【作者】 刘艳丽；

【导师】 麻彤辉；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2008， 博士

【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。更多还原

【Abstract】 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.更多还原