秀丽隐杆线虫感觉传导的分子机制研究和神经突触囊泡循环关键基因的筛选及鉴定
发布时间:2018-03-03 04:14
本文选题:机械敏感通道 切入点:氯通道 出处:《浙江大学》2016年博士论文 论文类型:学位论文
【摘要】:在生物体中,机械敏感通道介导许多感觉方式,比如听力,触摸和本体感觉[1]。机械力作用到机械敏感通道时,通道自身分子构象会发生结构变化,从而将机械力转化成电信号,将机体感觉系统的信号传递下去。在多细胞生物体中已知的机械敏感通道还很少,所以寻找新的机械敏感通道成为非常关键的问题。在本研究中,通过线虫在体膜片钳技术,我们首次发现雄虫特异性神经元CEM能够直接响应机械刺激,并且CEM上的机械敏感通道是氯离子选择性的。不仅如此,我们还发现当离体分化培养线虫单细胞时,从CEM上依然可以记录到机械响应电流,并且电流性质与在体时一致。CEM响应机械刺激时,导致细胞内的钙水平升高,我们推测生理状态下,CEM胞内氯离子浓度高于胞外,这也在外源表达组胺门控的氯离子通道的方式下得到证实。与此同时,药理学实验显示,CEM上机械门控的氯离子通道能够被NFA、DCPIB所抑制。经过一系列的筛选和鉴定,我们也得到了 一些候选基因,但是CEM上介导机械刺激的通道具体是什么还需要进一步的研究。我们首次鉴定出生物体中存在直接的机械敏感阴离子通道,我们的工作为鉴定新的阴离子选择性的机械敏感通道奠定了基础。生物体利用特异的感觉神经元来感受不同的疼痛刺激,包括伤害性的化学和机械刺激。然而,这些神经元如何感受和区分不同的刺激还不清楚。通过在体钙成像和分子遗传学操作,我们研究了线虫尾感神经元PHA/PHB对不同感觉刺激的响应及其可能的分子机制。我们的结果表明,PHA/PHB神经元是多功能感觉神经元,不仅对包括1-octanol、SDS、甘油及高浓度的IAA在内的有害化学刺激有响应,而且对伤害性机械刺激也有响应。我们发现TRPV家族的OSM-9和CNG通道亚基TAX-4对于PHA/PHB神经元的化学感受是非常重要的。此外,PHA/PHB神经元能够通过其他神经元释放的神经肽间接被重金属铜离子所抑制。我们还表明了OSM-9,还未被报道是已知的机械敏感通道,在PHA/PHB神经元的机械感受中起作用。综上所述,我们的工作证明了多功能神经元PHA/PHB是怎样被环境中众多的刺激所激活和调节的,并且为多功能信号比如在更高等的生物体中的伤害刺激感受机制的理解提供了基础。神经突触囊泡(synaptic vesicles,SV)存储神经递质,神经元在接受刺激后,突触囊泡与突触前膜融合,并释放神经递质,递质与突触后的受体结合,引起突触后产生动作电位。然后囊泡通过内吞作用回收,开始新一轮的循环过程。尽管对于囊泡的研究已经很多,但是它的分泌和内吞机制研究的还并不是很清楚。在本文中,我们希望利用线虫作为模式生物,通过全基因组的RNAi筛选找到更多参与调控突触囊泡循环的新基因。我们利用在神经系统中表达SNB-1::pHluorin(SpH)融合蛋白的RNAi敏感型线虫作为探针线虫,并用流式线虫仪COPAS进行高通量的荧光筛选。经过多次筛选验证,我们得到177个上调基因和97个下调基因,而且76%的基因在进化上是保守的。我们最终选取了一个未知功能的基因B0035.1/ZNF207,继续研究它在神经突触囊泡循环中的作用。b0035.1是进化上非常保守的基因,具有C2H2锌指蛋白结构域,并且在神经系统和部分肌肉细胞的细胞核内表徸,b0035.1突变体线虫的运动轨迹异常并且运动速率变慢,具有明显的Aldicarb抗性,SpH的荧光亮度增大,SNB-1在轴突定位缺陷,内源性兴奋性突触后电流频率降低以及诱发性兴奋性突触后电流减小。结合CHIP-seq和RNA-seq的结果,我们经过qPCR检测,最终发现,rab-11.1的RNA表达量在突变体线虫中降低,rab-11.1与b0035.1RNAi品系有相同的SpH荧光表型,并且叠加作用时没有产生叠加效应,证明b0035.1与rab-11.11在同一通路上起作用。这些数据说明了 B0035.1可能通过影响rab-11.1基因的功能,进而影响了神经突触囊泡的循环过程。
[Abstract]:In organisms, mechanosensitive channel mediated by many ways such as feeling, hearing, touch and proprioception [1]. mechanical force to the mechanosensitive channel, channel its molecular conformation occur in the structural changes, which will be mechanical power into electrical signals, the signal body sensory systems pass down the mechanosensitive channel known in multicellular organisms in a little, so finding new mechanosensitive channel become a key issue. In this study, the in vivo nematode patch clamp technique, we first found that male specific neurons CEM can direct response to mechanical stimulation, and mechanosensitive channel CEM is chloride ion selective. Moreover, we also found that when in vitro differentiation of nematode single cell, from CEM is still able to record the mechanical response of current and current properties and in vivo.CEM response to mechanical stimulation That led to the increase in intracellular calcium levels, we speculate that the physiological condition, chloride ion concentration of CEM in cells is higher than that of extracellular, the exogenous expression of chloride channel histamine gated mode is confirmed. At the same time, pharmacological experiments showed that CEM gated chloride ion channel machinery can be NFA, DCPIB suppression. After screening and identification of a series, we also got some candidate genes, but CEM mediated mechanical stimulation channel specifically what needs further study. We first identify the existence of mechanical sensitivity directly sense of anion channel was born in an object, laid the foundation for our work for the identification of new anion selectivity mechanosensitive channel. Organisms use specific sensory neurons to different feelings of pain, including chemical and mechanical nociceptive stimulation. However, these neurons to feel and distinguish not The same stimulus is not clear. The in vivo calcium imaging and molecular genetics, we investigated nematode response to different PHA/PHB tail sympathetic neurons to sensory stimulation and its possible molecular mechanisms. Our results suggest that PHA/PHB neurons are multifunctional sensory neurons, not only including 1-octanol, SDS, glycerol and high concentrations of harmful chemical IAA, the stimulus response, but also in response to noxious mechanical stimulation. We found that the TRPV family OSM-9 and CNG channel subunit TAX-4 is very important for the PHA/PHB chemosensory neurons. In addition, PHA/PHB neurons can release neuropeptides by other neurons indirectly by copper ions inhibited. We also show that OSM-9 that has not been reported is a mechanosensitive channel known, play a role in mechanoreceptive PHA/PHB neurons. In summary, our work demonstrates the versatility How many neurons in PHA/PHB were stimulated in the environment that is activated and regulated, and provide the basis for the multi function signal in higher organisms in damage to stimulate feelings of the understanding of the mechanism of synaptic vesicles. (synaptic vesicles, SV) memory neurotransmitter neurons during stimulation, synaptic vesicle fuse with the presynaptic membrane, and the release of neurotransmitters, neurotransmitter and combination of postsynaptic receptors, induced postsynaptic action potentials. Then vesicle recycling through endocytosis, start a new round of cycle. Although there has been lots of vesicles, but its secretory and endocytic mechanisms are still not very clear. In this paper, we hope to use elegans as a model organism, through genome-wide RNAi screening to find more new genes involved in synaptic vesicle recycling. We use the expression of SNB-1 in the nervous system :: pHluorin (SpH) fusion protein RNAi sensitive nematode as probe nematode, and screened by high-throughput flow cytometry nematode device COPAS. After several rounds of screening test, we get 177 upregulated genes and 97 downregulated genes, and 76% genes are conserved during evolution. We finally select gene B0035.1/ZNF207 is an unknown function, continue to study it in the synaptic vesicle cycle.B0035.1 is an evolutionarily conserved gene, with C2H2 zinc finger domain, and Christmas in the nervous system and muscle cell nucleus, trajectory of b0035.1 mutant nematode and abnormal movement rate is slow, with the obvious resistance to Aldicarb, SpH fluorescence intensity increases, SNB-1 in axonal defect location, the frequency of the current reduction of the endogenous excitatory postsynaptic and evoked excitatory postsynaptic currents decreased by CHI. P-seq and RNA-seq results, we passed the test of qPCR, eventually found that the expression of rab-11.1 RNA in the mutant nematodes decreased, rab-11.1 and b0035.1RNAi strains have the same SpH fluorescence phenotype, and additive effect without the additive effects, that b0035.1 and rab-11.11 play a role in the same pathway. These data indicate that B0035.1 may the effect of rab-11.1 gene function, thereby affecting the circulation of synaptic vesicles.
【学位授予单位】:浙江大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:Q42
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1 邹文娟;秀丽隐杆线虫感觉传导的分子机制研究和神经突触囊泡循环关键基因的筛选及鉴定[D];浙江大学;2016年
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