本文關(guān)鍵詞：不規(guī)則鈣振蕩通過累積波寬和振幅調(diào)節(jié)轉(zhuǎn)錄和基因表達　出處：《華中科技大學》2012年博士論文　論文類型：學位論文

  更多相關(guān)文章： 不規(guī)則鈣振蕩 受體刺激 人臍靜脈內(nèi)皮細胞 單細胞 RelA-GFP STAT3-GFP 不規(guī)則鈣振蕩 受體刺激 轉(zhuǎn)錄因子NFκB STAT3 IL-8 VEGF IκBα JAK-2 人臍靜脈內(nèi)皮細胞

【摘要】：鈣振蕩是普遍存在的一種鈣信號形式,可觀察于幾乎所有的非興奮性細胞。鈣振蕩頻率依賴性的調(diào)節(jié)機制已經(jīng)被廣泛接受,包括調(diào)節(jié)轉(zhuǎn)錄因子活性,細胞增殖、分化、遷移等。Dolmetsch等人早在1998年利用鈣鉗技術(shù)成功闡明了鈣振蕩調(diào)節(jié)轉(zhuǎn)錄因子活性的頻率依賴性,頻率依賴性的調(diào)節(jié)方式顯著提高了鈣信號的特異性和工作效率。與此同時,鈣振蕩頻率協(xié)同H202高效率的調(diào)節(jié)轉(zhuǎn)錄因子NFκB的轉(zhuǎn)錄活性。轉(zhuǎn)錄因子NFAT磷酸化與非磷酸化的動力學研究解釋了鈣振蕩頻率如何調(diào)節(jié)了NFAT的激活進而影響NFAT的轉(zhuǎn)錄活性。研究者們對鈣振蕩特異性調(diào)節(jié)機制的研究一致的體現(xiàn)了頻率依賴性的特點。然而,鈣振蕩頻率依賴性調(diào)節(jié)的本質(zhì)是什么?對此已有學者做了深入的研究,研究結(jié)果表明鈣振蕩頻率調(diào)節(jié)的內(nèi)在機制是累積波寬(cumulated spike duration),累積波寬依賴性機制表現(xiàn)為鈣離子濃度在某一閾值水平時的累積時間,相同時間內(nèi),規(guī)則鈣振蕩頻率越大其累積波寬越大,頻率越小其累積波寬越小,因此轉(zhuǎn)錄因子轉(zhuǎn)錄活性的頻率依賴性實質(zhì)是累積波寬依賴性。經(jīng)過不斷深入的探索,我們已經(jīng)對鈣振蕩參數(shù)調(diào)節(jié)下游生物學效應的機制有了深刻的認識。新的問題又出現(xiàn),以往對鈣振蕩的研究主要基于人工鈣振蕩模型的建立,所建立的人工模型均為規(guī)則的鈣振蕩,然而在眾多生理或病理生理狀況下,刺激所誘發(fā)的細胞胞漿鈣振蕩表現(xiàn)為不規(guī)則的形式：振幅不齊,頻率不均一,波寬(spike duration)不一致,并在細胞間表現(xiàn)鈣振蕩的異質(zhì)性。例如缺氧后再灌注誘發(fā)內(nèi)皮細胞不規(guī)則鈣振蕩,雌激素作用鼠卵母細胞誘發(fā)不規(guī)則鈣振蕩,組胺刺激Hela細胞誘發(fā)不規(guī)則鈣振蕩,趨化因子刺激中性粒細胞誘發(fā)不規(guī)則鈣振蕩,卵巢粒層細胞呈現(xiàn)高度不規(guī)則自發(fā)性鈣振蕩,膀胱平滑肌細胞不規(guī)則自發(fā)性鈣振蕩。于是生物相關(guān)性鈣振蕩表現(xiàn)為不規(guī)則性和異質(zhì)性。那么基于規(guī)則鈣振蕩模型(低生物相關(guān)性)得出的結(jié)論是否同樣適用于不規(guī)則鈣振蕩呢?于是應用10pM組胺刺激細胞,同時監(jiān)測胞漿鈣振蕩和轉(zhuǎn)錄因子核轉(zhuǎn)位,在單細胞水平研究生物相關(guān)性的不規(guī)則和異質(zhì)性鈣振蕩調(diào)節(jié)下游轉(zhuǎn)錄因子活性的內(nèi)在機制。為確切驗證單細胞水平的研究結(jié)論,研究不規(guī)則鈣振蕩參數(shù)和下游生物學效應的因-效關(guān)系,我們應用鈣鉗技術(shù)灌流人臍靜脈內(nèi)皮細胞在群體細胞水平建立振幅相同、頻率相同,累積波寬不同和振幅相同、累積波寬相同,頻率不同的同質(zhì)性的不規(guī)則鈣振蕩模型,對轉(zhuǎn)錄因子NFκB、STAT3轉(zhuǎn)錄活性及下游基因IL-8、VEGF的表達進行檢測,發(fā)現(xiàn)振幅相同、頻率相同,累積波寬不同時,NFκB及下游基因IL-8表達不同；振幅相同、累積波寬相同,頻率不同時,NFκB、STAT3轉(zhuǎn)錄活性及下游基因IL-8、VEGF表達相同。單細胞水平的異質(zhì)性不規(guī)則鈣振蕩和群體細胞水平同質(zhì)性不規(guī)則鈣振蕩的研究結(jié)果表明生物相關(guān)性不規(guī)則鈣振蕩通過累積波寬和振幅調(diào)節(jié)下游生物學效應。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R363

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