目的检测正常大鼠肝脏不同部位胆管细胞胆汁酸转运蛋白表达的差异。方法用酶消化辅助机械分离方法获得SD大鼠胆管组织并分为大、小胆管组织,应用免疫组织化学、Real time PCR及Western blot等方法检测大鼠胆道系统顶侧钠依赖性胆汁酸转...目的检测正常大鼠肝脏不同部位胆管细胞胆汁酸转运蛋白表达的差异。方法用酶消化辅助机械分离方法获得SD大鼠胆管组织并分为大、小胆管组织,应用免疫组织化学、Real time PCR及Western blot等方法检测大鼠胆道系统顶侧钠依赖性胆汁酸转运体(ASBT)、回肠胆汁酸结合蛋白(IBABP)和基底侧有机溶质转运体α(OSTα)表达,分析其定位及分布的差异。结果 ASBT表达于胆管细胞顶侧胞膜,IBABP表达于细胞浆,Ostα表达于胆管细胞基底侧胞膜。ASBT和IBABP主要表达于大胆管上皮细胞,大胆管组织中ASBT和IBABP的表达量显著高于小胆管组织(P<0.05);而Ostα则同时表达于大胆管和小胆管上皮细胞,其表达量在大、小胆管组织中无显著差异(P>0.05)。结论胆管细胞对胆汁酸的重吸收主要发生于大胆管。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
目的探讨胆汁酸盐输出泵(bile salt export pump,BSEP)、多药耐药相关蛋白2(multidrug resistant protein 2,MRP2)和多药耐药糖蛋白3(multidrug resistance associated protein 3,MDR3)在原发性胆汁性胆管炎(primary biliary cholangiti...目的探讨胆汁酸盐输出泵(bile salt export pump,BSEP)、多药耐药相关蛋白2(multidrug resistant protein 2,MRP2)和多药耐药糖蛋白3(multidrug resistance associated protein 3,MDR3)在原发性胆汁性胆管炎(primary biliary cholangitis,PBC)患者肝组织中的表达特点。方法收集2009年1月至2019年12月于南昌市第九医院住院且经肝组织病理诊断为PBC的46例患者临床资料,根据PBC严重程度分为PBC早期组(Ⅰ~Ⅱ期,31例)和PBC晚期组(Ⅲ~Ⅳ期,15例),比较两组患者血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate transaminase,AST)、总胆汁酸(total bile acid,TBA)、总胆红素(total bilirubin,TBil)、直接胆红素(direct bilirubin,DBil)、碱性磷酸酶(alkaline phosphatase,ALP)、γ-谷氨酰转移酶(gamma-glutamyltransferase,GGT)、高密度脂蛋白(high-density lipoprotein,HDL)、总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白(low-density lipoprotein,LDL)等的差异。选取10例慢性乙型肝炎病毒(hepatitis B virus,HBV)携带者作为对照组。对所有入选病例肝组织进行BSEP、MDR3、MRP2免疫组织化学标记,观察各组肝组织病理形态及3种转运蛋白表达差异。结果PBC晚期组患者血清ALP(中位数:404 U/L vs 281 U/L)、GGT(中位数:437 U/L vs 245 U/L)、TC(中位数:6.58 mg/L vs 4.50 mg/L)、TG(中位数:1.72 mg/L vs 1.24 mg/L)、LDL(中位数:3.61 mg/L vs 2.27 mg/L)水平均显著低于PBC早期组,差异有统计学意义(P均<0.05)。两组患者年龄、性别、血清ALT、AST、TBA、TBil、DBil、HDL水平及AMA阳性率差异均无统计学意义(P均>0.05)。与PBC早期组相比,PBC晚期组患者炎症活动度和纤维化程度均较重,差异有统计学意义(χ^(2)=14.71,P=0.0006;χ^(2)=20.57,P<0.001)。PBC早期组与PBC晚期组患者肝细胞CK7和CK19染色阳性率无统计学差异[54.84%(17/31)vs 46.67%(7/15),χ^(2)=0.271、P=0.755;74.19%(23/31)vs 86.67%(13/15),连续校正χ^(2)=0.337、P=0.562]。PBC组患者肝组织中BSEP高表达率显著低于对照组[54.76%(23/42)vs 100.00%(10/10);χ^(2)=5.311,P=0.021],MDR3和MRP2高表达率差异无统计学意义[91.18%(31/34)vs 100.00%(10/10),P=1.000;69.7%(23/33)vs 100.00%(10/10);χ^(2)=2.433,P=0.119]。PBC晚期组患者BSEP高表达率显著低于PBC早期组[68.97%(20/29)vs 23.08%(3/13);χ^(2)=7.630,P=0.008],MDR3和MRP2阳性高表达率差异无统计学意义[91.30%(21/23)vs 90.91%(10/11),P=1.000;68.18%(15/22)vs 72.73%(8/11),P=1.000]。BSEP、MRP2、MDR3在胆汁淤积区阳性表达减少越显著,肝细胞胆汁淤积肿胀及羽毛样变性越明显。结论BSEP在PBC患者肝组织中表达降低,且在PBC晚期组患者肝组织表达率显著降低,提示PBC胆汁淤积与毛细胆管膜侧BSEP蛋白表达缺陷有关,并且PBC疾病进展可能与BSEP表达减少有关。展开更多
Mutations of the bile salt export pump (BSEP) or the multidrug resistance P-glycoprotein 3 (MDR3) are linked to impaired bile salt homeostasis and lead to progressive familial intrahepatic cholestasis (PFIC)-2 and -3,...Mutations of the bile salt export pump (BSEP) or the multidrug resistance P-glycoprotein 3 (MDR3) are linked to impaired bile salt homeostasis and lead to progressive familial intrahepatic cholestasis (PFIC)-2 and -3, respectively. The regulation of bile salt transporters in PFIC is not known. Expression of hepatobiliary transporters in livers of ten patients with a PFIC phenotype was studied by quantitative reverse transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy. PFIC was diagnosed by clinical and laboratory findings. All patients could be assigned to PFIC-2 or PFIC-3 by the use of BSEP-and MDR3-specific antibodies and by MDR3 gene-sequencing. Whereas in all PFIC-2 patients, BSEP immunoreactivity was absent from the canalicular membrane, in three PFIC-3 livers, canalicular MDR3 immunoreactivity was detectable. Serum bile salts were elevated to 276 ±233 and to 221 ±109 μmol/L in PFIC-2 and PFIC-3, respectively. Organic anion transporting polypeptide OATP1B1, OATP1B3, and MRP2 mRNA and protein levels were reduced, whereas sodium taurocholate cotransporting polypeptide (NTCP) was only reduced at the protein level, suggesting a posttranscriptional NTCP regulation. Whereas MRP3 mRNA and protein were not significantly altered, MRP4 messenger RNA and protein were significantly increased in PFIC. In conclusion, PFIC-2 may be reliably diagnosed by immunofluorescence, whereas the diagnosis of PFIC-3 requires gene-sequencing. Several mechanisms may contribute to elevated plasma bile salts in PFIC: reduced bile salt uptake via NTCP, OATP1B1, and OATP1B3, decreased BSEP-dependent secretion into bile, and increased transport back into plasma by MRP4. Upregulation of MRP4, but not of MRP3, might represent an important escape mechanism for bile salt extrusion in PFIC.展开更多
文摘目的检测正常大鼠肝脏不同部位胆管细胞胆汁酸转运蛋白表达的差异。方法用酶消化辅助机械分离方法获得SD大鼠胆管组织并分为大、小胆管组织,应用免疫组织化学、Real time PCR及Western blot等方法检测大鼠胆道系统顶侧钠依赖性胆汁酸转运体(ASBT)、回肠胆汁酸结合蛋白(IBABP)和基底侧有机溶质转运体α(OSTα)表达,分析其定位及分布的差异。结果 ASBT表达于胆管细胞顶侧胞膜,IBABP表达于细胞浆,Ostα表达于胆管细胞基底侧胞膜。ASBT和IBABP主要表达于大胆管上皮细胞,大胆管组织中ASBT和IBABP的表达量显著高于小胆管组织(P<0.05);而Ostα则同时表达于大胆管和小胆管上皮细胞,其表达量在大、小胆管组织中无显著差异(P>0.05)。结论胆管细胞对胆汁酸的重吸收主要发生于大胆管。
文摘Mutations of the bile salt export pump (BSEP) or the multidrug resistance P-glycoprotein 3 (MDR3) are linked to impaired bile salt homeostasis and lead to progressive familial intrahepatic cholestasis (PFIC)-2 and -3, respectively. The regulation of bile salt transporters in PFIC is not known. Expression of hepatobiliary transporters in livers of ten patients with a PFIC phenotype was studied by quantitative reverse transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy. PFIC was diagnosed by clinical and laboratory findings. All patients could be assigned to PFIC-2 or PFIC-3 by the use of BSEP-and MDR3-specific antibodies and by MDR3 gene-sequencing. Whereas in all PFIC-2 patients, BSEP immunoreactivity was absent from the canalicular membrane, in three PFIC-3 livers, canalicular MDR3 immunoreactivity was detectable. Serum bile salts were elevated to 276 ±233 and to 221 ±109 μmol/L in PFIC-2 and PFIC-3, respectively. Organic anion transporting polypeptide OATP1B1, OATP1B3, and MRP2 mRNA and protein levels were reduced, whereas sodium taurocholate cotransporting polypeptide (NTCP) was only reduced at the protein level, suggesting a posttranscriptional NTCP regulation. Whereas MRP3 mRNA and protein were not significantly altered, MRP4 messenger RNA and protein were significantly increased in PFIC. In conclusion, PFIC-2 may be reliably diagnosed by immunofluorescence, whereas the diagnosis of PFIC-3 requires gene-sequencing. Several mechanisms may contribute to elevated plasma bile salts in PFIC: reduced bile salt uptake via NTCP, OATP1B1, and OATP1B3, decreased BSEP-dependent secretion into bile, and increased transport back into plasma by MRP4. Upregulation of MRP4, but not of MRP3, might represent an important escape mechanism for bile salt extrusion in PFIC.
