【作者】 韩重阳；

【导师】 王晓良；

【作者基本信息】 中国协和医科大学 ， 分子药理学， 2007， 博士

【摘要】 细胞膜离子通道在许多疾病的发生、发展过程中发生基因、蛋白表达及功能水平上的改变，并引起一系列临床症状，离子通道还是许多疾病的药物靶点，本论文旨在研究原发性红斑肢痛症和脑缺血这两种疾病中不同离子通道的变异、调节和功能变化在疾病中的作用。第一部分原发性红斑肢痛症相关的离子通道功能改变原发性红斑肢痛症是一种常染色体显性遗传病，主要临床表现为四肢末端反复发作的阵发性对称性灼痛，热刺激或运动后加重，遇冷后可部分缓解，现在尚无很好的治疗药物。其病因和病生理机制长期以来并不清楚，最近研究发现电压依赖性钠离子通道Nav1.7突变可导致原发性红斑肢痛症的疼痛等相关症状。本研究中，我们采用全细胞膜片钳技术先后研究了在中国人群中发现的两例原发性红斑肢痛症的Nav1.7通道突变L858F和V872G的电生理功能学变化，并探讨了温度降低缓解原发性红斑肢痛症疼痛的机理。一、本研究中的L858F突变的受累者为两名儿童，其父亲为无症状的嵌合体。我们对稳定转染野生型Nav1.7和Nav1.7-L858F突变体的细胞的钠通道各项电生理学参数进行了比较。我们发现：与野生型相比，L858F突变使得稳态半数激活电位向超极化方向移动约9mV，稳态半数失活电位向去极化方向移动约3mV，L858F突变还使得Nav1.7通道的去活化速度显著变慢，此外通道的复活速度在突变后显著加快，而关闭态失活速度则显著变慢，L858F突变还显著增大Nav1.7通道的斜坡电流，约为野生型的4倍。这些结果提示L858F突变使得Nav1.7通道发生“功能增强”的变化，从而使得表达Nav1.7的外周感觉神经元电兴奋性增强，原发性红斑肢痛症患者表现出相关的疼痛症状。二、降温可以部分缓解原发性红斑肢痛症的疼痛症状，因此我们在对L858F突变体电生理学功能研究的基础上，又观察了温度变化对Nav1.7通道野生型和L858F突变体门控特性的影响。在实验中我们分别选择了16℃、25℃和35℃作为参照温度。研究发现：对于Nav1.7野生型通道和L858F突变体，温度降低都使得通道电流密度降低，去活化速度变慢，以及斜坡电流增大。此外温度降低后野生型通道的稳态半数激活电位没有发生显著性变化，而L858F突变体的稳态半数激活电位却向去极化方向移动，所以低温下突变体的激活阈值更接近野生型，这可能是降温缓解原发性红斑肢痛症疼痛的生理基础。三、本研究中V872G突变所累及的患者为一散发病例，我们对稳定转染该突变体的细胞株和转染野生型Nav1.7通道的细胞株电生理学研究发现：V872G突变使得Nav1.7通道稳态半数激活电位向超极化方向移动约3mV，稳态半数失活电位向去极化方向移动约7mV，突变后通道的去活化速度变慢，复活速度加快，而关闭态失活速度变慢，此外V872G突变还使得Nav1.7通道的斜坡电流增大至野生型的2.5倍。因此我们认为Nav1.7通道的V872G突变也是一种“功能增强”的突变，导致感觉神经元异常放电，患者产生疼痛反应。总之，我们的研究表明在原发性红斑肢痛症中新发现的Nav1.7通道的L858F和V872G突变都是使得钠通道发生“功能增强”的变化，表达Nav1.7通道的神经元电兴奋性增强，导致患者表现出相关的疼痛症状。温度降低后使得突变体和野生型钠通道激活阈值差异减小，疼痛得以缓解。第二部分瞬时受体电位通道亚型在大鼠脑缺血中的基因表达及药理学研究瞬时受体电位通道(Transient Receptor Potential Channel，TRP Channel)是一类重要的非选择性阳离子通道，因其分布广泛，参与细胞众多基本生理功能，近年来成为离子通道领域研究的热点。脑缺血是脑血管疾病中最主要的类型，严重危害着人类的健康。脑缺血发生时以及缺血后再灌注可引起神经元缺氧、氧自由基爆发和酸中毒等众多病理生理学变化。以往研究报道瞬时受体电位通道TRPM2和TRPM7亚型可被包括缺氧和氧自由基等在内的多种因素所调节，在神经元死亡中起着重要的作用。为此我们应用大脑中动脉阻断方法建立大鼠急性短暂性脑缺血模型和持续性脑缺血模型，采用Real-Time PCR的方法分别检测了这两种病理损伤过程中大鼠皮层和海马不同时间点的TRPM2和TRPM7通道mRNA表达的变化。对大鼠大脑皮层的研究发现：在短暂性脑缺血中，TRPM2 mRNA在缺血2h后再灌24h时表达显著降低，减少了66.3％，TRPM7 mRNA在再灌2h、12h和24h时表达都显著降低，分别减少了47.2％，50.9％和68.9％。；在持续性脑缺血中，TRPM2 mRNA在缺血2h、12h和24h时表达都显著下降，分别减少了27.5％，55.9％和19.6％，TRPM7 mRNA在缺血2h和24h时表达亦显著下降，分别减少了26.6％和34.4％。对大鼠大脑海马的研究发现，在短暂性脑缺血中TRPM2 mRNA表达在缺血2h后再灌的各个时间点都没有显著性变化，TRPM7 mRNA在再灌6h时表达显著升高，增加了141.5％；在持续性脑缺血中TRPM2 mRNA在缺血2h和6h时表达显著升高，分别增加了47.5％和31.3％，TRPM7 mRNA亦在缺血2h和6h时表达显著性升高，分别增加了1188.5％和347.4％。结果表明无论TRPM2还是TRPM7在大鼠大脑皮层缺血和海马缺血的病理过程中的作用都不相同，特别是TRPM7可能在海马缺血中起着非常重要的作用。鉴于TRPM7通道的重要生理功能和病生理作用，我们构建了四环素调控的稳定表达TRPM7通道的细胞株。四环素诱导后TRPM7通道高表达，并产生TRPM7通道特异性电流，电流的大小对四环素剂量和诱导时间呈现出一定的依赖性。同时我们采用激光共聚焦成像技术观察了左旋丁基苯酞(1-NBP)，我们课题组自主研发的新型抗脑缺血药物，对表达TRPM7通道细胞的胞内钙离子的影响。结果发现1-NBP可以降低细胞内钙离子浓度，提示1-NBP对TRPM7通道可能具有一定的抑制作用。更多还原

【Abstract】 As the important transmembrane proteins, ion channels play a key role in the lifeof cells. And the alterations on the expressions and functions of ion channels havebeen found in lots of diseases. Meanwhile, ion channels are also the drug targets formany diseases. In the present thesis, we investigated the expressions and functions ofdifferent types of ion channels within two kinds of diseases, primary erythermalgiaand cerebral ischemia, in order to illuminate the roles of these channels in the relateddisorders.Part I Functional Studies of Ion Channels related with Primary ErythermalgiaPrimary erythermalgia (PEM) is a kind of autosomal dominant disease,characterized by intermittent burning pain with redness and heat in the extremities. Itcould be induced by heat or exercise, whereas keeping the involved extremities at anyicy cold temperature is the most effective way to relieve pain. It’s known little on thepathogenesis and the mechanism of PEM in the past. In recently, it’s reported that themutation of sodium channel Navl.7 could cause the syndromes of pain within PEM.In the present study, we carried on the electrophysiological studies of two newmutations which were found in Chinese PEM patients. Meanwhile, we investigatedthe physiological basis for the phenomenon that the pain within PEM can bealleviated by cooling.L858F mutation, a single amino acid substitution in Navl.7 was present in twochildren whose parents were asymptomatic. The asymptomatic father was geneticallymosaic for the mutation. The whole-cell patch clamp technique was used to comparethe electrophysiological characters of Nav1.7 wild-type channels and L858F mutants.Compared with wild-type, activation of L858F mutant channels was shifted by-9 mV,whereas steady-state inactivation was shifted by +3mV. There was a marked decreasein the rate of deactivation of L858F mutant channels, and the same change for the rate of closed-state inactivation. On the rate of recovery, L858F mutant channels showedthe significant faster than the wild-type channels. L858F also increased the rampcurrents which elicited by slow, small depolariztions, with 4 times larger than that ofwild-type channels. Our results suggested that L858F mutation could conferhyperexcitability on peripheral sensory neurons, and underlie PEM.Attacks of pain in PEM are alleviated by cooling of the limbs, but thephysiological basis for this phenomenon is not understood. Therefore, we investigatedthe influence of cooling on the biophysical properties of Nav1.7 wild-type and L858Fmutant channels. Whole-cell voltage-clamp measurements on wild-type or L858Fmutant channels expressed in HEK-293 cells revealed that cooling decreases currentdensity, slows deactivation and increases ramp currents for both wild-type and mutant.However, cooling differentially shifts the midpoint of steady-state activation in adepolarizing direction for L858F but not for wild type channels, which brings thethreshold of activation of the mutant channels closer to that of wild-type Navl.7 atlower temperatures. And we think that is likely to contribute to the alleviation ofpainful symptoms upon cooling in affected limbs of patient with PEM.V872G, a new mutation of Navl.7 was found in a Chinese girl with PEM. LikeL858F mutant, whole-cell patch clamp analysis was employed to characterizebiophysical properties of wild-type and the mutant channels in HEK-293 cells. Theresults showed that this new mutation produced a hyperpolarizing shift of about 3 mVin activation and a depolarizing shift of about 7 mV in steady-state inactivation. Alsolike L858F mutant, V872G mutant could decrease the rate of deactivationsignificantly and increase the rate of recovery. The rate of closed-state inactivation ofV872G was decreased significantly than that of wild-type. Meanwhile, the rampcurrent of V872G was about 2.5 times larger than that of wild-type. These changesshould increase excitability of nociceptive dorsal root ganglion neurons in which themutant channels are distributed, thus contributes to pain.In summary, we found that both L858F and V872G mutation present in patientswith PEM cause Nav1.7 ’gain of function’, which contributed to symptom productionin it. And the decreases in the difference of the activation between wild-type and mutants may contribute to the clinical observation that cooling alleviates pain.PartⅡGene Expressions of Transient Receptor Potential Channel Subtypes inRat Brain with Cerebral Ischemia & Relevant Pharmacological StudiesTransient Receptor Potential Channel (TRP channel), an important superfamilyof non-selective cation channels, with wide distribution and various functions, hasbeen one of the focuses in the research of ion channels recently.Stroke, including occlusive stroke, is one of the leading causes of death in theworld. It has been known that many pathophysiological mechanisms are responsiblefor the injury after cerebral ischemia and reperfusion, such as anoxia of neurons,bursting of free radicals and cumulation of acid substances.TRPM2 and TRPM7, two members of TRPM channel subfamily, have beendemonstrated to be regulated by anoxia and free radicals, and play an important rolein the death of neurons. In the present study, we used MCAO (middle cerebral arteryocclusion) to mimic the transient cerebral ischemia and permanent cerebral ischemia.And we observed the mRNA expressions ofTRPM2 & TRPM7 in rat brain cortex andhippocampus at different time points in these two pathophysiological conditions.Real-time PCR was employed to investigate the mRNA levels of TRPM2 &TRPM7 at 2h, 6h, 12h and 24h of reperfusion after 2h of MCAO during transientcerebral ischemia and the mRNA levels of TRPM2 & TRPM7 after 2h, 6h, 12h and24h of MCAO during permanent cerebral ischemia. In cortex, our results showed thatduring transient ischemia, the mRNA levels of TRPM2 was decreased by 66.3% at24h, and the mRNA levels of TRPM7 was decreased by 47.2%, 50.9% and 68.9% at2h, 12h and 24h respectively. And during permanent ischemia, the mRNA levels ofTRPM2 was decreased by 27.5%, 55.9% and 19.6% at 2h, 12h and 24h respectively,and the mRNA levels of TRPM7 was decreased by 26.6% and 34.4% at 2h and 24hrespectively. In hippocampus, we got the different results from the cortex. We foundthat during transient cerebral ischemia, there was not any alteration in the mRNAlevels of TRPM2 at these four time points, whereas the mRNA levels of TRPM7 was increased by 141.5% at 6h. Meanwhile, during permanent ischemia, the mRNA levelsof TRPM2 was increased by 47.5% and 31.3% at 2 h and 6h respectively, and themRNA levels of TRPM7 was increased by 1188.5% and 347.4% at 2h and 6h too.Our results suggested both TRPM2 and TRPM7 may play different roles duringthe ischemia of cortex and hippocampus, especially, TRPM7 may have importantroles during the ischemia of hippocampus.On the basis of the important physiological and pathophysiological functions ofTRPM7 channel, we constructed the stable cell lines transfected with TRPM7 gene.The expression of TRPM7 channel in the cell lines was regulated by tetracycline. Werecorded the characteristic TRPM7 channel currents in the cell lines after beingtreated with tetracycline and the amplitude of TRPM7 channel currents wasdependent on the dose and the treating-time of tetracycline. At the end, we studied theeffects of 1-NBP, a new anti-ischemia medicine, which invented by our research group,on the concentration of intracellular calcium with confocal assay. The results showedthat 1-NBP could decrease the concentration of intracellular calcium. And theseimplied that 1-NBP may have the inhibition effect on TRPM7 channel.更多还原