【作者】 葛红；

【导师】 麻彤辉； 杨红；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2009， 硕士

【摘要】 囊性纤维化跨膜电导调节因子(cystic fibrosis transmembrane conductance regulator, CFTR)是一种CAMP/PKA依赖的上皮氯离子通道,同时又是某些膜转运蛋白的调节因子。属于ATP结合盒(ATP binding cassette, ABC)转运蛋白超家族的成员。CFTR在哺乳动物中所有与分泌和吸收有关的上皮组织(例如小肠、气道、胰腺、汗腺、输精管细胞、心肌细胞、血管平滑肌细胞)中广泛表达。CFTR的主要功能是介导跨上皮细胞Cl-转运、控制氨敏感的上皮细胞Na+通道(epithelial Na+ channel, ENaC)、促进HCO3-分泌及调节K+通道等,因此CFTR与多种生理和病理状态密切相关。CFTR功能障碍会引起致死性遗传疾病囊性纤维化病(cystic fibrosis, CF)、特发性慢性胰腺炎(idiopathic chronic pancreatiti, ICP)、干眼病(keratoconjunctivitis sicca, KCS)、习惯性便秘(habitual constipation)等;而霍乱毒素和耐热性致病性大肠杆菌内毒素导致的分泌型腹泻(secretary diarrhea)、多囊肾病(polycystic kidney)等则是CFTR活动过强引起的。因此寻找能够调节野生型和突变型CFTR功能的小分子调节剂的工作近年来受到了广泛重视并取得了较大进展,目前仍是CFTR有关研究的重要目标之一。我们利用含?F508-CFTR(或wt-CFTR)的表达质粒与一种对卤族元素碘离子高度敏感的荧光绿蛋白突变体EYFP-H148Q的表达质粒共转染Fischer大鼠甲状腺上皮细胞,得到稳定表达该蛋白的两种细胞系(FRT/?F508-CFTR /EYFP-H148Q和FRT/wt-CFTR /EYFP-H148Q-I152L)。以此为筛选模型对49种生物碱类化合物和20种挥发油类化合物进行筛选,目的是为了筛选出wt-CFTR氯离子通道激活剂和?F508-CFTR氯离子通道激活剂及蛋白质质膜转运障碍纠正剂,并对其分子药理学特征进行系统研究。我们得到了8种生物碱类化合物和6种挥发油类化合物对wt-CFTR均具有明显的激活作用。由于前期本实验室已对生物碱中有明显活性的菏叶碱和白鲜碱有系统的研究,因此我们对其余六种生物碱类化合物进行进一步的研究分析,同时我们也分析了挥发油类化合物中活性最强的对羟基苯甲酸丁酯。结果表明,六种生物碱类化合物和对羟基苯甲酸丁酯对野生型CFTR Cl-通道具有明显的激活作用,其中盐酸罂粟碱、尼莫地平、对羟基苯甲酸丁酯对?F508突变型通道开放障碍也有明显的激活作用,同时胡椒碱也表现出了一定的活性。此外,六种生物碱类化合物和对羟基苯甲酸丁酯对G551D突变型CFTR Cl-通道都无激活作用,也不能纠正?F508-CFTR蛋白胞内转运的障碍。上述七种单体化合物对野生型和突变型CFTR Cl-通道的激活作用具有作用迅速、可逆、剂量依赖的特点,初步机制分析结果表明它们可能是通过与CFTR直接结合而发挥作用的。本研究中七种单体化合物对CFTR Cl-通道具有激活作用不仅为阐明CFTR的结构与功能之间的关系,以及作为先导化合物开发探索治疗与CFTR突变有关疾病等方面具有重要用途,而且为进一步明确这七种化合物其它方面的药理机制奠定了基础。更多还原

【Abstract】 The CAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a family of proteins called traffic ATPases or ABC transporters. Its mutation will cause the most prevalence lethal hereditary disease-cystic fibrosis (CF). It is most prominently expressed in the apical membrane of various epithelia in mammal involved secretion and absorption (such as intestine, airways, pancreas, sweat ducts), which works as Cl- channel and also a regulator of other anion channels. Therefore, CFTR is closely related to many physiological and pathological conditions. Impaired function of CFTR Cl- channels in CF disrupts transepithelial ion transport, thus leads to the wide-ranging manifestations of the disease such as idiopathic chronic pancreatitis (ICP), keratoconjunctivitis sicca (KCS), habitual constipation. Evidence also showed that over active of CFTR is associated with such diseases as secretory diarrhea and polycystic kidney disease. Identification of small-molecule modulators to regulate the function of wild-type and mutant CFTR have been attracted many attention in recent years and have obtained substantial progress, which is still one of most important fields of CFTR study.A stably transfected Fischer thyroid epithelial (FRT) cell lines co-expressing ?F508-CFTR or wt-CFTR and a green fluorescent protein mutant with ultra-high halide sensitivity (EYFP-H148Q) was generated as assay for the drug screening. Using this cell model, 8 alkaloid compounds and 6 volatile compounds were screened for CFTR-mediated halide transport. Previously, we have a systematic study to Holland and Dictamnine which have significantly activition, so we make further research and analysis on the remaining six compounds of alkaloid compounds, at the same time we also analyzedButyl-phydroxybenzoate (Bpb) which has strongest activity in the volatile oil compounds. As a result, the six alkaloid compounds and Bpb can attivate wild-type CFTR chloride channel gating. Papaverine hydrochloride, Nimodipine, Bpb and Piperine can stimulated ?F508-CFTR mediate anion transport, but had no effect on ?F508-CFTR misprocessing defect or G551D-CFTR channel gating defect. Pharmacological properties of the seven compounds were characterized systematically in terms of the dose-dependent, reversibility, activition time-course and so on. The study provided clues that they activate CFTR chloride channel through a direct binding mechanism.This studies indicated that we regard the seven compounds not only as important tools in better understanding the structure and function of CFTR but aslo as a lead compound to develop pharmacological therapy of CFTR-related disease. On the other hand, it lays a foundation for them at further exploring other aspects pharmacology mechanism.更多还原