不规则钙振荡通过累积波宽和振幅调节转录和基因表达

发布时间：2018-01-11 09:04

本文关键词：不规则钙振荡通过累积波宽和振幅调节转录和基因表达　出处：《华中科技大学》2012年博士论文　论文类型：学位论文

【摘要】：钙振荡是普遍存在的一种钙信号形式,可观察于几乎所有的非兴奋性细胞。钙振荡频率依赖性的调节机制已经被广泛接受,包括调节转录因子活性,细胞增殖、分化、迁移等。Dolmetsch等人早在1998年利用钙钳技术成功阐明了钙振荡调节转录因子活性的频率依赖性,频率依赖性的调节方式显著提高了钙信号的特异性和工作效率。与此同时,钙振荡频率协同H202高效率的调节转录因子NFκB的转录活性。转录因子NFAT磷酸化与非磷酸化的动力学研究解释了钙振荡频率如何调节了NFAT的激活进而影响NFAT的转录活性。研究者们对钙振荡特异性调节机制的研究一致的体现了频率依赖性的特点。然而,钙振荡频率依赖性调节的本质是什么?对此已有学者做了深入的研究,研究结果表明钙振荡频率调节的内在机制是累积波宽(cumulated spike duration),累积波宽依赖性机制表现为钙离子浓度在某一阈值水平时的累积时间,相同时间内,规则钙振荡频率越大其累积波宽越大,频率越小其累积波宽越小,因此转录因子转录活性的频率依赖性实质是累积波宽依赖性。经过不断深入的探索,我们已经对钙振荡参数调节下游生物学效应的机制有了深刻的认识。新的问题又出现,以往对钙振荡的研究主要基于人工钙振荡模型的建立,所建立的人工模型均为规则的钙振荡,然而在众多生理或病理生理状况下,刺激所诱发的细胞胞浆钙振荡表现为不规则的形式：振幅不齐,频率不均一,波宽(spike duration)不一致,并在细胞间表现钙振荡的异质性。例如缺氧后再灌注诱发内皮细胞不规则钙振荡,雌激素作用鼠卵母细胞诱发不规则钙振荡,组胺刺激Hela细胞诱发不规则钙振荡,趋化因子刺激中性粒细胞诱发不规则钙振荡,卵巢粒层细胞呈现高度不规则自发性钙振荡,膀胱平滑肌细胞不规则自发性钙振荡。于是生物相关性钙振荡表现为不规则性和异质性。那么基于规则钙振荡模型(低生物相关性)得出的结论是否同样适用于不规则钙振荡呢?于是应用10pM组胺刺激细胞,同时监测胞浆钙振荡和转录因子核转位,在单细胞水平研究生物相关性的不规则和异质性钙振荡调节下游转录因子活性的内在机制。为确切验证单细胞水平的研究结论,研究不规则钙振荡参数和下游生物学效应的因-效关系,我们应用钙钳技术灌流人脐静脉内皮细胞在群体细胞水平建立振幅相同、频率相同,累积波宽不同和振幅相同、累积波宽相同,频率不同的同质性的不规则钙振荡模型,对转录因子NFκB、STAT3转录活性及下游基因IL-8、VEGF的表达进行检测,发现振幅相同、频率相同,累积波宽不同时,NFκB及下游基因IL-8表达不同；振幅相同、累积波宽相同,频率不同时,NFκB、STAT3转录活性及下游基因IL-8、VEGF表达相同。单细胞水平的异质性不规则钙振荡和群体细胞水平同质性不规则钙振荡的研究结果表明生物相关性不规则钙振荡通过累积波宽和振幅调节下游生物学效应。
[Abstract]:Calcium oscillations are a common form of calcium signal, can be observed in almost all non excitable cells. The regulation mechanism of calcium oscillation frequency dependence has been widely accepted, including the regulation of transcription factor activity, cell proliferation, differentiation, migration,.Dolmetsch et al in 1998 using the calcium clamp technique successfully explains the regulation of calcium oscillation the transcription factor activity of the frequency dependent regulation of frequency dependence significantly increased the calcium signal specificity and efficiency. At the same time, calcium oscillation frequency CO regulation of transcription factor NF kappa B transcription activity of H202 high efficiency. Kinetics of transcription factor NFAT phosphorylation and non phosphorylation explains how to adjust the frequency of calcium oscillation the activation of NFAT and affect the transcriptional activity of NFAT. The study on calcium oscillation in specific regulatory mechanisms consistent reflects the characteristics of frequency dependence. However, What is the nature of the calcium oscillation frequency dependent regulation? The scholars have done in-depth research, the results show that the intrinsic mechanism of calcium oscillation frequency adjustment is the cumulative wave width (cumulated spike duration), the cumulative wave width dependent mechanism shows the cumulative time of calcium ion concentration in a certain threshold level at the same time. The rules of calcium oscillation frequency is larger, the cumulative wave width is larger, the smaller the cumulative frequency wave width is smaller, so the transcription activity of the frequency dependence is cumulative wave width dependence. Through continuous in-depth exploration, a deep understanding of the mechanism we have regulated the downstream biological effects on calcium oscillation parameters. New problems arise, the previous research on calcium oscillation is mainly based on the establishment of artificial calcium oscillation model, artificial model for calcium oscillation rules, however, in many physiological or pathological physiology Under the condition, intracellular calcium oscillations evoked by stimulation showed irregular forms: amplitude uneven, frequency is not uniform, wave width (spike duration) are not consistent, and the heterogeneity of calcium oscillations in the cell. For example, after hypoxia reperfusion induced endothelial cell irregular calcium oscillations, estrogen effect of mouse oocytes the mother cell induced by irregular calcium oscillations, histamine stimulated Hela cells induced by irregular calcium oscillations, chemokine stimulated neutrophils induced by irregular calcium oscillations, ovarian granulosa cells appear to be highly irregular spontaneous calcium oscillations in bladder smooth muscle cells, irregular spontaneous calcium oscillations. Then the biological relevance of calcium oscillations showed irregular and heterogeneous based on the rules. Then the calcium oscillation model (low biological relevance) whether the conclusion is also applicable to irregular calcium oscillations? So the application of 10pM histamine stimulated cells, while monitoring cytosolic calcium oscillations And the transcription factor nuclear translocation mechanism regulating downstream transcription factor activity in the study on irregular biological relevance of single cell level and heterogeneity of calcium oscillations. To study the exact verification of the single cell level conclusion, effect of irregular calcium oscillation parameters and downstream biological effects, we used calcium clamp perfusion of people human umbilical vein endothelial cells in the cell population level establishes the same amplitude and the same frequency, different amplitude and cumulative wave width, cumulative wave width, irregular homogeneity of different frequency of calcium oscillation model, the transcription factor NF kappa B, STAT3 transcriptional activity and downstream gene IL-8, VEGF expression was detected and found the same amplitude. The same frequency, cumulative wave width is not at the same time, the different expression of NF kappa B and downstream gene IL-8; the same amplitude, the cumulative wave width of same frequency at the same time, NF kappa B, STAT3 transcriptional activity and downstream gene IL-8, VEGF At the same level, heterogenous irregular calcium oscillations at single cell level and homogenous irregular calcium oscillations at population level showed that biologically related irregular calcium oscillations modulate downstream biological effects by accumulating wave width and amplitude.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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