本文選題：機(jī)械敏感通道　切入點(diǎn)：氯通道　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：在生物體中,機(jī)械敏感通道介導(dǎo)許多感覺方式,比如聽力,觸摸和本體感覺[1]。機(jī)械力作用到機(jī)械敏感通道時(shí),通道自身分子構(gòu)象會(huì)發(fā)生結(jié)構(gòu)變化,從而將機(jī)械力轉(zhuǎn)化成電信號(hào),將機(jī)體感覺系統(tǒng)的信號(hào)傳遞下去。在多細(xì)胞生物體中已知的機(jī)械敏感通道還很少,所以尋找新的機(jī)械敏感通道成為非常關(guān)鍵的問題。在本研究中,通過線蟲在體膜片鉗技術(shù),我們首次發(fā)現(xiàn)雄蟲特異性神經(jīng)元CEM能夠直接響應(yīng)機(jī)械刺激,并且CEM上的機(jī)械敏感通道是氯離子選擇性的。不僅如此,我們還發(fā)現(xiàn)當(dāng)離體分化培養(yǎng)線蟲單細(xì)胞時(shí),從CEM上依然可以記錄到機(jī)械響應(yīng)電流,并且電流性質(zhì)與在體時(shí)一致。CEM響應(yīng)機(jī)械刺激時(shí),導(dǎo)致細(xì)胞內(nèi)的鈣水平升高,我們推測(cè)生理狀態(tài)下,CEM胞內(nèi)氯離子濃度高于胞外,這也在外源表達(dá)組胺門控的氯離子通道的方式下得到證實(shí)。與此同時(shí),藥理學(xué)實(shí)驗(yàn)顯示,CEM上機(jī)械門控的氯離子通道能夠被NFA、DCPIB所抑制。經(jīng)過一系列的篩選和鑒定,我們也得到了 一些候選基因,但是CEM上介導(dǎo)機(jī)械刺激的通道具體是什么還需要進(jìn)一步的研究。我們首次鑒定出生物體中存在直接的機(jī)械敏感陰離子通道,我們的工作為鑒定新的陰離子選擇性的機(jī)械敏感通道奠定了基礎(chǔ)。生物體利用特異的感覺神經(jīng)元來(lái)感受不同的疼痛刺激,包括傷害性的化學(xué)和機(jī)械刺激。然而,這些神經(jīng)元如何感受和區(qū)分不同的刺激還不清楚。通過在體鈣成像和分子遺傳學(xué)操作,我們研究了線蟲尾感神經(jīng)元PHA/PHB對(duì)不同感覺刺激的響應(yīng)及其可能的分子機(jī)制。我們的結(jié)果表明,PHA/PHB神經(jīng)元是多功能感覺神經(jīng)元,不僅對(duì)包括1-octanol、SDS、甘油及高濃度的IAA在內(nèi)的有害化學(xué)刺激有響應(yīng),而且對(duì)傷害性機(jī)械刺激也有響應(yīng)。我們發(fā)現(xiàn)TRPV家族的OSM-9和CNG通道亞基TAX-4對(duì)于PHA/PHB神經(jīng)元的化學(xué)感受是非常重要的。此外,PHA/PHB神經(jīng)元能夠通過其他神經(jīng)元釋放的神經(jīng)肽間接被重金屬銅離子所抑制。我們還表明了OSM-9,還未被報(bào)道是已知的機(jī)械敏感通道,在PHA/PHB神經(jīng)元的機(jī)械感受中起作用。綜上所述,我們的工作證明了多功能神經(jīng)元PHA/PHB是怎樣被環(huán)境中眾多的刺激所激活和調(diào)節(jié)的,并且為多功能信號(hào)比如在更高等的生物體中的傷害刺激感受機(jī)制的理解提供了基礎(chǔ)。神經(jīng)突觸囊泡(synaptic vesicles,SV)存儲(chǔ)神經(jīng)遞質(zhì),神經(jīng)元在接受刺激后,突觸囊泡與突觸前膜融合,并釋放神經(jīng)遞質(zhì),遞質(zhì)與突觸后的受體結(jié)合,引起突觸后產(chǎn)生動(dòng)作電位。然后囊泡通過內(nèi)吞作用回收,開始新一輪的循環(huán)過程。盡管對(duì)于囊泡的研究已經(jīng)很多,但是它的分泌和內(nèi)吞機(jī)制研究的還并不是很清楚。在本文中,我們希望利用線蟲作為模式生物,通過全基因組的RNAi篩選找到更多參與調(diào)控突觸囊泡循環(huán)的新基因。我們利用在神經(jīng)系統(tǒng)中表達(dá)SNB-1::pHluorin(SpH)融合蛋白的RNAi敏感型線蟲作為探針線蟲,并用流式線蟲儀COPAS進(jìn)行高通量的熒光篩選。經(jīng)過多次篩選驗(yàn)證,我們得到177個(gè)上調(diào)基因和97個(gè)下調(diào)基因,而且76%的基因在進(jìn)化上是保守的。我們最終選取了一個(gè)未知功能的基因B0035.1/ZNF207,繼續(xù)研究它在神經(jīng)突觸囊泡循環(huán)中的作用。b0035.1是進(jìn)化上非常保守的基因,具有C2H2鋅指蛋白結(jié)構(gòu)域,并且在神經(jīng)系統(tǒng)和部分肌肉細(xì)胞的細(xì)胞核內(nèi)表徸,b0035.1突變體線蟲的運(yùn)動(dòng)軌跡異常并且運(yùn)動(dòng)速率變慢,具有明顯的Aldicarb抗性,SpH的熒光亮度增大,SNB-1在軸突定位缺陷,內(nèi)源性興奮性突觸后電流頻率降低以及誘發(fā)性興奮性突觸后電流減小。結(jié)合CHIP-seq和RNA-seq的結(jié)果,我們經(jīng)過qPCR檢測(cè),最終發(fā)現(xiàn),rab-11.1的RNA表達(dá)量在突變體線蟲中降低,rab-11.1與b0035.1RNAi品系有相同的SpH熒光表型,并且疊加作用時(shí)沒有產(chǎn)生疊加效應(yīng),證明b0035.1與rab-11.11在同一通路上起作用。這些數(shù)據(jù)說明了 B0035.1可能通過影響rab-11.1基因的功能,進(jìn)而影響了神經(jīng)突觸囊泡的循環(huán)過程。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q42

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