猪囊性纤维化跨膜电导调节因子及其△F508突变体的细胞生物学和分子药理学研究

详细信息    本馆镜像全文|  推荐本文 |  |   获取CNKI官网全文

英文题名：Cell Biology and Molecular Pharmacology of Pig Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and △F508-pCFTR 作者：刘艳丽 论文级别：博士 学科专业名称：细胞生物学 中文关键词：囊性纤维化跨膜电导调节因子(CFTR) ; 囊性纤维化(CF) ; 动物模型 ; 抑制剂 英文关键词：cystic fibrosis transmembrane conductance regulator (CFTR) ; cystic fibrosis (CF) ; animal models ; inhibitor 学位年度：2008 导师：麻彤辉 学科代码：071009 学位授予单位：东北师范大学 论文提交日期：2008-10-01

摘要

CFTR(cystic fibrosis transmembrane conductance regulator,CFTR)即囊性纤维化跨膜电导调节因子,是一种cAMP依赖的Cl~-通道,广泛表达于呼吸道、胰腺、胃肠道、睾丸、汗腺和唾液腺等多种分泌型和吸收型上皮,主要参与电解质、液体转运以及调节其它膜通道蛋白的功能。其基因突变导致白种人中常见的致死性常染色体隐性遗传疾病即囊性纤维化病(cystic fibrosis,CF)。其中最为常见的突变类型是AF508(第508位氨基酸苯丙氨酸缺失),有90%的CF病人其一条染色体都携有AF508突变。CF疾病的主要特征是反复的肺部感染、结构破坏和纤维修复,最终导致肺功能不可逆性的丧失,这也是CF疾病高发病率和致死性的主要原因。由于缺少合适的动物模型,CF发病机理并不清楚。尽管在CFTR基因克隆后,建立了许多CF小鼠转基因模型,并且这些小鼠也重现了CF病人的肠道病变。但由于小鼠与人在气道细胞生物学、粘膜下腺表达量以及小鼠气道可能存在其它的非CFTR介导的氯离子转运,CF转基因小鼠模型未能表现作为CF病人主要致死原因的肺部病变。因此,需要寻找其它的动物建立CF模型。
     猪在解剖学、生理学上和人有极大的相似性,比较适合建立CF动物模型。此外,由于猪具有较长的寿命,使得对于CF肺疾病的发生、治疗手段的长期治疗效果以及副作用的研究成为可能。现在很多研究者致力于构建猪CFTR基因敲除猪和AF508突变猪CF模型。但由于基因敲除猪也可能发生类似于基因敲除鼠的肠梗阻,在未发生肺部疾病前过早死亡,使后续研究难以进行。此外,我们实验室和美国Ostedgaad等的近期研究发现,猪AF508-CFTR在稳定转染细胞模型中能够正常加工成熟转运到质膜,提示猪AF508转基因模型将不会出现预期的表型。用CFTR特异性抑制剂诱导猪CF模型是另一个可行的策略。但现今发现的人CFTR特异性抑制剂CFTR_(inh)-172对猪CFTR的亲和力差,抑制效果并不明显,并且本实验室前期研究发现,该药不能诱导猪CF模型的产生。另一种CFTR抑制剂GlyH-101虽然对猪有较好的抑制效果,但其在动物体内分布和代谢情况并不清楚,是否适合用来做药物诱导猪CF模型也不清楚。因此,需要寻找高亲和力、高特异性、在猪体内主要CF病变器官分布较高的猪CFTR抑制剂来诱导猪CF模型。
     本文的目的是系统研究猪△F508-CFTR的细胞生物学和电生理特征,并通过高通量筛选发现新的猪CFTR特异性、高亲和力抑制剂,为建立猪CF药理模型奠定基础。首先利用PCR方法我们获得了猪CFTR基因,并将其连入真核表达载体pcDNA3.1Zeo。对猪CFTR基因进行点突变,获得了猪△F508-CFTR,也将其连入真核表达载体pcDNA3.1Zeo。将这两个构建好的重组质粒与对碘离子敏感的绿色荧光蛋白突变体EYFP-H148Q-1152L稳定转染FRT(fisher大鼠甲状腺上皮细胞),建立了猪CFTR高通量筛选细胞模型。利用自己制备的针对猪CFTR NBD2结构域的多克隆抗体,对瞬时及稳定转染野生型和△F508-pCFTR的Cos7细胞及FRT细胞进行免疫印记及免疫荧光分析,发现△F508-pCFTR蛋白大部分(77.8%)都具有成熟型糖基化形式,能够被正常转运上膜。通过Cl~-电流荧光测定、短路电流分析以及全细胞膜片钳电流测定,发现△F508-pCFTR具有依赖于cAMP激活的Cl~-转运功能。较野生型相比,△F508-pCFTR对cAMP激动剂forskolin的敏感性降低10倍(EC50 5μM vs EC50 0.5pM);其forskolin激活的最大电流为野生型的61%;对人CFTR抑制剂CFTR_(inh)-172和GlyH-101的灵敏性更高,1μM CFTR_(inh)-172对△F508-pCFTR和野生型CFTR的抑制百分率分别为40%和20%。这些结果都表明猪△F508突变是一种“温和性”突变,突变蛋白能正常转运到达顶质膜,只是功能较野生型有所降低。以往的研究表明,10%的人△F508-CFTR转运到达顶质膜就可以避免CF症状的发生,因此△F508转基因猪CF模型不会出现预期表型。
     此外,对GlyH-101在小鼠体内的分布、代谢情况进行了初步研究,发现其在小鼠肺内分布并不明显,并很快被清除干净。据此我们推断其在猪体内也具有类似分布代谢情况,提示GlyH-101并不适合作为药物来诱导猪CF模型。因此,需要寻找对猪更有效的特异性抑制剂。
     利用构建的FRT/EYFP-H148Q-I152L/pCFTR稳定转染细胞模型,通过高通量筛选技术对含10万个结构多样的小分子组合化学库进行抑制剂的筛选,发现了3个新的猪CFTR特异性抑制剂。其中活性最好的PI1,能够以剂量依赖的方式抑制FRT单层上皮细胞CPT-cAMP激活的猪CFTR短路电流,其IC50为13.34μM,且这种作用具有快速、可逆、无毒的特点。在小鼠体内活性试验中,可有效抑制霍乱毒素诱导的肠道液体分泌(80%)。
     通过以上研究,进一步证实猪△F508突变CFTR的细胞生物学行为与人△F508-pCFTR有显著差异,提示猪△F508转基因模型将不会出现预期的表型。此外,我们对10万个小分子组合化学库筛选,发现了3个新结构的猪CFTR抑制剂,但其亲和力不高,提示我们从小分子组合化学库获得猪CFTR高亲和力的抑制剂可能性不大。下一步计划对本实验室近期从500种常用中草药建立的一个含40000个组分的小分子库进行系统筛选,从结构更为复杂多样的天然产物中寻找猪CFTR的高亲和力、特异性抑制剂。
The cystic fibrosis transmembrane conductance regulator(CFTR) is a cAMP-activated Cl~- channel expressed in secretory and absorptive epithelia in the airways,pancreas, gastrointestinal tract,testis,sweat gland,salivary gland and other tissues.It is involved in transport of electrolyte,fluid and regulation of other membrane channel proteins.Mutations in CFTR cause cystic fibrosis(CF),the most common lethal autosomal recessive genetic disease involving multisystem disorders in the Caucasian population.Deletion of the codon encoding the phenylalanine residue at position 508(ΔF508) in CFTR is the most common mutation of CF and appears in one allele of～90%CF patients.The disease is characterized by recurrent lung infections with opportunistic pathogens,which results in progressive and irreversible loss of lung function.The airway disease is currently the source of most CF morbidity and mortality.A major impediment to understand CF pathogenesis and to develop new treatment is the limitation of current animal models.Although many lines of CF mice have been generated by targeting the mouse CFTR gene after cloning of CFTR,these CF mouse models manifest some intestinal phenotypes of CF patients.They have failed to develop the spontaneous lung infections seen in patients with CF.This failure may be due to differences in airway cell biology,the abundance of submucosal glands in the airway,and the alternative expression or activation of pathways that control non-CFTR Cl~- channels in airways.This has prompted researchers in the CF field to attempt to identify or generate models for human CF in other animals.The pig represents another potentially useful alternative species for generating a CF model due to the similarities between its lung anatomical and physiological features and human lung's.Further more,the longevity of pigs also offers opportunities for investigating the pathogenesis of lung disease,the long-term therapeutic efficacy of treatments and adverse effects of interventions that might only become apparent with time. Recent advances were reported in generating transgenic pig models withΔF508 and null mutations of CFTR.However,the null mutation of CF pig maybe die from intestinal obstruction at early age like the CF mice and this presents a technical challenge when trying to use these pigs for experimentation.In another way,a recent study reported by Osedgaad et al indicated partial cellular processing and significant plasma membrane targeting of porcineΔF508-CFTR,raising cautions on the phenotypic consequences of theΔF508 pig model.Then there is considerable interest in development pig CF models by using pig CFTR specific, high-affinity inhibitors.However,the human CFTR inhibitor CFTR_(inh)-172 produced a significantly lower level of inhibition of pig CFTR and our previous work failed to create pig CF models by in vivo administration of CFTR_(inh)-172.Another specific human CFTR inhibitor GlyH101 is also a potent inhibitor of pig CFTR,but its organ distribution in animals is not clear.Therefore we are not sure if GlyH101 is suitable for developing CF pig models.What we need is a high-affinity,specific inhibitor of pig CFTR to create CF lung phenotypes in pig.
     The purpose of this study was to characterize the biochemical and electrophysiological features of pigΔF508-CFTR and to discover new pig CFTR inhibitors by high throughput screening.
     Firstly,we cloned the cDNA sequence encoding full-length porcine CFTR(pCFTR) by RT-PCR and subcloned it to expression vector pcDNA3.1Zeo to form recombinant plasmid pcDNA3.1Zeo/pCFTR.TheΔF508 mutation of pig CFTR was generated in pcDNA3.1ZeopCFTR by site-directed mutagenesis to form pcDNA3.1ZeoΔF508-pCFTR using a PCR-based strategy.Then we established stably cotransfected FRT cell lines coexpressing the wildtype or mutant pig CFTRs and iodide-sensitive green fluorescent analog EYFP-H148Q-I152L as cell-based assay models for functional analysis and high-throughput screening.Western blot analysis and immunofluorescence of Cos7 and FRT cell by using the generated pig CFTR polyclonal antibody against the NBD2 domain revealed predominant plasma membrane targeting(77.8%) ofΔF508-pCFTR protein.Functinal measurements by fluorometric assay,short circuit current assay and whole-cell patch-clamp assay indicated thatΔF508-pCFTR produced a cAMP-activated Cl~- current.However,the low sensitivity ofΔF508-pCFTR to cAMP agonist forskolin and high sensitivity to CFTR inhibitor suggested reduced channel activity ofΔF508-pCFTR.It was estimated that plasma membrane targeting of 10%ΔF508-CFTR protein could avoid the CF phenotype in human subjects.The mild processing defect of porcineΔF508-CFTR arouses suspicion that the ongoing transgenicΔF508-CFTR pig model may not develop desired CF phenotypes.
     Secondly,we analyze the distribution of GlyH101 in mouse tissues and found low distribution and rapid clearance of GlyH101 in mouse lung after intraperitoneal injection, which suggested that GlyH101 is not suitable for developing pig CF models.
     Lastly,we performed high-throughput screening of 100,000 synthetic small molecules to identify new chemical scaffolds with pig CFTR inhibitory activity.We obtained 3 new pig CFTR inhibitors PI1,PI2 and PI3.The most potent CFTR inhibitor was PI1 with IC50 value about 14μM.The inhibition was rapid,reversible,and nontoxic.Further more,it could inhibit mouse intestinal fluid secretion induced by cholera toxin.
     The present study confirmed the species difference in cellular processing ofΔF508-CFTR.However,our results indicated rather mild processing defect ofΔF508-pCFTR. It was estimated that plasma membrane targeting of 10%ΔF508-CFTR protein could avoid the CF phenotype in human subjects.The mild processing defect of porcineΔF508-CFTR arouses suspicion that the ongoing transgenicΔF508-CFTR pig model may not develop desired CF phenotypes.
     We identified 3 new pig CFTR inhibitors from 100,000 synthetic small molecules. However,the poor affinity of these inhibitors potentially precluded their usefulness in generating CF phenotypes in pig.Future direction to discover potent pig CFTR inhibitors will involve screening of a natural compounds library recently established in our laboratory to identify high-affinity pig CFTR inhibitors instead of small molecules.

引文

[1] Rommens J M, Iannuzzi M C, Kerem B, et al. Identification of the cystic fibrosis gene:Chromosome walking and jumping[J]. Science, 1989, 245: 1059-1065.
    
    [2] Riordan J R, Rommens J M, Kerem B, et al. Identification of the cystic fibrosis gene:Cloning and characterization of complementary DNA[J]. Science, 1989, 245: 1066-1073.
    
    [3] Kerem B, Rommens J M, Buchanan J A, et al. Identification of the cystic fibrosis gene:Genetic analysis[J]. Science, 1989, 245: 1073-1080. [4] Romeo G, Devoto M, Galietta LJV. Why is the cystic fibrosis gene so frequent? Hum Genet. 1989, 84: 1-5.
    [5] Zielenski J, Rozmahel R, Bozon D, et al. Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene[J]. Genomics, 1991, 10: 214-228.
    [6] Sheppard D N, Rich D P, Ostedgaard L S, et al. Mutations in CFTR associated with mild disease form C10 channels with altered pore properties[J]. Nature, 1993, 362: 160-164.
    [7] Mcdonough S, Davidson N, Lester H A, et al. Novel pore ation and open-channel block[J]. Neuron, 1994, 13: 623-634.
    [8] Cheung M, Akabas M H. Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment[J]. Biophys J, 1996, 70: 2688-2695.
    [9] Ishihar H, Wesh M J Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis[J]. Am J Physiol, 1997, 273(Cell Physiol 42):1278-1289.
    [10] Linsdell P, Tabcharani J A, Hanrahan J W. Multiion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel [J]. J Gen Physiol, 1997, 110: 365-377.
    [11] Linsdell P, Zheng S X, Hanrahan J W. Possible contribution of the peptide backbone to CFTR anion selectivity (Abstract)[J]. Pediatr. Pulmonol, 1997, 14: 214.
    [12] Mansoura M K, Smith S S, Choi A D, et al. Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore[J]. Biophys J, 1998, 74: 1320-1332.
    [13] Tabcharani J A, Linsdell A P, Hanrahan J W. Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels[J] . J Gen Physiol, 1997, 110: 341-354.
    [14] Anderson M P, Berger H A, RICH D P, et al. Nucleoside triphosphates are required to open the CFTR chloride channel[J]. Cell, 1991, 67: 775-784.
    [15] Hwang T C, Nagel G, Nairn A C, et al. Regulation of the gating of CFTR Cl channels by phosphorylation and ATP hydrolysis[J]. Proc Natl Acad Sci. USA, 1994, 91: 4698-4702.
    [16] Baukrowitz T, Hwang T C, Nairn A C, et al. Coupling of CFTR Cl channel gating to an ATP hydrolysis cycle[J]. Neuron, 1994, 12: 473-482.
    [17] Venglarik C J, Schultz B D, Frizzell R A, et al. ATP alters current fluctuations of cystic fibrosis transconductance regulator: evidence for a three-state activation mechanism[J]. J Gen Physiol, 1994, 104: 123-146.
    [18] Carson MR, Travis SM, Welsh MJ. The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity[J]. J Biol Chem, 1995, 270: 1711-1717.
    [19] Ko Y H, Pederson P L. The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase[J]. J Biol Chem,1995, 270: 22093-22096.
    [20] Gunderson K L, Kopito R R. Conformational states of CFTR associated with channel gating:the role ATP binding and hydrolysis[J]. Cell, 1995, 82: 231-239.
    [21] Li C, Ramjeesingh M, Wang W, et al. ATPase activity of the cystic fibrosis transmembrane conductance regulator[J]. J Biol Chem, 1996, 271: 28463-28468.
    [22] Baukrowitz T, Hwang T C, Nairn A C, et al. Coupling of CFTR Cl channel gating to an ATP hydrolysis cycle[J]. Neuron, 1994, 12: 473-482.
    [23] Gadsby D C, Nagel G A, Hwang T C. The CFTR chloride channel of mammalian heart [J]. Annu Rev Physiol, 1995, 57: 387-416.
    [24] Gadsby D C, Nairn A C. Regulation of CFTR channel gating[J]. Trends BioChemSci, 1994,19: 513-518.
    [25] Carson M R, Travis SM, Welsh M J The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity[J]. J Biol Chem, 1995, 270: 1711-1717.
    [26] Smith S S , Steinle E D, Pecoraro V, et al. CFTR: probing the pore with permeantions[J], Pediatr Pulmonol, 1997, 14: 220.
    [27] Berger H A, Anderson M P, Gregory R J, et al. Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel[J]. J Clin Invest, 1991. 88: 1422-1431.
    [28] Picciotto M R, Cohn J A, Bertuzzi G, et al. Phosphorylation of the cystic fibrosis transmembrane conductance regulator[J]. J Biol Chem, 1992, 267: 12742-12752.
    [29] Chan X B, Tabcharani J A, Hou Y X, et al. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites[J]. J Biol Chem, 1993, 268: 11304-11311.
    [30] Hwang T C, Horie M, Gadsby D C. Functionally distinct phospho-f orms underlie incremental activation of protein kinase regulated C10 conductance in mammalian heart[J]. J Gen Physiol, 1993, 101: 629-650.
    [31] Rich D P, Berger H A, Cheng S H, et al. Regulation of the cystic fibrosis transmembrane conductance regulator Cl" channel by negative charge in the R domain[J]. J Biol Chem268:20259-20267, 1993.
    [32] Wilkinson D J, Strong T V, Mansoura M K, et al. CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain[J]. Am J Physiol,1997, 273 (Lung Cell. Mol. Physiol 17):127-133.
    [33] Winter M C , Welsh M J Stimulation of CFTR activity by its phosphorylated Rdomain[J]. Nature, 1997, 389: 294-296.
    [34] Picciotto M R, Cohn J A, Bertuzzi G, et al. Phosphorylation of the cystic fibrosis transmembrane conductance regulator[J]. J Biol Chem, 1992, 267: 12742-12752.
    [35] Berger H A, Anderson M P, Gregory RJ, et al. Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel[J]. J Clin Invest, 1991, 88: 1422- 1431.
    [36] Chan X B, Tabcharani J A, Hou Y X, et al. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites[J]. J Biol Chem, 1993, 268: 11304-11311.
    [37] Hwang T C, HorieM, Gadsby D C. Functionally distinct phospho-forms underlie incremental activation of protein kinase regulated Cl~- conductance in mammalian heart[J]. J Gen Physiol, 1993, 101: 629-650.
    [38] Rich D P, Berger H A, Cheng S H, et al. Regulation of the cystic fibrosis transmembrane conductance regulator Cl channel by negative charge in the R domain[J]. J Biol Chem,1993, 268: 20259-20267.
    
    [39] Tabcharani J A, Chang X B, Riodan J R, et al. Phosphorylation-regulated Cl channel in CHO cells stably expressing the cystic fibrosis gene[J]. Nature, 1991, 352: 628-631.
    
    [40] Wilkinson D J, Strong T V, Mansoura M K, et al. CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain[J]. Am J Physiol,1997, 273(Lung Cell Mol Physiol 17): 127-133.
    
    [41] BergerHA, Rich D P, et al. Regulation of the cystic fibrosis transmembrane conductance regulator C10 channel by negative charge in the R domain[J]. Jpn J Physiol, 1994,44, 2: S167-S172
    [42] Ma J, Zhao J, et al. Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel[J]. J Biol Chem, 1997, 272: 28133-28141.
    [43] Rich D P, Gregory R J, et al. Effect of deleting the R domain on CFTR-generated chloride channels[J]. Science, 1991, 253: 205-207.
    [44] Rich D P, Gregory R J, et al. Effect of deletion mutations on the function of CFTR chloride channels[J]. Receptors Channels, 1993, 1: 221-232.
    [45] Winter M C, and Welsh M J Stimulation of CFTR activity by its phosphorylated R domain[J]. Nature, 1997, 389: 294-296.
    [46] Chang X B, Hou Y X, Jensen T J, et al. Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion[J]. J Biol Chem, 1994,269: 18572-18575.
    [47] Cotten J F, Ostedgaard L S, et al. Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator[J]. J Biol Chem, 1996, 271: 21279-21284.
    
    [48] Gadsby D C, Nagel G, et al. The CFTR chloride channel of mammalian heart. Annu Rev Physiol, 1995, 57: 387-416.
    [49] Price M P, Ishihara H, et al. Function of Xenopus CFTR Cl~- channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR[J]. J Biol Chem, 1996, 271:25184-25191.
    [50] Sheppard D N, Rich D P, et al. Mutations in CFTR associated with mild disease form altered pore properties. Nature, 1993, 362: 160-164.
    [51] Cantiello H F. Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator[J]. Exp Physiol, 1996, 81(3): 505-514.
    [52] Benharouga M, Sharma M, So J, et al. The role of the C terminus and Na+/H+ exchanger regulatory factor in the functional expression of cystic fibrosis transmembrane conductance regulator in nonpolarized cells and epithelia[J]. J Biol Chem, 2003, 278(24):22079-22089.
    [53] Kwon S H, Pollard H, Guggino W B. Knockdown of NHERF1 enhances degradation of temperature rescued DeltaF508 CFTR from the cell surface of human airway cells[J]. Cell Physiol Biochem, 2007, 20(6): 763-772
    [54] Rossmann H, Jacob P, Baisch S, etal. The CFTR associated protein CAP70 interacts with the apical Cl-/HCO3- exchanger DRA in rabbit small intestinal mucosa. Biochemistry[J]. 2005, 44(11): 4477-4487.
    [55] TandonC, De Lisle RC, Boulatnikov I, et al. Interaction of carboxyl-terminal peptides of cytosolic-tail of apactin with PDZ domains of NHERF/EBP50 and PDZK-1/CAP70[J]. Mol Cell Biochem, 2007, 302(1-2): 157-167.
    [56] Wolde M, Fellows A, Cheng J, et al. Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression : an RNA interference and structure-based mutagenetic approach[J]. J Biol Chem, 2007, 282(11): 8099-8109.
    [57] Piserchio A, Fellows A,Madden DR, et al. Association of the cystic fibrosis transmembrane regulator with CAL: structural features and molecular dynamics. Biochemistry[J]. 2005,44(49): 16158-16166.
    [58] Naren A P, Quick M W, Collawn J F, et al. Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein-protein interactions[J]. Proc Natl Acad Sci, 1998,95(18): 10972-10977.
    [59] Naren A P, Di A, Cormet-Boyaka E, et al . Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents[J]. JClin Invest. 2000, 105(3): 377-386.
    [60] Tata F, Stanier P, Wicking C, et al. Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene[J]. Genomics, 1991, 10 (2), 301-307.
    [61] Fiedler MA, Nemecz Z K, ShullGE. Cloning and sequence analysis of rat cystic fibrosis transmembrane conductance regulator[J]. Am J Physiol, 1998, 262: 779-784.
    [62] Tebbutt S J, WardleCJ, Hi 11 D F, etal. Molecular analysis of the ovine cystic fibrosis transmembrane conductance regulator gene[J]. Proc Natl Acad Sci. USA, 1995, 92(6),2293-2297.
    [63] Diamond G, Scanlin T F, Zasloff M A, et al. A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites[J]. J Biol Chem, 1991 266 (33), 22761-22769.
    [64] David N, Sheppard and Michael J Welsh. Structure and function of the CFTR chloride channel[J]. Physiol Reviews. 1999, 79: S23-S45.
    [65] Carol A. Bertrand and Raymond A. Frizzell. The role of regulated CFTR trafficking in epithelial secretion[J]. Am J Physiol Cell Physiol, 2003, 285: C1-C18.
    [66] B. Nilius and G. Droogmans. Amazing chloride channels: an overview[J]. Acta Physiol Scand, 2003, 177: 119-147.
    [67] Horowitz B, Tsung S S, Hart P, et al. Alternative splicing of CFTR Cl~- channels in heart[J]. Am J Physiol, 1993, 264: 2214-2220.
    [68] Gadsby D C, Nagel G, Hwang TC. The CFTR chloride channel of mammalian heart[J]. Annu Rev Physiol, 1995, 57: 387-416.
    [69] WarthJD, Collier ML, Hart P, Geary Y, GelbandCH, Chapman T, Horowitz B, Hume JR. CFTR chloride channels in human and simian heart [J]. Cardiovasc Res, 1996, 31(4): 615-624.
    [70] Hart P, Warth J D, Levesque P C, et al. Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart[J]. Proc Natl. Acad. Sci, 1996, 93, 6343-6348.
    [71] Tilly B C, Bezstarosti K, Boomaars W E, Marino CR, et al. Expression and regulation of chloride channels in neonatal rat cardiomyocytes [J]. Mol Cell Biochem, 1996,12-26;157(1-2): 129-135.
    [72] James A F, TominagaT, OkadaY, et al. Distribution of cAMP-activated chloride current and CFTR mRNA in the guinea pig heart[J]. Circ Res, 1996, 79(2):201-7.
    [73] Gao Z, Sun H Y, Lau C P, et al. Evidence for cystic fibrosis transmembrane conductance regulator chloride current in swine ventricular myocytes[J]. J Mol Cell Cardiol, 2007,42(1): 98-105.
    [74] Robert R, Norez C, Becq F. Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cl transport of mouse aortic smooth muscle cells[J]. J Physiol, 2005, 568(Pt 2): 483-495.
    [75] Robert R, Thoreau V, Norez C, et al. Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation[J]. J Biol Chem,2004 May 14, 279(20): 21160-8.
    [76] Brochiero E, Dagenais A, Prive A, et al. Evidence of a functional CFTR Cl(-) channel in adult alveolar epithelial cells[J]. Am J Physiol Lung Cell Mol Physiol, 2004, 287(2):L382-L392.
    [77] Herndndez-Gonzalez E O, Trevino CL, Castellano L E, etal. Involvement of CFTR in Mouse Sperm Capacitation[J]. J Biol Chem, 2007, 282(33): 24397-24406.
    [78] Xu W M, Shi Q X, Chen W Y, et al. Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility[J]. Proc Natl Acad Sci USA,2007, 104(23):9816-9821.
    [79] Zheng X Y, Chen G A, Wang H Y. Expression of cystic fibrosis transmembrane conductance regulator in human endometrium[J]. Hum Reprod, 2004, 19(12): 2933-2941.
    [80] Grubb B R, Rogers T D, Kulaga H M, et al. Olfactory epithelia exhibit progressive functional and morphological defects in CF mice[J]. Am J Physiol Cell Physiol, 2007,293(2): C574-C583.
    [81] Dif F, Marty C, Baudoin C, et al. Severe osteopenia in CFTR-null mice[J]. Bone. 2004,35 (3): 595-603.
    [82] Winter M C , Welsh M J Stimulation of CFTR activity by its phosphorylated R domain[J]. Nature, 1997, 389: 294-296.
    [83] Vais H, Zhang R, Reenstra W W. Dibasic phosphorylation sites in the R domain of CFTR have stimulatory and inhibitory effects on channel activation[J]. Am J Physiol Cell Physiol, 2004, 287(3): 737-745.
    [84] Berger H A, Anderson M P, Gregory R J, et al. Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel[J]. J Clin Invest, 1991, 88: 1422-1431.
    [85] Schwiebert EM, Egan M E, Hwang T H, et al. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP[J]. Cell, 1995, 81(7): 1063-1073.
    [86] Jovov B, Ismailov I, Benos D J Cystic fibrosis transmembrane conductance regulator is required for protein kinase A activation of an outwardly rectified anion channel purified from bovine tracheal epithelia[J]. J Biol Chem, 1995, 270(4): 1521-1528.
    [87] Jovov B, Ismailov II, Berdiev B K, et al. Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels[J]. J Biol Chem, 1995, 270(49): 29194-29200.
    [88] Stuuts M J, Canessa C M, Olsen J C, et al. CFTR as a cAMP-dependent regulator of sodium channels[J]. Science, 1995, 269: 847-850.
    [89] Mall M, Hipper A, GregerR, et al. Wild type but not ΔF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes[J]. FEBS Lett, 1996, 381: 47-52.
    
    [90] McNicholas C M, Wang W, Ho K, et al. Regulation of R0MK1 K channelactivity involves phosphorylation processes[J]. Proc Natl Acad Sci USA, 1994, 91: 8077-8081.
    
    [91] McNicholas C M, Guggino W B, Schwiebert E M, et al. Sensitivity of a renal K+ channel (R0MK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator[J]. Proc Natl Acad Sci USA, 1996, 93: 8083-8088.
    
    [92] McNicholas C M, Nason M W, Guggino W B, et al. A functional CFTR-NBF1 is required for R0MK2-CFTR interaction[J]. Am J Physiol 1997, 273: F843-F848.
    
    [93] Lu M, Leng Q, Egan M E, et al. CFTR is required for PKA-regulated ATP sensitivity of Kir1. 1 potassium channels in mouse kidney[J]. J Clin Invest, 2006, 116(3): 797-807.
    [94] Ishida-Takahashi A, Otani, etal. Cystic fibrosis transmembrane conductance regulator mediates sulphonylurea block of the inwardly rectifying K+ channel Kir[J]. J Physiol,Lond, 1998, 508: 23-30.
    [95] Schreiber R, Greger R, Nitschke R, et al. Cystic fibrosis tranmerabrane conductance regulator activates water conductance in Xenopus oocytes[J]. Pflugers Arch, 1997,434: 841-847.
    
    [96] FULMER S B.SCHWIEBERT E M, et al. Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents[J]. Proc Natl Acad Sci USA, 1995, 92: 6832-6836.
    [97] BOUCHER R C, STUTTS M J, et al. Na~+ transport in cystic fibrosis epithelia. Abnormal basal rate and response to adenylate cyclase activation[J]. J Clin Invest,1986, 78:1245-1252.
    [98] Knowles M R, Stutts M J, et al. Abnormal ion permeation through cystic fibrosis respiratory epithelium[J]. Science, 1983, 221: 1067-1070.
    [99] LING B, ZUCKERMAN J B, LIN C, et al. Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line[J]. J Biol Chem1997, 272: 594-600.
    [100] ISMAIL0V I I, AWAYDA M S, et al. Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator[J]. J Biol Chem, 1996, 271: 4725-4732.
    [101] Gergely L, Lukacs, and Peter R, Durie. Pharmacologic approaches to correcting the basic defect in cystic fibrosis[J]. N Engl J Med. 2003, 349(15): 1433-41.
    [102] Ameen NA, Alexis J, and Salas P. Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract[J]. Histochem Cell Biol,2000, 114: 69-75.
    [103] Ameen NA, Martensson B, Bourguinon L, et al. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo[J]. J Cell Sci,1999, 112: 887-894.
    [104] MoyerBD, Loffing J, Schwiebert EM, etal. Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in Madin-Darby canine kidney cells[J]. J Biol Chem, 1998, 273:21759-21768.
    [105] Puchelle E, Gaillard D, Ploton D, et al. Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium[J]. Am J Respir Cell Mol Biol, 1992, 7: 485-491.
    [106] Webster P, Vanacore L, Nairn AC, et al. Subcellular localization of CFTR to endosomes in a ductal epithelium[J]. Am J Physiol Cell Physiol, 1994, 267: C340-C348.
    [107] Howard M, Jiang X, Stolz D B, et al. Forskolin-induced apical membrane insertion of virally expressed, epitope-tagged CFTR in polarized MDCK cells[J]. Am J Physiol Cell Physiol, 2000, 279: C375-C382.
    [108] DalemansW, Hinnrasky J, Slos P, etal. Immunocytochemical analysis reveals differences between the subcellular localization of normal and delta Phe508 recombinant cystic fibrosis transmembrane conductance regulator[J]. Exp Cell Res, 1992, 201: 235-240.
    [109] Bradbury NA, Jilling T, Berta G, et al. Regulation of plasma membrane recycling by CFTR[J]. Science, 1992, 256: 530-532.
    [110] Bradbury N A, JillingT, Kirk K L, etal. Regulated endocytosis in a chloride secretory epithelial cell line[J]. Am J Physiol Cell Physiol, 1992, 262: C752-C759.
    [111] Prince LS, Workman RB Jr, and Marchase RB. Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel [J]. Proc Natl Acad Sci USA, 1994,91: 5192-5196.
    [112] Schwiebert E M.Gesek F, Ercolani L, et al. Heterotrimeric G proteins, vesicle trafficking,and CFTR Cl channels[J]. Am J Physiol Cell Physiol, 1994, 267: C272-C281.
    [113] Hamosh A, Rosenstein B J, Cutting G R. CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells[J]. Hum Mol Genet, 1992,1: 542-544.
    [114] Hamosh A, Trapnell B C, Zeitlin P L, et al. Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic f ibrosis[J]. J Clin Invest, 1991, 88: 1880-1885.
    [115] Hentze M W, and Kulozik A E. A perfect message: RNA surveillance and nonsense-mediated decay[J]. Cell, 1999, 96: 307-10.
    [116] Hull J, Shackleton S, and Harris A. Abnormal mRNA splicing resulting from three different mutations in the CFTR gene[J]. Hum Mol Genet, 1993, 2: 689-92.
    [117] Thomas P J, Pedersen P L. Effects of the ΔF508 mutation on the structure, function,and folding of the first nucleotide-binding domain of CFTR[J]. J Bioenerg Biomembr,1993, 25: 11-19.
    [118] Cheng S H, Gregory R J, Marshall J, et al. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis[J]. Cell, 1990, 63: 827-834.
    [119] Ward C L, Kopito R R. Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins[J]. J Biol Chem, 1994, 269(41): 25710-25718.
    [120] Ward C L, Omura S, Kopito R R. Degradation of CFTR by the ubiquitinproteasome pathway[J]. Cell, 1995, 83: 121-127.
    [121] Xiong X, Chong E, Skach W R. Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductanceregulator is linked to retrograde translocation from the ER membrane [J]. J Biol Chem, 1999, 274: 2616-2624.
    [122] Yang Y, Janich S, Cohn J A, et al. The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment[J]. Proc Natl Acad Sci, 1993, 90: 9480-9484.
    [123] Ward C L, Kopito R R. Intracellular turnover of cystic fibrosis transmembrane conductance regulator: inefficient processing and rapid degradation of wild type and mutant proteins[J]. J Biol Chem, 1994, 269: 25710-25718.
    [124] Ward C L, Omura S, Kopito R R. Degradation of CFTR by the ubiquitinproteasome pathway[J]. Cell, 1995, 83: 121-127.
    [125] Xiong X, Chong E, Skach W R. Evidence that endoplasmic reticulum (ER) associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane [J]. J Biol Chem, 1999, 274: 2616-2624.
    [126] Yang Y, Engelhardt J F, Wilson J M. Ultrastructural localization of variant forms of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial of xenografts[J]. Am J Respir Cell Mol Biol 1994, 11: 7-15.
    [127] Yang Y, Devor D C, Engelhardt J F, et al. Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR[J]. Hum Mol Genet 1993, 2: 1253-1261
    [128] Choo-KangLR, and Zeitlin P L. Type I, II, III, IV, and V cystic fibrosis transmembrane conductance regulator defects and opportunities for therapy[J]. Curr Opin Pulm Med,2000, 6: 521-529.
    [129] Anderson M P and Walsh M J. Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains[J]. Science, 1992, 257: 1701-1704.
    [130] Sheppard D N, Rich D P, Ostedgaard LS, et al. Mutations in CFTR associated with mild disease form Cl channels with altered pore properties[J]. Nature 1993, 362: 160-164.
    [131] AkabasMH, KaufmannC, Cook T A, et al. Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator[J]. J Biol Chem, 1994,269: 14865-14868.
    [132] Vankeerbergher A, Wei L, Jaspers M, et al. Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator[J]. Hum Mol Genst, 1998a, 7: 1761-1769.
    [133] Vankeerbergher A, Wei L, Teng H, et al. Characterization of mutations located in exon 18 of the CFTR gene[J]. FEBS Lett, 1998b, 437: 1-4.
    [134] Highsmith WE Jr, Burch LH, Zhou Z, et al. Identification of a splice site mutation (2789-5G-A) associated with small anounts of normal CFTR mRNA and mild cystic fibrosis[J]. Hum Mutat 1997, 9:332-338.
    [135] Zielenski J, Markiewicz D, Lin SP, et al. Skipping of exon 12 as a consequence of a point mutation (1898-5G→T) in the cystic fibrosis transmembrane conductance regulator gene found in a consanguineous Chinese family[J]. Clin Genet, 1995, 47: 124-132.
    [136] Chiba-Falek O, Kerem E, Shoshani T, et al. The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10 kb C -T mutation[J]. Genomics,1998, 53: 276-283.
    [137] Chiba-Falek O, Parad R B, Kerem E, et al. Variable levels of normal RNA in different fetal organs carrying a cystic fibrosis transmembrane conductance regulator splicing mutation[J]. Am J Respir Crit Care Med, 1999, 159: 1998-2002.
    [138] Nissim-Rafinia M,Kerem B. Splicing regulation as a potential genetic modifier[J] .Trends Genet, 2002, 18: 123-127.
    [139] Welsh M J, and Smith A E. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis[J]. Cell, 1993, 73: 1251-1254.
    [140] Farrell P M. Improving the health of patients with cystic fibrosis through newborn screening . Wisconsin Cystic Fibrosis Neonatal Screening Study Group[J] . Adv Pediatr. 2000, 47: 79-115.
    [141] Knowles M, Gatzy J, Boucher R. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis[J]. N Engl J Med, 1981, 305(25): 1489-1495.
    [142] Boucher R C, Cotton C U, Gatzy J T, et al. Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures[J]. J Physiol, 1988, 405:77-103.
    [143] WyckoffTJ, Thomas B, Hassett D J, et al. Static growth of mucoid Pseudomonas aeruginosa selects for non-mucoid variants that have acquired flagellum-dependent motility[J]. Microbiology, 2002, 148: 3423-30.
    [144] WhiteleyM, BangeraMG, Bumgarner R E, et al. Gene expression in Pseudomonas aeruginosa biofilms[J]. Nature, 2001, 413: 860-4.
    [145] Sagel S D, SontagMK, Wagener JS et al. Induced sputum inflammatory measures correlate with lung function in children with cystic fibrosis[J]. J Pediatr 2002, 141: 811-7.
    [146] Konstan M W, Davis P B. Pharmacological approaches for the discovery and development of new anti-inflammatory agents for the treatment of cystic fibrosis[J]. Adv Drug Deliv Rev 2002, 54: 1409-23.
    [147] Koehler D R, Downey G P, Sweezey N B, et al. Lung inflammation as a therapeutic target in cystic fibrosis[J]. Am J Respir Cell Mol Biol, 2004, 31: 377-81.
    [148] Muir A, SoongG, Sokol S, etal. Toll-like receptors in normal and cystic fibrosis airway epithelial cells[J]. Am J Respir Cell Mol Biol, 2004, 30: 777-83.
    [149] Freedman S D, Blanco P G, Zaman M M, et al. Association of cystic fibrosis with abnormalities in fatty acid metabolism[J]. N Engl J Med, 2004, 350: 560-9.
    [150] Garred P, Pressler T, Madsen H 0, et al. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis[J]. J Clin Invest, 1999, 104: 431-7.
    [151] Puchelle E, De Bentzmann S, Hubeau C, et al. Mechanisms involved in cystic fibrosis airway inflammation [J]. Pediatr Pulmonol 2001, Suppl 23: 143—5.
    [152] Sagel S D, and Accurso F J. Monitoring inflammation in CF: cytokines[J]. Clin Rev Allergy Immunol, 2002, 23: 41-57.
    [153] HilliardJB, KonstanMW, DavisPB. Inflammatory mediators in CF patients[J]. Methods Mol Med, 2002, 70: 409-31.
    [154] Frangolias D D, Ruan J, Wilcox P J, et al. Alpha 1-antitrypsin deficiency alleles in cystic fibrosis lung disease[J]. Am J Respir Cell Mol Biol, 2003, 29: 390-6.
    [155] Gilljam H, Stenlund C, Ericsson-Hollsing A, et al. Passive smoking in cystic fibrosis[J]. Respir Med, 1990 , 84(4): 289-291.
    [156] Rubin B K. Exposure of children with cystic fibrosis to environmental tobacco smoke[J]. N Engl J Med, 1990, 323(12): 782-788.
    [157] Bakker W. Nutritional state and lung disease in cystic fibrosis[J]. Neth J Med, 1992,41(3-4): 130-6.
    [158] Poller W, Faber J P, Scholz S, et al. Sequence analysis of the cystic fibrosis gene in patients with disseminated bronchiectatic lung disease . Application in the identification of a cystic fibrosis patient with atypical clinical course[J]. Klin Wochenschr, 1991, 69: 657-63.
    [159] Pignatti P F, Bombieri C, Marigo C, et al. Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis[J]. Hum Mol Genet. 1995, 4: 635-9.
    [160] Pignatti P F, Bombieri C, BenetazzoM, et al. CFTR gene variant IVS8-5T in disseminated bronchiectasis[J]. Am J Hum Genet. 1996, 58: 889-92.
    [161] GirodonE, Cazeneuve C, Lebargy F, et al. CFTR gene mutations in adults with disseminated bronchiectasis[J]. Eur J Hum Genet. 1997, 5: 149-55.
    [162] Bombieri C, Benetazzo M, Saccomani A, et al. Complete mutational screening of the CFTR gene in 120 patients with pulmonary disease[J]. Hum Genet. 1998, 103: 718-22.
    [163] TzetisM, Efthymiadou A, StrofalisS, etal. CFTR gene mutations—including three novel nucleotide substitutions—and haplotype background in patients with asthma ,disseminated bronchiectasis and chronic obstructive pulmonary disease[J]. Hum Genet,2001, 108: 216-21.
    [164] Schroeder S A, Gaughan D M, and Swift M. Protection against bronchial asthma by CFTR delta F508 mutation: a heterozygote advantage in cystic fibrosis[J]. Nat Med 1995,1: 703-5.
    [165] MennieM, Gilfillan A, Brock DJ, etal. Heterozygotes for the delta F508 cystic fibrosis allele are not protected against bronchial asthma[J]. Nat Med, 1995, 1: 978-9.
    [166] Dahl M, Tybjaerg-Hansen A, Lange P, etal. DeltaF508 heterozygosity in cystic fibrosis and susceptibility to asthma[J]. Lancet, 1998, 351: 1911-13.
    [167] Lazaro C, de Cid R, Sunyer J, et al. Missense mutations in the cystic fibrosis gene in adult patients with asthma[J]. Hum Mutat, 1999, 14: 510-19.
    [168] Casals T, Ramos MD, Gimenez J, et al. High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes[J]. Hum Genet. 1997, 101: 365-70.[169] Marchand E, Verellen-Dumoulin C, Mairesse M, et al. Frequency of cystic fibrosis transmembrane conductance regulator gene mutations and 5T allele in patients with allergic bronchopulmonary aspergillosis[J]. Chest. 2001, 119: 762-7.
    [170] Castellani C, Quinzii C, Altieri S, et al. A pilot survey of cystic fibrosis clinical manifestations in CFTR mutation heterozygotes[J]. Genet Test. 2001, 5: 249-54.
    [171] Hinson KFW, Moon AJ, PlummerNS. Bronchopulmonary aspergillosis: a review and a report of eight new cases[J]. Thorax, 1952, 7: 317-33.
    [172] Geller DE, Kaplowitz H, Light MJ, et al. Allergic bronchopulmonary aspergillosis in cystic fibrosis : reported prevalence , regional distribution , and patient characteristics. Scientific Advisory Group, Investigators, and Coordinators of the Epidemiologic Study of Cystic Fibrosis[J]. Chest, 1999, 116: 639-46.
    [173] Mastella G, Rainisio M, Harms H K, et al. Allergic bronchopulmonary aspergillosis in cystic fibrosis. A European epidemiological study. Epidemiologic Registry of Cystic Fibrosis[J]. Eur Respir J, 2000, 16: 464-71.
    [174] Starke I D. Asthma and allergic aspergillosis in monozygotic twins[J]. Br J Dis Chest,1985, 79: 295-300.
    [175] Shah A, Khan Z U, Chaturvedi S, et al. Concomitant allergic Aspergillus sinusitis and allergic bronchopulmonary aspergillosis associated with familial occurrence of allergic bronchopulmonary aspergillosis[J]. Ann Allergy, 1990, 64: 507-12.
    [176] Kopelman H, Durie P, Gaskin K, et al . Pancreatic fluid secretion and protein hyperconcentration in cystic fibrosis[J]. N Engl J Med, 1985, 312(6):329-334.
    [177] Fried M D, Durie P R, Tsui L C, et al. The cystic fibrosis gene and resting energy expenditure[J]. J Pediatr, 1991, 119(6): 913-916.
    [178] Lanng S, Thornsteinsson B, Lund-Anderen C, et al. Diabetes mellitus in Danish cystic fibrosis patients: prevalence and late diabetic complications[J]. Acta Paediatr, 1994,83: 72-77.
    [179] Holl R W, Wolf A, Thon A, et al. Insulin resistance with altered secretory kinetics and reduced proinsulin in cystic fibrosis patients[J]. J Pediatr Gastroenterol Nutr,1997, 25(2):188-193.
    [180] KeremE, Corey M, Kerem BS, etal. The relation between genotype and phenotype in cystic fibrosis analysis the common mutation (F508)[J]. N Eng J Med, 1990, 323: 1517-1522.
    [181] Kristidis P, Bozon D, Corey M, et al. Genetic determination of exocrine pancreatic function in cystic fibrosis[J]. Am J Hum Genet, 1992, 50: 1178-1184.
    [182] Hamosh A, Corev M. Correlation between genotype and phenotype in patients with cystic fibrosis[J]. N Eng J Med, 1993, 329: 1308-1313.
    [183] HolslcawD, Eckstein H, and Nixon HH. Meconium ileus: a 20 year review of 109 cases[J]. Am J Dis Child 1965, 109:101-13.
    [184] Kerera E, Corey M, Kerem B, et al. Clinical and genetic comparisons of patients with cystic fibrosis, with or without meconium ileus[J]. J Pediatr 1989, 114: 767-73.
    [185] ShwachmanH, and Antonowicz I. Studies on meconium. In: Gastroenterology and Nutrition in Infancy, edited by E. Lebenthal[J]. New York: Raven, 1981, 83-93.
    [186] Park R W , Grand R J . Gastrointestinal manifestations of cystic fibrosis : a review[J]. Gastroenterology, 1981, 81(6): 1143-1161.
    [187] Knowles M R, StuttsMJ, Spock A, etal. Abnormal ion permeation through cystic fibrosis respiratory epithelium[J]. Science, 1983, 221: 1067-1070.
    [188] Quinton P M. Chloride impermeability in cystic fibrosis[J]. Nature, 1983 Feb 3,301 (5899):421-2.
    [189] Dray X, Bienvenu T, Desmazes-Dufeu N, et al. Distal intestinal obstruction syndrome in adults with cystic fibrosis[J]. Clin Gastroenterol Hepatol, 2004, 2(6):498-503.
    [190] Kristidis P, Bozon D, Corey M, et al. Genetic determination of exocrine pancreatic function in cystic fibrosis[J]. Am J Hum Genet, 1992, 50: 1178-84.
    [191] The Cystic Fibrosis Genotype-Phenotype Consortium. Correlation between genotype and phenotype in patients with cystic fibrosis[J]. N Engl J Med, 1993, 329: 1308-13.
    [192] Zielenski J, Corey M, Rozmahel R, et al. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13[J]. Nat Genet, 1999, 22: 128-9.
    [193] Rohlfs E M, Shaheen N J, Silverman L M. Is the hemochromatosis gene a modifier locus for cystic fibrosis? [J]. Genet Test, 1998, 2: 85-8.
    [194] Cohn J A, Strong T V, Picciotto M R, et al. Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells[J]. Gastroenterology, 1993, 105(6): 1857-1864.
    [195] Peters R H, French P J, van Doorninck J H, et al. CFTR expression and mucin secrction in cultured mouse gallbladder epithelial cells[J]. Am J Physiol, 1996, 271(6 Pt 1):1074-1083.
    [196] Roy C C, Weber A M, Morin C L, et al. Abnormal biliary lipid composition in cystic fibrosis. Effect of pancreatic enzymes[J]. NEnglJMed, 1977 , 15; 297(24): 1301-1305.
    [197] Nagel R A, Westaby D, Javaid A, et al. Liver disease and bile duct abnormalities in adults with cystic fibrosis[J]. Lancet, 1989, 2(8677): 1422-1425.
    [198] O'BrienS, KeoganM, Casey M, etal. Biliary complications of cystic fibrosis[J]. Gut,1992, 33(3):387-391.
    
    [199] Lanng S, Thornsteinsson B, Lund-Anderen C, et al. Diabetes mellitus in Danish cystic fibrosis patients: prevalence and late diabetic complications[J]. Acta Paediatr, 1994,83: 72-77.
    [200] Ferrari M, Colombo C, Sebastio G, et al. Cystic fibrosis patients with liver disease are not genetically distinct[J]. Am J Hum Genet, 1991, 48: 815-16.
    [201] Duthie A, Doherty DG, Williams C, et al. Genotype analysis for delta F508, G551D and R553X mutations in children and young adults with cystic fibrosis with and without chronic liver disease[J]. Hepatology, 1992, 15: 660-4.
    [202] Colombo C, Apostolo MG, Ferrari M, et al. Analysis of risk factors for the development of liver disease associated with cystic fibrosis[J]. J Pediatr, 1994, 124: 393-9.
    [203] Wilschanski M, Rivlin J, Cohen S, et al. Clinical and genetic risk factors for cystic fibrosis-related liver disease[J]. Pediatrics, 1999, 103: 52-7.
    [204] Waters DL, Dorney SF, Gruca MA, etal. Hepatobiliary disease in cystic fibrosis patients with pancreatic sufficiency[J]. Hepatology, 1995, 21: 963-9.
    [205] Duthie A, Doherty DG,Donaldson PT, et al.The major histocompatibility complex influences the development of chronic liver disease in male children and young adults with cystic fibrosis[J]. J Hepatol, 1995, 23: 532-7.
    [206] Gabolde M, Hubert D, Guilloud-Bataille M, et al. The mannose binding lectin gene influences the severity of chronic liver disease in cystic fibrosis[J]. J Med Genet,2001, 38: 310-11.
    [207] Kaplan E, ShwachmanH, Perlmutter AD, etal. Reproductive failure in males with cystic fibrosis[J]. N Engl J Med, 1968, 279: 65-9.
    [208] Dumur V, Gervais R, Rigot J M, et al. Abnormal distribution of CF delta F508 allele in azoospermic men with congenital aplasia of epididymis and vas deferens[J]. Lancet,1990, 336:512.
    [209] Culard J F, Desgeorges M, Costa P, et al. Analysis of the whole CFTR coding regions and splice junctions in azoospermic men with congenital bilateral aplasia of epididymis or vas deferens[J]. Hum Genet, 1994, 93: 467-70.
    [210] MercierB, VerlingueC, LissensW, et al. Is congenital bilateral absence of vas deferens a primary form of cystic fibrosis? [J]. Analyses of the CFTR gene in 67 patients. Am J Hum Genet, 1995, 56: 272-277.
    [211] Costes B, Girodon E, Ghanem N, et al. Frequent occurrence of the CFTR intron 8 (TG)n 5T allele in men with congenital bilateral absence of the vas deferens[J]. Eur J Hum Genet, 1995, 3:285-93.
    [212] Casals T, Bassas L, Ruiz-Romero J, et al. Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected[J]. Hum Genet, 1995, 95: 205-11.
    
    [213] ChillonM, Casals T, MercierB, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens[J]. N Engl J Med, 1995, 332: 1475-80.
    [214] Zielenski J, Patrizio P, Corey M, et al. CFTR gene variant for patients with congenital absence of vas deferens[J]. Am J Hum Genet, 1995, 57: 958-60.
    
    [215] Dodge J A. Male fertility in cystic fibrosis[J]. Lancet, 1995, 346: 587-88.
    [216] Chu C S, Trapne11 B C, Murtagh J J, et al. Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium[J]. EMBO J, 1991, 10: 1355-13363.
    [217] Chu CS, Trapnell BC, Curristin S, et al. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA[J]. Nat Genet, 1993, 3: 151—6.
    [218] Strong TV, Wilkinson DJ, MansouraMK, et al. Expression of an abundant alternatively spliced form of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is not associated with a cAMP-activated chloride conductance[J]. Hum Mol Genet, 1993,2: 225-30.
    [219] Kiesewetter S,Macek M Jr, Davis C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background[J]. Nat Genet, 1993, 5: 274-8.
    [220] Stead R J, Hodson M E, Batten J C, et al. Amenorrhoea in cystic fibrosis[J]. Clinical Endocrinology, 1987, 26: 187-195.
    [221] Reiter E O , Stern R C , and Root AW. The reproductive endocrine system in cystic fibrosis[J]. American Journal of Disease in Childhood, 1981, 135:422-426.
    [222] Kartner N, AugustinasO, Jensen T J, et al. Mislocalization of delta F508 CFTR in cystic fibrosis sweat gland[J]. Nat Genet, 1992, 1: 321-327.
    [223] Tizzano E F, Chitayat D, and Buchwald M. Cell-specific localization of CFTR mRNA shows developmentally regulated expression in human fetal tissues[J]. Hum Mol Genet, 1993,3: 219-224.
    [224] Sato F, Sato K. cAMP-dependent Cl(-) channel protein (CFTR) and its mRNA are expressed in the secretory portion of human eccrine sweat gland[J]. J Histochem Cytochem, 2000,48(3):345-354.
    [225] Stern R C,Boat T F, Abramowsky C R, et al. Intermediate-range sweat chloride concentration and Pseudomonas bronchitis: a cystic fibrosis variant with preservation of exocrine pancreatic function[J]. JAMA, 1978, 239: 2676-2680.
    [226] Oppenheimer E H, and Esterly J R. Pathology of cystic fibrosis; review of the literature and comparison with 146 autopsied cases[J]. Perspect Pediatr Pathol, 1975, 2: 241-78.
    [227] Mandel I D, Kutscher A, Denning C R, et al. Salivary studies in cystic fibrosis[J]. Am J Dis Child, 1967, 110: 431-438.
    [228] BlomfieldJ, Rush A R, AllarsHM, etal. Parotid gland function in children with cystic fibrosis and child control subjects[J]. Pediatr Res, 1976, 10: 574-578.
    
    [229] Mangos J A, and Donnelly W H. Isolated parotid acinar cells from patients with cystic fibrosis. Morphology and composition[J]. J Dent Res, 1981, 60: 19-25.
    [230] Boat T F, Weisman U N, Pallavicini J C. Purification and properties of the calcium precipitable protein in submaxillary saliva of normal and cystic fibrosis subjects[J]. Pediatr Res, 1974, 8: 531-534.
    [231] Wotman S, Mercadante J, Mandel ID, et al. The occurrence of calculus in normal children,children with cystic fibrosis and children with asthma[J]. J Periodontol, 1973, 44:278-280.
    [232] Slomiany B L, Aono M, Murty V L, et al. Lipid composition of submandibular saliva from normal and cystic fibrosis individuals[J]. J Dent Res, 1982, 61: 1163-1166.
    [233] Castellani C, GomezLiraM, Frulloni L, etal. Analysis of the entire coding region ofthe cystic fibrosis transmembrane regulator gene in idiopathic pancreatitis[J]. Hum Mutat,2001, 18: 166.
    [234] Ockenga J, SfuhrmannM, BallmannM, etal. Mutations of the cystic fibrosis gene, but not cationic trypsinogengene , are associated with recurrent or chronic idiopathic pancreatitis[J]. Am J Gastroenterol, 2000, 95: 2061-2067.
    [235] AudrezetMP, Chen J M, Le Marechal C, et al. Determination of the relative contribution of three genes: the cystic fibrosis transmembrane conductance regulator gene, the cationic trypsinogengene, and the pancreatic secretory trypsin inhibitor gene-to the etiology of idiopathic chronic pancreatitis[J]. Eur J Hum Genet, 2002, 10: 100-106.
    [236] Maire F, Bienvenu T, Ngukam A, et al. Frequency of CFTR gene mutations in idiopathic pancreatitis[J]. Gastroenterol Clin Biol, 2003, 27: 398-402.
    [237] Cohn J A, Friedman K J, Noone P G, et al. Relation between Mutations of the cystic fibrosis gene and idiopathic pancreatitis[J]. N Engi J Med, 1998, 339: 653-658.
    [238] Kostuch M, Rudzki S, Semczuk A, et al. CFTR gene mutations in patients suffering from acute pancreatitis[J]. Med Sci Monit, 2002, 8: BR369-BR372.
    [239] Pezzilli R, Morselli-Labate A M, Mantovani V, et al. Mutations of the CFTR gene in pancreatic disease[J]. Pancreas, 2003, 27: 332-336.
    [240] Wang X, MoylanB, Leopold DA, etal. Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population[J]. JAMA, 2000,284: 1814-19.
    [241] Raman V, Clary R, Siegrist KL, et al. Increased prevalence of mutations in the cystic fibrosis transmembrane conductance regulator in children with chronic rhinosinusitis[J]. Pediatrics, 2002, 109: E13.
    [242] HytonenM, Pat jas M, Vento SI, etal. Cystic fibrosis gene mutations AF508 and 394delTT in patients with chronic sinusitis in Finland[J]. Acta Otolaryngol, 2001, 121: 945-7.
    [243] Bombieri C, Luisetti M, Belpinati F, etal. Increased frequency of CFTR gene mutations in sarcoidosis: a case/control association study[J]. Eur J Hum Genet, 2000, 8: 717-20.
    [244] Vlietinck A J, De Bruyne T, Apers S, et al. Plant-derived leading compounds for chemotherapy of human immunodeficiency virus (HIV) infection[J]. Planta Med, 1998,64: 97-109.
    [245] Qais AL-Awqati. Alternative treatment for secretory diarrhea revealed in a new class of CFTR inhibitors[J]. J Clin Invest, 2002, 110: 1599-1601.
    [246] Granger D N, Barrowman J A, and Kvietys P R. The small intestine. In: Clinical Gastrointestinal Physiology[J]. Philadelphia, PA: Saunders, chapt. 1985, 7.
    [247] BuresiMC, BuretAG, HollenbergMD, et al. Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway[J]. FASEB J, 2002, 16: 1515-1525.
    [248] Uribe J M, McCole D F, Barrett K E. Interferon-gamma activates EGF receptor and increases TGF-alpha in T84 cells: implications for chloride secretion[J]. Am J Physiol, 2002,283: 923-931.
    [249] Oprins J C, van der Burg C, Meijer H P, et al. Tumour necrosis factor alpha potentiates ion secretion induced by histamine in a human intestinal epithelial cell line and in mouse colon: involvement of the phospholipase D pathway[J]. Gut, 2002, 50: 314-321.
    [250] Kottgen M, Loffler T, Jacobi C, et al. P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport[J]. J Clin Invest, 2003, 111:371-379.
    [251] Crane J K, Olson R A, Jones H M, et al. Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator[J]. Am J Physiol, 2002,283: 74-86.
    [252] de Zoysa I, Feachem R. Interventions for the control of diarrhoeal diseases among young children: rotavirus and cholera immunization[J]. Bull World Health Organ 1985, 63:569-583.
    [253] Morris A P, Scott J K, Ball J M, et al. NSP4 elicits age-dependent diarrhea and Ca2R-mediated IS influx into intestinal crypts of CF mice[J]. Am J Physiol, 1999,277: 431-444.
    [254] Quinton P M. Physiological basis of cystic f ibrosis: a historical perspective[J]. Physiol Rev, 1999, 79, Suppl: S3-S22
    [255] Peter Igarashi and Stefan Somlo. Genetics and Pathogenesis of Polycystic Kidney Disease[J]. J Am Soc Nephrol, 2002, 13: 2384-2398.
    [256] Koichi Nakanishi, William E. Sweeney J R, et al. Role of CFTR in Autosomal Recessive Polycystic Kidney Disease[J]. J Am Soc Nephrol, 2001, 12: 719-725.
    [257] Patricia D. Wilson. Mechanisms of disease of polycystic kidney disease[J]. N Engl J Med, 2004, 350: 151-164.
    
    [258] The European Polycystic Kidney Disease Consortium. The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16[J]. Cell,1994, 77: 881-894.
    [259] Mochizuki T, Wu G, Hayashi T, et al. PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein[J]. Science, 1996, 272: 1339-42.
    [260] Wilson P D, Sherwood A C, Palla K, et al. Reversed polarity of Na(+)-K(+)-ATPase:mislocation to apical plasma membranes in polycystic kidney disease epithelia[J]. Am J Physiol 1991, 260: F420-F430.
    [261] LebeauC, HanaokaK, Moore-HoonML, et al. BSC2 is the basolateral chloride transporter in a subset of cysts in autosomal dominant polycystic kidney disease (ADPKD)[J]. J Am Soc Nephrol 1999, 10: 36A.
    [262] Wilson P D. Cystic fibrosis transmembrane conductance regulator in the kidney: Clues to its role[J]? Exp Nephrol, 1999, 7: 284-289
    [263] Chauvet V, Deen P, Guggino W B, et al. Expression of cystic fibrosis transmembrane conductance regulator (CFTR) and aquaporins (AQP) 1 and 2 in autosomal dominant polycystic kidney disease (ADPKD)[J]. J Am Soc Nephrol, 1999, 10: 415A.
    [264] Grantham J J, Ye M, Gattone V H, Sullivan L P. In vitro fluid secretion by epithelium from polycystic kidneys[J]. J Clin Invest, 1995, 95: 195-202.
    [265] Grantham J J, UchicM, CragoeEJJr, et al. Chemical modification of cell proliferation and fluid secretion in renal cysts[J]. Kidney Int, 1989, 35: 1379-1389.
    [266] Brill S R, Ross K E, Davidow C J, et al. Immunolocalization of ion transport proteins in uman autosomal dominant polycystic kidney epithelial cells[J]. Proc Natl Acad Sci USA, 1996, 93: 10206-10211
    [267] Lebeau C, Hanaoka K, Morre-Hoon M L, et al. BSC2 is the basolateral Na-K-2CL cotransporter in a subset of cysts in autosomal dominant polycystic kidney disease[J]. J Am Soc Nephrol, 1999, 36A-37A.
    [268] Sullivan L P, Wallace D P, Grantham J J. Chloride and fluid secretion in polycystic kidney disease[J]. J Am Soc Nephrol, 1998, 9: 903-916.
    [269] McAteer J A, Evan A P, Gardner K D. Morphogenetic clonal growth of kidney epithelial cell line MDCK[J]. Anat Rec, 1987, 217: 229-239
    [270] Grantham J J, Mangoo-Karim R, Uchic M E, et al. Net fluid secretion by mammalian renal epithelial cells: Stimulation by cAMP in polarized cultures derived from established renal cells and from normal and polycystic kidneys[J]. Trans Assoc Am Physicians,1989, 102: 158-162
    [271] Davidow C J, Maser R L, Rome L A, et al. The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro[J]. Kidney Int, 1996, 50: 208-218.
    
    [272] Sweeney W E, Avner E D, Elmer HL, et al. CFTR is required for cAMP-dependent: In vitro renal cyst formation[J]. J Am Soc Nephrol, 1998, 9: 38A.
    [273] O' Sullivan D A, Torres V E,GabowP A, et al. Cystic fibrosis and the phenotypic expression of autosomal dominant polycystic kidney disease[J]. Am J Kidney Dis, 1998, 32:976-983.
    [274] Cotton C U, Avner E D. PKD and CF: An interesting family provides insight into the molecular pathophysiology of polycystic kidney disease[J]. Am J Kidney Dis, 1998,32: 1081-1083.
    [275] Howard M, Frizzell R A, Bedwell D M. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations[J]. Nature Med, 1996, 4: 467-469.
    [276] Bedwell D M, Kaenjak A, Benos D et al. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line[J]. Nature Med, 1997, 3: 1280-1284.
    [277] Wilschanski M, Famini C, Blau H, et al. A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations[J]. Am J Respir Crit Care Med, 2000, 161: 860-865.
    [278] Clancy J P, BebokZ, Ruiz F, et al. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis[J]. Am J Respir Crit Care Med, 2000,163: 1683-1692.
    [279] Wilschanski M, Yahav Y, Yaacov Y, etal. Gentamicininduced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations[J]. N Engl J Med, 2003, 349:1433-1441.
    [280] Denning G M, Anderson M P, Amara J F, et al. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive [J]. Nature, 1992, 358:761-764.
    [281] Brown C R, Hong-Brown L Q, Biwersi J, et al. Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein[J]. Cell Stress. Chaperones 1996, 1: 117-125.
    [282] Egan M E, Schweibert E M, Guggino W B. Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells[J]. Am J Physiol (Cell Physiol),1995, 268: C243-C251.
    [283] Rubenstein R C, Egan M E, Zeitlin P L. In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR[J]. J Clin Invest, 1997, 100: 2457-2465.
    [284] Rubenstein RC, Zeitlin PL. Sodium 4-phenylbutyrate downregulates hsc 70: implications for intracellular trafficking of deltaF508-CFTR[J]. Am J Physiol Cell Physiol, 2000,278(2): C259-C267
    [285] Rubenstein R C, Lyons B M. Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation[J]. Am J Physiol Lung Cell Mol Physiol 2001, 281: L43-L51.
    
    [286] Choo-Kang, L and Zeitlin, PL. Induction of Hsp 70 promotes AF508 CFTR trafficking[J]. Am J Physiol Lung Cell Mol Physiol, 2001, 281: L58-L68.
    [287] Rubenstein R C, Egan M E, Zeitlin P L. In vitro pharmaco-logic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells dipropylxan-containing DF508-CFTR[J]. J Clin Invest 1997, 100:2457-2465.
    [288] Zeitlin PL, Diener-West M, Rubenstein R C, et al. Evidence of CFTR function in cystic fibrosis after systemic administration of 4-phenylbutyrate[J]. Mol Ther, 2002, 6:119-126.
    [289] ArispeN, Ma J, Jacobson K A, et al. Direct activation of cystic fibrosis transmembrane conductance regulator channels by 8-cyclopentyl- 1, 3- dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX)[J]. J Biol Chem, 1998, 273: 5727-5734.
    [290] Pollard H B. Role of CPX in promoting trafficking and chloride channel activity of wildtype and mutant CFTR[J]. Pediatr Pulmonol 1997, S14: 128-131.
    [291] Konstan M W, Aitken M L, Ahrens R C, et al. Pharmacokinetics of CPX (8-cyclopentyl-1,3-dipropylxanthine) in adults with cystic fibrosis[J]. Pediatr Pulmonol, 1998, S17:277
    [292] McCarty N A, Standaert T A, Teresi M, et al. A phase I randomized, multicenter trial of CPX in adult subjects with mild cystic fibrosis[J]. Pediatr Pulmonol, 2002 Feb,33(2): 90-98.
    [293] ReenstraWW, Yurko-MauroK, DamA, etal. CFTR chloride channel activation by genistein:the role of serine/threonine protein phosphatases[J]. Am J Physiol 1996, 271: 650-657.
    [294] Yang I C, Cheng T H, Wang F, et al. Modulation of CFTR chloride channels by calyculin A and genistein[J]. Am J Physiol 1997, 272: 142-155.
    [295] Weinreich F, Wood P G, Riordan J R, et al. Direct action of genistein on CFTR[J]. Pflugers Arch, 1997, 434: 484-491.
    [296] RandakC, Auerswald E A, Assfalg-Machleidt I, et al. Inhibition of ATPase, GTPase and adenylate kinase activities of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein[J]. Biochem J, 1999, 340:227- 235.
    [297] Illek B, Zhang L, Lewis N C, et al. Defective function of the cystic fibrosis causing missense mutation G551D is recovered by genistein[J]. Am J Physiol, 1999, 277: C833-C839.
    [298] Illek B, Fischer H. Flavonoids stimulate Cl- conductance of human airway epithelium in vitro and in vivo[J]. Am J Physiol 1998, 275: L902-L910.
    [299] Shoskes DA, Zeitlin SI, Shahed A, et al. Quercetin in men with category III chronic prostatitis : a preliminary prospective , doubleblind , placebo-controlled trial[J]. Urology 1999, 54: 960-963.
    [300] Kelley TJ, Al-Nakkash L, Drumm ML. CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells[J]. Am J Respir Cell Mol Biol.1995, 13: 657-664.
    [301] Schultz BD, Singh AK, Devor DC, et al . Pharmacology of CFTR chloride channel activity[J]. Physiol Rev 1999, 79: S109-S144.
    [302] Maitra R,Shaw C, Stanton B A, et al. Increases in CFTR mRNA expression,protein trafficking and functional membrane expression by systemic chemotherapy drugs[J]. Pediatr Pulmonol,1999, 19: 187.
    [303] Cafferata E G, Gonzalez-Guerrico A M, Giordano L, et al. Interleukin-1beta regulates CFTR expression in human intestinal T84 cells[J]. Biochim Biophys Acta, 2000, 1500:241-248.
    [304] Nissim-Rafinia M, Aviram M, Randell S H, et al. Restoration of the CFTR function by splicing modulation[J]. EMB0 Letts, 2004, 5(11): 1071-1077.
    [305] Schwiebert E M, Cid-Soto L P, Stafford D, et al. Analysis of C1C-2 channels as an alternative pathway for chloride conduction in cystic fibrosis airway cells[J]. Proc Natl Acad Sci USA, 1998, 95: 3879-3884.
    [306] Devor D C, Pilewski J M. UTP inhibits Na+ absorption in wild-type and deltaF508 CFTR-expressing human bronchial epithelia[J]. Am J Physiol, 1999, 276: C827-837.
    [307] Fulmer S B, Schwiebert E M, Morales M M, et al. Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents[J]. Proc Natl Acad Sci USA, 1995,92: 6832-6836.
    [308] Griesenbach U, Geddes D M, Alton EW. Update on gene therapy for cystic fibrosis[J]. Curr Opin Mol Ther, 2003, 5: 489-494.
    [309] Harvey B-G, Leopold P L, Hackett NR et al. Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus[J]. J Clin Invest, 1999, 104: 1245-1255.
    [310] Flotte T R, Zeitlin P L, Reynolds T C, et al. Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study[J]. Hum Gene Ther, 2003,14: 1079-1088.
    [311] Flotte T, Carter B, Conrad C, et al. A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease[J]. Hum Gene Ther,1996, 7:1145-1159.
    [312] Aitken M L, Moss R B, Waltz D A, et al. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease[J]. Hum Gene Ther, 2001,12: 1907-1916.
    [313] Moss, R. B. et al. Repeated adencrassociated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: amulticenter, double-blind, placebo-controlled trial[J]. Chest, 2004, 125:509-521.
    [314] Limberis, M. et al. Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer[J]. Hum. Gene Ther. 2002, 13: 1961-1970.
    [315] Yonemitsu, Y. et al. Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat[J]. Biotechnol. 2000, 18: 970-973.
    [316] Kobinger, G. P. et al. Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo[J]. Nat. Biotechnol. 2001, 19: 225-230.
    [317] Alton, E. W. et al. Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebocontrolled trial[J]. Lancet,1999, 353: 947-954.
    [318] Ruiz, F. E. et al. A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis[J]. Hum. Gene Ther. 2001, 12: 751-761.
    [319] Gill , D . R . et al . The development of gene therapy for diseases of the lung[J]. Cell. Mol. Life Sci. 2004, 61: 355-368.
    [320] Klink, D. T. et al. Gene therapy of cystic fibrosis (CF) airways: a review emphasizing targeting with lactose[J]. GlycoconJ J 2001, 18: 731-740.
    [321] Wiseman, J W. et al. A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo[J]. Gene Ther. 2003, 10: 1654-1662.
    [322] Stern M, Caplen NJ, Browning JE et al. The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro[J]. Gene Ther, 1998, 5: 91-98.
    [323] Lisby D A, Ballard P L, Fox W W, et al. Enhanced distribution of adenovirus-mediated gene transfer to lung parenchyma by perfluorochemical liquid [J]. Hum Gene Ther, 1997,8: 919-928.
    [324] Johnson L G,Vanhook M K, Coyne C B,et al.Safety and efficiency of modulating paracellular permeability to enhance airway epithelial gene transfer in vivo[J]. Hum. Gene Ther,2003, 14: 729-747.
    [325] Knowles M R, Clarke L L, and Boucher R C. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis[J]. N Engl J Med, 1991, 325: 575-577.
    [326] Devor D C , Singh A K , Bridges R J , et al . Modulation of Cl~- secretion by benzimidazolones. II. Coordinate regulation of apical G_(cl) and basolateral G_k[J]. Am J Physiol Lung Cell Mol Physiol, 1996, 271: L785-L795.
    [327] GelmanM S and Kopito R R. Rescuing protein conformation: prospects for pharmacological therapy in cystic fibrosis[J]. J Clin Invest, 2002, 110: 1591-1597.
    [328] Dasgupta B and King M. Reduction in viscoelasticity in cystic fibrosis sputum in vitro using combined treatment with nacystelyn and rhDNase[J]. Pediatr Pulmonol, 1996, 22:161-166.
    [329] Johnson C A, Butler S M, Konstan M W, et al. Estimating effectiveness in an observational study: A case study of dornase alfa in cystic fibrosis. Investigators and coordinators of the epidemiologic study of cystic fibrosis[J]. J Pediatr, 1999, 134: 734-739.
    [330] Weller P H. Implications of early inflammation and infection in cystic fibrosis: a review of new and potential interventions[J]. Pediatr Pulmonol, 1997, 24: 143-146.
    [331] Campbell P W III, Saiman L. Use of aerosolized antibiotics in patients with cystic fibrosis[J]. Chest, 1999, 116: 775-788.
    [332] Frederisksen B, LanngS, Koch C, etal. Improved survival in the Danish center-treated cystic fibrosis patients: results of aggressive treatment[J]. Pediatr Pulmonol, 1996,21: 153-158.
    [333] Ramsey B W, Pepe M S, QuanJM, etal. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic fibrosis Inhaled Tobramycin Study Group[J]. N Engl J Med, 1999, 340: 23-30.
    [334] Barbara E Goodman and William H Percy. CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice[J]. Adv Physiol Educ, 2005, 29: 75-82.
    [335] Richard B Moss. New approaches to cystic fibrosis[J]. Hospital Practice, 2001.
    [336] Glass RI , Libel M , and Brandling-Bennett A D . Epidemic cholera in the Americas[J]. Science, 1992, 256: 1524-1525.
    [337] Da Cunha Ferreira RMC and Cash R A. History of the development of oral rehydration therapy[J]. Clin Ther, 1990, Suppl 12: 2-13.
    [338] World Health Organization . Cholera fact sheet [EB/OL] . http ://www. who. int/mediacentre/factsheets/fsl07/en/ [March 2000].
    [339] Duggan C, Fontaine 0, Pierce N F, et al. Scientific rationale for a change in the composition of oral rehydration solution[J]. JAMA, 2004, 291: 2628-26354.
    [340] Ma T, Thiagarajah J R, Yang H, et al. Thiazolidinone CFTR inhibitor identified by highthroughput screening blocks cholera toxin-induced intestinal fluid secretion[J]. J Clin Invest, 2002, 110:1651-1658.
    [341] Chatchai Muanprasat N D. Sonawane, Danieli Salinas, et al. Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy[J]. J Gen. Physiol, 2004, 124: 125-137.
    [342] Thiagarajah J R, Broadbent T, Hsieh E, et al. Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor[J]. Gastroenterology, 2004, 126: 511-519.
    [343] Davidson D J and Dorin J R. The CF mouse: an important tool for studying cystic fibrosis[J]. Expert Rev Mol Med, 2001, (http: //wwwermm. cbcu. cAm ac. uk/01002551h. htm)
    [344] Scholte B J,Donald J Davidson,Martina Wilke,et al. Animal models of cystic fibrosis[J]. J Cyst. Fibros. 2004, 3 (Suppl. 2):183-190.
    [345] Grubb B R and Boucher R C. Pathophysiology of gene-targeted mouse models for cystic fibrosis[J]. Physiol Rev, 1999, 79 (Suppl. 1):S193-S214.
    [346] Colledge W H, Abella B S, Southern K W, et al. Generation and characterization of a delta F508 cystic fibrosis mouse model[J]. Nat Genet, 1995, 10(4):445-452.
    [347] Zeiher B G, Eichwald E, Zabner J, et al. A mouse model for the delta-F508 allele of cystic-fibrosis[J]. J Clin Invest, 1995, 96: 2051-2064.
    [348] van Doorninck J H, French P J, Verbeek E, et al. A mouse model for the cystic fibrosis delta-F508 mutation[J]. EMBO J, 1995, 14: 4403-4411.
    [349] Kent G, Oliver M, Foskett J K, et al. Phenotypic abnormalities in long-term surviving cystic fibrosis mice[J]. Pediatr. Res, 1996, 40: 233-241.
    [350] Delaney S J, Alton E W, Smith S N, et al. Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations[J]. EMBO J, 1996, 15,955-963.
    [351] Zielenski J. Genotype and phenotype in cystic fibrosis[J]. Respiration, 2000, 67:117-133.
    [352] Rowntree R K, and Harris A. The phenotypic consequences of CFTR mutations[J]. Ann Hum Genet, 2003, 67 (PT 5):471-485.
    [353] Bob J Scholtel, William H Colledge, Martina Wilke, et al. Cellular and animal models of cystic fibrosis, tools for drug discovery[J]. Drug Discovery Today: Disease Models,2006, 3: 251-259.
    [354] Gray M A, Winpenny J P, Verdon B, et al. Chloride channels and cystic fibrosis of the pancreas[J]. Biosci. Rep, 1995, 15, 531-541
    [355] Grubb B R, Paradiso A M, Boucher R C. Anomalies in ion transport in CF mouse tracheal epithelium[J]. Am J Physiol, 1994, 267: 293-300.
    [356] Kent G, Iies R, Bear C E, et al. Lung disease in mice with cystic fibrosis[J]. J Clin Invest, 1997 Dec 15, 100(12):3060-9.
    [357] Plopper C G, Hill L H, Mariassy AT. Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A study of man with comparison of 15 mammalian species[J]. Exp Lung Res, 1980, 1: 171-180.
    [358] Plopper C G, Mariassy A T, Wilson DW, et al. Comparison of nonciliated tracheal epithelial cells in six mammalian species: ultrastructure and population densities[J]. Exp Lung Res, 1983, 5: 281-294.
    [359] Choi H K, Finkbeiner W E, Widdicombe J H. A comparative study of mammalian tracheal mucous glands[J]. J Anat 2000, 197: 361-372.
    [360] Turk J R, Henderson KK, VanvickleGD, et al. Arterial endothelial function in a porcine model of early stage atherosclerotic vascular disease[J]. Int J Exp Pathol, 2005,86: 335-345.
    [361] DysonMC, Alloosh M, VuchetichJP, etal. Components of metabolic syndrome and coronary artery disease in female Ossabaw swine fed excess atherogenic diet[J]. Comp Med, 2006,56: 35-45.
    [362] Bellinger D A, Merricks E P, Nichols T C. Swine models of type 2 diabetes mellitus:insulin resistance, glucose tolerance, and cardiovascular complications[J]. ILAR J,2006, 47: 243-258.
    [363] Larsen M O, Rolin B. Use of the Gottingen minipig as a model of diabetes, with special focus on type 1 diabetes research[J]. ILAR J, 2004, 45: 303-313.
    [364] Wallock-Montelius L M, Villanueva J A, Chapin R E, et al. Chronic ethanol perturbs testicular folate metabolism and dietary folate deficiency reduces sex hormone levels in the Yucatan micropig[J]. Biol Reprod, 2007, 76: 455-465.
    [365] Simon G A, Maibach H I. The pig as an experimental animal model of percutaneous permeation in man: qualitative and quantitative observations-an overview[J]. Skin Pharmacol Appl Skin Physiol, 2000, 13: 229-234.
    [366] Winter G D. Some factors affecting skin and wound healing[J]. J Tissue Viability,2006, 16: 20-23.
    [367] Standaert T A, Boitano L, Emerson J, et al. Standardized procedure for measurement of nasal potential difference: an outcome measure in multicenter cystic fibrosis clinical trials[J]. Pediatr Pulmonol, 2004, 37: 385-392.
    [368] Crews A, Taylor A E, Ballard S T. Liquid transport properties of porcine tracheal epithelium[J]. J Appl Physiol, 2001, 91: 797-802.
    [369] Fang X, Fukuda N, Barbry P, et al. Novel role for CFTR in fluid absorption from the distal airspaces of the lung[J]. J Gen Physiol, 2002, 119: 199-207.
    [370] Ballard S T, Trout L, Garrison J, et al. Ionic mechanism of forskolininduced liquid secretion by porcine bronchi[J]. Am J Physiol Lung Cell Mol Physiol 2006, 290: L97-104.
    [371] Ballard S T, InglisSK. Liquid secretion properties of airway submucosal glands[J]. J Physiol 2004, 556: 1-10.
    [372] Ballard ST, Trout L, Mehta A, InglisSK. Liquid secretion inhibitors reduce mucociliary transport in glandular airways[J]. Am J Physiol Lung Cell Mol Physiol 2002, 283:L329-L335.
    [373] Inglis S K, Corboz M R, Taylor A E, Ballard S T. Regulation of ion transport across porcine distal bronchi[J]. Am J Physiol, 1996, 270:L289-L297.
    [374] Song Y, Salinas D, Nielson DW, et al. Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis[J]. Am J Physiol Cell Physiol, 2006, 290: C741-C749.
    [375] Ballard ST, Fountain JD, Inglis SK, et al. Chloride secretion across distal airway epithelium: relationship to submucosal gland distribution[J]. Am J Physiol, 1995,268: L526-L531.
    [376] Ballard ST, Trout L, Bebok Z, et al. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands[J]. Am J Physiol, 1999, 277: L694-L699.
    [377] Crews A, Taylor AE, Ballard ST. Liquid transport properties of porcine tracheal epithelium[J]. J Appl Physiol, 2001, 91: 797-802.
    [378] Vacciana A, EbeigbeAB, Hasse J, et al. Evidence for preservation of epithelial function in cryopreserved porcine and human bronchi[J]. Exp Physiol 1994, 79: 409-414.
    [379] Farmer S J, Fliss A E, Simmen R C. Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancyassociated porcine uterine protein related to human antileukoproteinase[J]. Mol Endocrinol, 1990, 4: 1095-1104.
    [380] Furutani Y, Kato A, Yasue H, et al. Evolution of the trappin multigene family in the Suidae[J]. J Biochem, 1998, 124: 491-502.
    [381] Johannisson A, Jonasson R, Dernfalk J, et al. Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP technology[J]. Cytometry, 2006, A 69: 391-395.
    [382] Marquette C H, Wermert D, Wallet F, et al. Characterization of an animal model of ventilator-acquired pneumonia[J]. Chest, 1999, 115: 200-209.
    [383] Budas G R, Churchill E N, Mochly-Rosen D. Cardioprotective mechanisms of PKC isozyme-selective activators and inhibitors in the treatment of ischemia-reperfusion injury[J]. Pharmacol Res, 2007, 55: 523-536.
    [384] Schwarzkopf K, Schreiber T, Gaser E, et al. The effects of xenon or nitrous oxide supplementation on systemic oxygenation and pulmonary perfusion during one-lung ventilation in pigs[J]. Anesth Analg, 2005, 100: 335-339.
    [385] Gushima Y, Ichikado K, Suga M, et al. Expression of matrix metalloproteinases in pigs with hyperoxia-induced acute lung injury[J]. Eur Respir J, 2001, 18: 827-837.
    [386] Brandler MD, Powell SC, Craig DM, et al. A novel inhaled organic nitrate that affects pulmonary vascular tone in a piglet model of hypoxia-induced pulmonary hypertension[J]. Pediatr Res, 2005 58: 531-536.
    [387] Mitchell HW, Turner D J, Noble PB. Cholinergic responsiveness of the individual airway after allergen instillation in sensitised pigs[J]. Pulm Pharmacol Ther, 2004, 17:81-88.
    [388] Jolliet P, Watremez C, Roeseler J, et al. Comparative effects of helium-oxygen and external positive end-expiratory pressure on respiratory mechanics, gas exchange, and ventilation-perfusion relationships in mechanically ventilated patients with chronic obstructive pulmonary disease[J].Intensive Care Med,2003,29:1442-1450.
    [389]Rogers C S,Hao Y,Rokhlina T,et al.Production of CFTR-null and CFTR-DeltaF508heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer[J].J Clin Invest,2008,118:1571-1577.
    [390]Ostedgaard L S,Rogers C S,Dong Q,et al.Processing and function of CFTR-DeltaF508are species-dependent[J].Proc Natl Acad Sci USA,2007,104:15370-15375.
    [391]Salinas D B,Pedemonte N,Muanprasat C,et al.CFTR involvement in nasal potential differences in mice and pigs studied using a thiazolidinone CFTR inhibitor[J].Am J Physiol Lung Cell Mol Physiol,2004,287:L936-L943.
    [392]Liu X,Luo M,Zhang L,et al.Bioelectric properties of chloride channels in human,pig,ferret,and mouse airway epithelia[J].Am J Respir Cell Mol Biol,2007,36:313-323.
    [393]何成彦.水和氯离子通道在人消化系统的表达和功能[D]:[博士学位论文].长春:东北师范大学,2004.
    [394]Galietta L V,Jayaraman S,Verkman A S.Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists[J].Am J Physiol Cell Physiol,2001,281:C1734-C1742.
    [395]Dalemans W,Barbry P,Champigny G,et al.Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation[J].Nature,1991,354:526-528.
    [396]Haws C M,Nepomuceno I B,Krouse M E,et al.Delta F508-CFTR channels:kinetics,activation by forskolin,and potentiation by xanthines[J].Am J Physiol,1996,270:C1544-C1555.
    [397]Hwang T C,Wang F,Yang I C,et al.Genistein potentiates wild-type and delta F508-CFTR channel activity[J].Am J Physiol,1997,273:C988-C998.
    [398]Wang F,Zeltwanger S,Hu S,et al.Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels[J].J Physiol,2000,524 Pt 3:637-648.
    [399]Davidson D J,Kilanowski F M,Randell S H,et al.A primary culture model of differentiated murine tracheal epithelium[J].Am J Physiol Lung Cell Mol Physiol,2000,524 Pt 3:L766-L778.
    [400]Kelley T J,Thomas K,Milgram L J,et al.in vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium[J].Proc Natl Acad Sci U S A,1997,94:2604-2608.
    [401]DeCarvalho A C,Gansheroff L J,Teem J L.Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508[J].J Biol Chem,2002,277:35896-35905.
    [402]Teem J L,Carson M R,Welsh M J.Mutation of R555 in CFTR-delta F508 enhances function and partially corrects defective processing[J]. Receptors Channels, 1996, 4: 63-72.
    [403] Johnson L G, Olsen J C, Sarkadi B, et al. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis[J]. Nat Genet 1992, 2: 21-25.
    [404] O' Donnell E K, Sedlacek R L, Singh A K, et al. Inhibition of enterotoxin-induced porcine colonic secretion by diarylsulfonylureas in vitro[J]. Am J Physiol, 2000, 279: 1104-1112.