AIM： To characterize H^＋ and HCO3^- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the AF508 CFTR mutation. METHODS： CFPAC-1 cells were seeded ...AIM： To characterize H^＋ and HCO3^- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the AF508 CFTR mutation. METHODS： CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells. RESULTS： Our results showed differential permeability to HCO3^＋/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na^＋/HCO3^- co-transporters （NBCs） and Cl^-/HCO3^- exchangers （AEs） were present on the basolateral membrane, and Na＋/H＋ exchangers （NHEs） on both the apical and basolateral membranes of the cells. Basolateral HCO3 uptake was sensitive to variations of extracellular K^＋ concentration, the membrane permeable carbonic anhydrase （CA） inhibitors acetazolamide （100 μmol/L） and ethoxyzolamide （100 μmol/L）, and was partially inhibited by H2-DIDS （600 μmol/L）. The membrane-impermeable CA inhibitor 1-N-（4-sulfamoylphenylethyl）-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3^- uptake.The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 μmol/L forskolin did not significantly influence Cl^-/HCO3^- exchange on the apical and basolateral membranes. The administration of 250 μmol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride （300 μmol/L） completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA. CONCLUSION： These data suggest that apart from the lack of CFTR and apical Cl^-/HCO3^- exchanger activity, CFPAC-1 cells express similar H^＋ and HCO3^- transporters to those observed in native animal tissue.展开更多
目的探讨马钱子碱对人胰腺癌CFPAC-1细胞凋亡的影响及其可能机制。方法以不同浓度马钱子碱干预CFPAC-1细胞。采用MTT比色法和流式细胞仪检测细胞增殖和凋亡;采用花青染料(JC-1)染色法检测细胞线粒体膜电位的变化;采用蛋白质印迹法检测Ba...目的探讨马钱子碱对人胰腺癌CFPAC-1细胞凋亡的影响及其可能机制。方法以不同浓度马钱子碱干预CFPAC-1细胞。采用MTT比色法和流式细胞仪检测细胞增殖和凋亡;采用花青染料(JC-1)染色法检测细胞线粒体膜电位的变化;采用蛋白质印迹法检测Bax、B cl-2蛋白表达情况。结果〇(对照组)及〇.4、0.8 m m ol/L马钱子碱组干预CFPAC-1细胞24、48、72 h后,细胞增殖抑制率分别为0;(30.23±0.55)%、(40.61±0.15)%、(46.98±1.27)%;(50.17±0.75)%、(61.23±0.91)%、(70.32±0.40)%,呈浓度及时间依赖性增加而升高,显著高于对照组,且不同时间两两组间比较差异均有统计学意义(/>值均<〇.〇5)。〇及0.4、0.8 mmol/L马钱子碱干预CFPAC-1细胞48 h后,细胞凋亡率分别为(2.92±0.46)%、(4.64±1.31)%、(13.09±0.65)%,随药物浓度增加而逐渐升高,其中0.8 mm ol/L干预组显著高于对照组,差异有统计学意义(P<0.05);随着药物浓度的增加,呈红色荧光的未凋亡活细胞逐渐减少,而呈绿色荧光的凋亡坏死细胞逐渐增多,提示CFPAC-1细胞线粒体膜电位受到重度破坏而降低;CFPAC-1细胞的B d-2蛋白表达量分别为(0.92±0.12)、(0.67±0.14)、(0.35±0.14)mmol/L,Bax蛋白表达量分别为(0.56±0.12)、(0•85±0.10)、(1.15±0.12)m m ol/L,随马钱子碱浓度增加,Bcl-2表达量显著下调,而Bax表达量显著上调,差异均有统计学意义(P值均<0.05)。结论马钱子碱可能通过线粒体凋亡途径上调Bax和下调Bcl-2表达,从而诱导人胰腺癌CFPAC-1细胞凋亡。展开更多
基金Supported by a Wellcome Trust Travelling Fellowship to Z.R.,No.069470 a Wellcome Trust IRDA Grant to P.H.,No.068096
文摘AIM： To characterize H^＋ and HCO3^- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the AF508 CFTR mutation. METHODS： CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells. RESULTS： Our results showed differential permeability to HCO3^＋/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na^＋/HCO3^- co-transporters （NBCs） and Cl^-/HCO3^- exchangers （AEs） were present on the basolateral membrane, and Na＋/H＋ exchangers （NHEs） on both the apical and basolateral membranes of the cells. Basolateral HCO3 uptake was sensitive to variations of extracellular K^＋ concentration, the membrane permeable carbonic anhydrase （CA） inhibitors acetazolamide （100 μmol/L） and ethoxyzolamide （100 μmol/L）, and was partially inhibited by H2-DIDS （600 μmol/L）. The membrane-impermeable CA inhibitor 1-N-（4-sulfamoylphenylethyl）-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3^- uptake.The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 μmol/L forskolin did not significantly influence Cl^-/HCO3^- exchange on the apical and basolateral membranes. The administration of 250 μmol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride （300 μmol/L） completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA. CONCLUSION： These data suggest that apart from the lack of CFTR and apical Cl^-/HCO3^- exchanger activity, CFPAC-1 cells express similar H^＋ and HCO3^- transporters to those observed in native animal tissue.
文摘目的探讨马钱子碱对人胰腺癌CFPAC-1细胞凋亡的影响及其可能机制。方法以不同浓度马钱子碱干预CFPAC-1细胞。采用MTT比色法和流式细胞仪检测细胞增殖和凋亡;采用花青染料(JC-1)染色法检测细胞线粒体膜电位的变化;采用蛋白质印迹法检测Bax、B cl-2蛋白表达情况。结果〇(对照组)及〇.4、0.8 m m ol/L马钱子碱组干预CFPAC-1细胞24、48、72 h后,细胞增殖抑制率分别为0;(30.23±0.55)%、(40.61±0.15)%、(46.98±1.27)%;(50.17±0.75)%、(61.23±0.91)%、(70.32±0.40)%,呈浓度及时间依赖性增加而升高,显著高于对照组,且不同时间两两组间比较差异均有统计学意义(/>值均<〇.〇5)。〇及0.4、0.8 mmol/L马钱子碱干预CFPAC-1细胞48 h后,细胞凋亡率分别为(2.92±0.46)%、(4.64±1.31)%、(13.09±0.65)%,随药物浓度增加而逐渐升高,其中0.8 mm ol/L干预组显著高于对照组,差异有统计学意义(P<0.05);随着药物浓度的增加,呈红色荧光的未凋亡活细胞逐渐减少,而呈绿色荧光的凋亡坏死细胞逐渐增多,提示CFPAC-1细胞线粒体膜电位受到重度破坏而降低;CFPAC-1细胞的B d-2蛋白表达量分别为(0.92±0.12)、(0.67±0.14)、(0.35±0.14)mmol/L,Bax蛋白表达量分别为(0.56±0.12)、(0•85±0.10)、(1.15±0.12)m m ol/L,随马钱子碱浓度增加,Bcl-2表达量显著下调,而Bax表达量显著上调,差异均有统计学意义(P值均<0.05)。结论马钱子碱可能通过线粒体凋亡途径上调Bax和下调Bcl-2表达,从而诱导人胰腺癌CFPAC-1细胞凋亡。
