【摘要】：生物光子學(xué)是光學(xué)與生物學(xué)高度交叉的前沿領(lǐng)域,飛秒激光技術(shù)的進(jìn)步,為生物光子學(xué)提供了一系列新技術(shù)、新方法、新思路、新理論。目前基于飛秒激光的生物光子學(xué)已經(jīng)在多光子成像、細(xì)胞顯微操作等領(lǐng)域得到了深入的研究和廣泛的應(yīng)用。然而,此前飛秒激光刺激對(duì)細(xì)胞的影響和損傷機(jī)制的研究相對(duì)粗糙,飛秒激光脈沖和細(xì)胞相互作用的機(jī)制本身,以及細(xì)胞對(duì)飛秒激光刺激的應(yīng)激反應(yīng)等問(wèn)題卻一直尚未清晰闡明。在這樣的大背景之下,本文研究了飛秒激光刺激對(duì)細(xì)胞過(guò)程的影響和調(diào)節(jié)機(jī)制,提出了新的飛秒激光調(diào)控細(xì)胞內(nèi)分子行為的方法,并進(jìn)一步發(fā)展了超快成像技術(shù)。本文首先綜述了基于飛秒激光的生物光子學(xué)的進(jìn)展、近年來(lái)研究的關(guān)鍵問(wèn)題與發(fā)展現(xiàn)狀。接著,作為相關(guān)生物分析的基礎(chǔ)理論,簡(jiǎn)要介紹了鈣離子的相關(guān)背景,包括鈣離子的存儲(chǔ)、生理作用、與之相關(guān)的分子信號(hào)等。進(jìn)一步地,介紹了基于飛秒激光器、共聚焦顯微鏡、CCD等設(shè)備構(gòu)成的顯微操作系統(tǒng),并討論了在此系統(tǒng)之上使用飛秒激光調(diào)控細(xì)胞內(nèi)鈣離子濃度變化的實(shí)驗(yàn)現(xiàn)象和相關(guān)機(jī)理。在以上內(nèi)容的基礎(chǔ)上,我們發(fā)現(xiàn),飛秒激光可以進(jìn)一步調(diào)節(jié)細(xì)胞內(nèi)的多種分子行為。通過(guò)精細(xì)地調(diào)節(jié)飛秒激光曝光刺激,可使其對(duì)He La細(xì)胞內(nèi)鈣離子進(jìn)行釋放或維持細(xì)胞的高鈣水平,這種機(jī)制可以使得鈣調(diào)磷酸酶充分將轉(zhuǎn)錄因子NFAT去磷酸化,從而激活NFAT的表達(dá)鏈路,使其在光刺激之后入核,啟動(dòng)相應(yīng)的下游基因的表達(dá)。我們?cè)趯?shí)驗(yàn)上實(shí)現(xiàn)了飛秒激光對(duì)轉(zhuǎn)錄因子NFAT入核過(guò)程的調(diào)控,作為原理性的驗(yàn)證,實(shí)驗(yàn)中使用了熒光素酶報(bào)告基因做了檢測(cè)。進(jìn)一步地,我們使用飛秒激光對(duì)細(xì)胞的刺激,可以直接調(diào)控細(xì)胞自身的TNF-α等基因的表達(dá)。更進(jìn)一步地,我們將這種技術(shù)應(yīng)用于人類間充質(zhì)干細(xì)胞(MSCs)中,實(shí)驗(yàn)上實(shí)現(xiàn)了飛秒激光對(duì)間充質(zhì)干細(xì)胞內(nèi)鈣離子濃度的調(diào)控,并且對(duì)干細(xì)胞內(nèi)的轉(zhuǎn)錄因子Runx2、Osterix等具有一定的影響,這種光刺激方法在誘導(dǎo)干細(xì)胞分化領(lǐng)域具有一定的應(yīng)用價(jià)值。然而,飛秒激光刺激對(duì)于細(xì)胞的損傷是不可忽略的。我們發(fā)現(xiàn)不管是飛秒激光的單次短時(shí)間刺激,還是低功率連續(xù)激光掃描,都會(huì)影響細(xì)胞自噬水平。我們系統(tǒng)性地觀測(cè)了不同的激光刺激對(duì)于細(xì)胞自噬的影響,分析了兩種刺激機(jī)制,論證了細(xì)胞自噬對(duì)于光刺激的敏感性。我們認(rèn)為,激光對(duì)于細(xì)胞的刺激,即使相對(duì)安全,不會(huì)對(duì)細(xì)胞活性造成較大影響,但依然會(huì)引起細(xì)胞自噬等過(guò)程。在飛秒激光的刺激下,線粒體可能是自噬體膜的來(lái)源之一。由于飛秒激光作用于細(xì)胞屬于超快行為,所以對(duì)超快行為的觀測(cè)具有著重要的意義。本文最后介紹了STEAM、STAMP、FIRE等幾種超快成像技術(shù),論述了這些技術(shù)的技術(shù)特點(diǎn)、發(fā)展現(xiàn)狀以及應(yīng)用場(chǎng)合等問(wèn)題。實(shí)驗(yàn)上參與設(shè)計(jì)搭建了新型FIRE成像系統(tǒng),得到了熒光成像圖樣,并對(duì)實(shí)驗(yàn)中遇到的一些細(xì)節(jié)問(wèn)題做了實(shí)驗(yàn)上的探索以及總結(jié)。本文的一系列工作探索了飛秒激光對(duì)細(xì)胞刺激帶來(lái)的影響,為光學(xué)方法對(duì)細(xì)胞過(guò)程和功能的調(diào)控提供了全新的理論和技術(shù)基礎(chǔ)。
[Abstract]:Biophotonics is the leading field of optical and biological high-crossing, and the progress of femtosecond laser technology provides a series of new technologies, new methods, new ideas and new theories for biophotonics. At present, the biological photonics based on femtosecond laser have been deeply studied and widely used in many fields such as multi-photon imaging, cell micro-operation and so on. However, the effects of the femtosecond laser stimulation on the cell and the mechanism of the damage are relatively rough, the mechanism of the femtosecond laser pulse and the cell interaction, and the stress response of the cell to the femtosecond laser stimulation have not been clearly set forth. Under such a background, the influence of femtosecond laser stimulation on the cell process and the regulation mechanism were studied. The new method of femtosecond laser to regulate the intracell molecular behavior was put forward, and the ultra-fast imaging technique was further developed. In this paper, the progress of the bio-photonics based on femtosecond laser is reviewed, and the key problems and the development status of the research are reviewed in recent years. Then, as the basic theory of the related biological analysis, the related background of the calcium ion is briefly introduced, including the storage of calcium ion, the physiological function, the related molecular signal and so on. In this paper, a micro-operating system based on femtosecond laser, confocal microscope and CCD is introduced, and the experimental phenomena and related mechanism of the change of calcium ion concentration in the cell using femtosecond laser are discussed. On the basis of the above, we have found that the femtosecond laser can further regulate the various molecular behaviors in the cell. by finely adjusting the femtosecond laser exposure stimulus, it can release or maintain the high calcium level of the cells on the calcium ions in the He La cells, allowing it to enter the nucleus after the light stimulus to initiate the expression of the corresponding downstream gene. In the experiment, the regulation of the nuclear process of the transcription factor NFAT by the femtosecond laser is realized, and the luciferase reporter gene is used as the detection in the experiment. Further, we use the femtosecond laser to stimulate the cell, which can directly control the expression of the TNF-1 gene of the cell itself. Further, we apply this technique to the human mesenchymal stem cells (MSCs), and the effect of the femtosecond laser on the calcium ion concentration in the mesenchymal stem cells is realized, and the transcription factor Runx2, Osterx and the like in the stem cells have a certain influence, The photostimulation method has certain application value in the field of inducing stem cell differentiation. However, femtosecond laser stimulation is not negligible for cell damage. We found that both a single short-time stimulus of a femtosecond laser or a low-power continuous laser scan would affect the level of autophagy. We systematically observed the effects of different laser stimuli on the autophagy of the cells, and analyzed the two stimulation mechanisms, and demonstrated the sensitivity of the autophagy to the photostimulation. In our opinion, the stimulation of the laser to the cells, even relatively safe, does not have a great effect on the cell activity, but it still causes the cell autophagy and the like. Under the stimulation of femtosecond laser, the mitochondria may be one of the source of autophagy. It is of great significance to observe the ultra-fast behavior because the femtosecond laser is applied to the super-fast behavior of the cell. In this paper, several super fast imaging technologies, such as STEAM, STAMP, FIRE, etc., are introduced in this paper. The technical features, development status and application of these technologies are discussed. In the experiment, a new type of FIRE imaging system was built, and the fluorescence imaging pattern was obtained, and some of the detailed problems encountered in the experiment were explored and summarized. A series of work in this paper explores the influence of femtosecond laser on the cell stimulation, and provides a new theoretical and technical basis for the regulation of the process and function of the cell by the optical method.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q27;TN249

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