).展开更多
目的利福平(Rifampicin,RIF)具有肝毒性,但其机制尚不清楚。本研究在RIF诱导的肝内胆汁淤积小鼠中,探讨RIF对肝细胞胆汁酸转运体胆汁酸输出泵(bile salt exportpump,Bsep)和多药抵抗相关蛋白-2(multidrug resistance-associated protein...目的利福平(Rifampicin,RIF)具有肝毒性,但其机制尚不清楚。本研究在RIF诱导的肝内胆汁淤积小鼠中,探讨RIF对肝细胞胆汁酸转运体胆汁酸输出泵(bile salt exportpump,Bsep)和多药抵抗相关蛋白-2(multidrug resistance-associated protein-2,Mrp2)表达和定位影响。方法 48只♀ICR小鼠随机分为4组,RIF1wk组:经灌胃给予RIF(200mg.kg-1.d-1),连续1周,于末次给药后6h取材;RIF6h组:单次灌胃给予RIF(200mg.kg-1)后6h取材;RIF1周对照组(CON1wk)与RIF6h对照组(CON6h):经灌胃给予等容积生理盐水。所有小鼠均收集血液和肝组织,常规生化检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、总胆红素(TB)和结合胆红素(DB),并检测小鼠血清和肝组织总胆汁酸(TBA)水平。HE染色分析肝组织病理改变。RT-PCR测定肝脏肝细胞胆汁酸转运体Bsep和Mrp2mRNA表达。免疫荧光法分析Bsep和Mrp2在肝细胞的位置。结果给予RIF1周后,小鼠血清TB由(1.25±0.69)μmol.L-1上升至(65.73±12.08)μmol.L-1,上升近70倍,DB由(0.77±0.40)μmol.L-1上升至(53.33±12.43)μmol.L-1,上升约80倍,ALP由(110.2±13.8)U.L-1上升至(279.5±80.4)U.L-1,上升约1.5倍,TBA由(3.15±0.89)μmol.L-1上升至(13.54±6.51)μmol.L-1,上升约5倍并伴有血清ALT和AST轻度升高;肝脏组织TBA由(0.15±0.04)μmol.g-1liver上升至(0.30±0.19)μmol.g-1liver,上升约2倍;肝脏组织HE染色显示肝细胞出现脂肪变性、轻度坏死和炎症。单次给予RIF6h后血清TB、DB、ALP、ALT、AST和TBA明显上升,但未观察到小鼠肝脏组织病理发生改变。免疫荧光分析显示,给予小鼠RIF1wk与单次给予RIF6h后肝细胞中Bsep和Mrp2的定位发生了改变。而无论单次给予RIF还是连续给药1周,肝细胞Bsep和Mrp2mRNA表达水平均未发生变化。结论肝细胞胆汁酸转运体Bsep和Mrp2定位改变可能是RIF诱发肝内胆汁淤积的重要机制。展开更多
目的通过考察茵栀黄颗粒(茵陈、栀子、金银花和黄芩)对肝细胞膜转运体多药耐药相关蛋白2(Multidrugresistance-associated protein 2,Mrp2/ABCC2)、Na+-牛黄胆酸共转运多肽(Na+/taurocholate cotransporter,Ntcp/SLC10A1)及胆盐输出泵(B...目的通过考察茵栀黄颗粒(茵陈、栀子、金银花和黄芩)对肝细胞膜转运体多药耐药相关蛋白2(Multidrugresistance-associated protein 2,Mrp2/ABCC2)、Na+-牛黄胆酸共转运多肽(Na+/taurocholate cotransporter,Ntcp/SLC10A1)及胆盐输出泵(Bile salt export pump,Bsep/ABCB11)表达的影响。方法胆管结扎术制备胆汁淤积大鼠模型;生化检测和HE染色,观察茵栀黄颗粒的干预效果;流式细胞仪测定肝脏转运体Ntcp、Bsep和Mrp2的表达。结果与模型组相比,茵栀黄组肝细胞的脂肪病变和水肿较模型组显著减轻(P<0.05),Mrp2的表达显著升高(P<0.01);但Ntcp、Bsep的表达无明显变化。结论肝细胞膜转运体Mrp2的表达升高可能是茵栀黄颗粒的退黄利胆分子机制之一。展开更多
目的通过测定孕酮、苯甲酸雌二醇诱发的孕鼠肝内胆汁淤积症模型肝脏、胎盘组织中法尼醇受体(farnesoid X receptor,FXR)及胆盐输出泵(bile salt export pump,BSEP)的表达情况,探讨雌孕激素诱导妊娠期肝内胆汁淤积症(Intrahepatic choles...目的通过测定孕酮、苯甲酸雌二醇诱发的孕鼠肝内胆汁淤积症模型肝脏、胎盘组织中法尼醇受体(farnesoid X receptor,FXR)及胆盐输出泵(bile salt export pump,BSEP)的表达情况,探讨雌孕激素诱导妊娠期肝内胆汁淤积症(Intrahepatic cholestasis of pregnancy,ICP)过程中FXR和BSEP的作用机制。方法孕15dSD大鼠60只随机分成对照组、实验组(A组、B组),分别给予生理盐水,孕酮,苯甲酸雌二醇。于用药前、用药后测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、总胆酸(TBA)总胆红素(TBIL)、直接胆红素(DBIL)、间接胆红素(IBIL)水平;测定胎鼠生长发育情况;H.E染色观察肝脏、胎盘组织形态学变化,免疫组化法测定肝脏及胎盘BSEP、FXR的表达情况。结果用药后,实验组各项血清生化指标明显高于对照组水平(P<0.05);对照组用药前后各项血清生化指标无明显变化(P>0.05);实验组用药后各项血清生化指标较用药前显著升高(P<0.05)实验组胎鼠体质量、死胎数与对照组相比较有显著差异(P<0.05)。H.E染色观察到A、B组孕鼠肝脏出现肝内胆汁淤积典型表现。免疫组化法检测到,实验组肝脏FXR表达升高,BSEP表达降低,与对照组相比差异具有显著性(P<0.05)。此外,在孕鼠胎盘也发现了少量FXR和BSEP的表达。结论雌激素具有明确的诱发孕鼠肝内胆汁淤积症的作用,孕激素也可诱导孕鼠肝内胆汁淤积症。雌激素诱导的机制可能是通过上调肝脏FXR的表达及下调肝脏BSEP的表达来影响胆汁酸的合成与代谢,进而导致了肝内胆汁淤积的发生;孕激素引起ICP,可能因为某些孕酮代谢产物诱导了BSEP的传输抑制作用致胆汁酸淤积;同时孕酮代谢障碍造成非结合胆红素增高也是引起胆汁淤积的原因之一。孕激素可以强化雌激素诱导ICP的作用。展开更多
基金Supported by Grants from the Gebert Rüf Foundation, the Forschungskredit of the University Zurichthe Swiss National Science Foundation, Grants PP00B-108511/1 and 31-64140.00
文摘AIM： To study the association of three common ABCB11 and ABCC2 polymorphisms （ABCB11： 1331T〉C→V444A; ABCC2： 3563T〉A → V1188E and 4544G 〉A → C1515Y） with intrahepatic cholestasis of pregnancy （ICP） and contraceptive-induced cholestasis （CIC）. METHODS： ABCB11 and ABCC2 genotyping data were available from four CIC patients and from 42 and 33 ICP patients, respectively. Allele-frequencies of the studied polymorphisms were compared with those in healthy pregnant controls and Caucasian individuals. Furthermore, serum bile acid levels were correlated with the presence or absence of the 1331 C allele. RESULTS： The ABCB11 1331T〉C polymorphism was significantly more frequent in cholestatic patients than in pregnant controls： C allele 76.2% （CI, 58.0-94.4） vs 51.3% （CI 35.8-66.7）, respectively （P = 0.0007）; and CC allele 57.1% （CI 36.0-78.3） vs 20% （CI 7.6-32.4）, respectively （P = 0.0065）. All four CIC patients were homozygous carriers of the C allele. In contrast, none of the studied ABCC2 polymorphism was overrepresented in ICP or CIC patients. Higher serum bile acid levels were found in carriers of the 1331CC genotype compared to carriers of the TT genotype. CONCLUSION： Our data support a role for the ABCB11 1331T〉C polymorphism as a susceptibility factor for the development of estrogen-induced cholestasis, whereas no such association was found for ABCC2. Serum bile acid and 7-glutamyl transferase levels might help to distinguish ABCB4- and ABCB11-related forms of ICP and CIC.
基金Supported by the German -Research Foun-dation-through the Clin-ical Research Group KFO217“Hepatobiliary tran-sport an-d liver diseases”the Collaborative Research Cen-tre 974“Commun-ication-an-d Systemic Relevan-ce in-Liver Damage an-d Regen-eration-”
文摘To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency. METHODSMutations in the gene encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney (HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatography-tandem mass spectrometry. Wild-type and mutant BSEP transport of [<sup>3</sup>H]-labeled taurocholate (TC) and taurochenodeoxycholate (TCDC) was assessed by vesicular transport assays. RESULTSA girl (at 2 mo) presented with pruritus, jaundice and elevated serum bile salts (BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919del and the nonsense mutation p.R1235X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy (age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives (< 5%). The patients’ native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919del and p.G1032R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively. CONCLUSIONIn summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2 (PFIC-2).
文摘目的通过考察茵栀黄颗粒(茵陈、栀子、金银花和黄芩)对肝细胞膜转运体多药耐药相关蛋白2(Multidrugresistance-associated protein 2,Mrp2/ABCC2)、Na+-牛黄胆酸共转运多肽(Na+/taurocholate cotransporter,Ntcp/SLC10A1)及胆盐输出泵(Bile salt export pump,Bsep/ABCB11)表达的影响。方法胆管结扎术制备胆汁淤积大鼠模型;生化检测和HE染色,观察茵栀黄颗粒的干预效果;流式细胞仪测定肝脏转运体Ntcp、Bsep和Mrp2的表达。结果与模型组相比,茵栀黄组肝细胞的脂肪病变和水肿较模型组显著减轻(P<0.05),Mrp2的表达显著升高(P<0.01);但Ntcp、Bsep的表达无明显变化。结论肝细胞膜转运体Mrp2的表达升高可能是茵栀黄颗粒的退黄利胆分子机制之一。
文摘目的通过测定孕酮、苯甲酸雌二醇诱发的孕鼠肝内胆汁淤积症模型肝脏、胎盘组织中法尼醇受体(farnesoid X receptor,FXR)及胆盐输出泵(bile salt export pump,BSEP)的表达情况,探讨雌孕激素诱导妊娠期肝内胆汁淤积症(Intrahepatic cholestasis of pregnancy,ICP)过程中FXR和BSEP的作用机制。方法孕15dSD大鼠60只随机分成对照组、实验组(A组、B组),分别给予生理盐水,孕酮,苯甲酸雌二醇。于用药前、用药后测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、总胆酸(TBA)总胆红素(TBIL)、直接胆红素(DBIL)、间接胆红素(IBIL)水平;测定胎鼠生长发育情况;H.E染色观察肝脏、胎盘组织形态学变化,免疫组化法测定肝脏及胎盘BSEP、FXR的表达情况。结果用药后,实验组各项血清生化指标明显高于对照组水平(P<0.05);对照组用药前后各项血清生化指标无明显变化(P>0.05);实验组用药后各项血清生化指标较用药前显著升高(P<0.05)实验组胎鼠体质量、死胎数与对照组相比较有显著差异(P<0.05)。H.E染色观察到A、B组孕鼠肝脏出现肝内胆汁淤积典型表现。免疫组化法检测到,实验组肝脏FXR表达升高,BSEP表达降低,与对照组相比差异具有显著性(P<0.05)。此外,在孕鼠胎盘也发现了少量FXR和BSEP的表达。结论雌激素具有明确的诱发孕鼠肝内胆汁淤积症的作用,孕激素也可诱导孕鼠肝内胆汁淤积症。雌激素诱导的机制可能是通过上调肝脏FXR的表达及下调肝脏BSEP的表达来影响胆汁酸的合成与代谢,进而导致了肝内胆汁淤积的发生;孕激素引起ICP,可能因为某些孕酮代谢产物诱导了BSEP的传输抑制作用致胆汁酸淤积;同时孕酮代谢障碍造成非结合胆红素增高也是引起胆汁淤积的原因之一。孕激素可以强化雌激素诱导ICP的作用。
