本文選題：細(xì)胞膜外基質(zhì)　切入點：金納米顆粒　出處：《湖南大學(xué)》2012年博士論文

【摘要】：前人已經(jīng)證明了顆粒尺寸大小、形貌、表面化學(xué)、表面電荷等物理化學(xué)性質(zhì)以及大顆粒的沉降、細(xì)胞周期等因素會影響納米顆粒的內(nèi)吞效率。但以往的研究中大家都普遍認(rèn)為細(xì)胞膜是細(xì)胞內(nèi)環(huán)境與外環(huán)境的唯一障礙。實際上，在大多真核細(xì)胞外都存在著一層細(xì)胞膜外基質(zhì)。因膜外基質(zhì)層透明不易被觀察，因此在以往細(xì)胞內(nèi)吞的研究中被認(rèn)為是物質(zhì)進(jìn)入細(xì)胞的障礙或被忽略，更有研究工作通過降解膜外基質(zhì)來提高小分子的內(nèi)吞效率。在納米顆粒的內(nèi)吞研究進(jìn)展中，膜外基質(zhì)對納米顆粒進(jìn)入細(xì)胞的影響至今是空白。因膜外基質(zhì)對細(xì)胞的貼壁、遷移、增殖、分化等生命活動起著很重要的作用，且存在于細(xì)胞膜外，是納米顆粒接觸細(xì)胞的第一層。因此，本論文在前人研究基礎(chǔ)上，以金納米顆粒作為探針，利用暗場顯微鏡研究了細(xì)胞膜外基質(zhì)對納米顆粒內(nèi)吞過程及內(nèi)吞效率的影響，，主要開展了以下工作： （1）第2章我們通過實時示蹤單個金納米顆粒的內(nèi)吞過程，根據(jù)顆粒的運動受限程度，顆粒進(jìn)入細(xì)胞可分為四個階段，這與以往別人的研究結(jié)果不一致，新發(fā)現(xiàn)了內(nèi)吞過程中膜外基質(zhì)層對顆粒運動的影響。通過單顆粒擴(kuò)散系數(shù)計算及暗場層層聚焦，發(fā)現(xiàn)顆粒在距離細(xì)胞膜幾微米范圍內(nèi)運動受限，速度減慢，并且膜外基質(zhì)層對顆粒擴(kuò)散運動的影響與細(xì)胞系及顆粒的表面化學(xué)無關(guān)。 （2）為了更加直接的證明膜外基質(zhì)對金納米顆粒內(nèi)吞的影響，我們在第3章中用自身具有厚膜外基質(zhì)的MG-63細(xì)胞作為厚膜外基質(zhì)的細(xì)胞模型，降解膜外基質(zhì)后的MG-63細(xì)胞作為薄膜外基質(zhì)的細(xì)胞模型。利用暗場顯微鏡半定量的證明了在相同的顆粒濃度及培養(yǎng)條件下，厚膜外基質(zhì)的細(xì)胞對顆粒具有更高的內(nèi)吞效率，暗場結(jié)果顯示厚膜外基質(zhì)細(xì)胞的各個層面都聚集了大量的顆粒。接著，利用電感耦合等離子體發(fā)射光譜儀(ICP-AES)直接定量的證明了在相同條件下，厚膜外基質(zhì)的細(xì)胞比薄膜外基質(zhì)的細(xì)胞對金納米顆粒具有更多的內(nèi)吞量。初步說明了膜外基質(zhì)促進(jìn)了顆粒的內(nèi)吞效率，相比之下，膜外基質(zhì)變薄之后，顆粒內(nèi)吞量大大降低，暗示膜外基質(zhì)在顆粒內(nèi)吞過程起著很重要的作用。 （3）前面的工作已經(jīng)證明了厚膜外基質(zhì)細(xì)胞可以促進(jìn)金納米顆粒的內(nèi)吞效率，暗場結(jié)果明顯顯示出顆粒在細(xì)胞膜外基質(zhì)和細(xì)胞內(nèi)都有大量的聚集。第4章主要研究膜外基質(zhì)促進(jìn)顆粒內(nèi)吞效率的機制。將與顆粒共培養(yǎng)之后的厚膜外基質(zhì)細(xì)胞和薄膜外基質(zhì)細(xì)胞樣品，用共聚焦顯微鏡對各個焦面上的顆粒進(jìn)行分層聚焦，發(fā)現(xiàn)厚膜外基質(zhì)細(xì)胞在膜外基質(zhì)層聚集了大量的粒子，而薄膜外基質(zhì)細(xì)胞中幾乎沒有聚集顆粒。再者，利用共聚焦顯微鏡實時示蹤了顆粒在厚膜外基質(zhì)細(xì)胞中的聚集過程，結(jié)果顯示在較短的時間內(nèi)有大量的顆粒在厚膜外基質(zhì)細(xì)胞中聚集，提高了細(xì)胞膜外的顆粒濃度，從而促進(jìn)了顆粒的內(nèi)吞量。接著，我們通過單顆粒示蹤發(fā)現(xiàn)顆粒在膜外基質(zhì)中的擴(kuò)散運動受限變慢，顆粒的擴(kuò)散系數(shù)接近0.1m2/s，這個值與很多膜受體在細(xì)胞膜上的擴(kuò)散系數(shù)接近，說明了顆粒擴(kuò)散系數(shù)慢有利于顆粒與膜上受體的結(jié)合，從而促進(jìn)了顆粒的內(nèi)吞效率。而薄膜外基質(zhì)情況下，顆粒的擴(kuò)散系數(shù)偏大，不易于顆粒與膜上受體的結(jié)合。內(nèi)吞較少。證明了膜外基質(zhì)通過兩方面促進(jìn)顆粒內(nèi)吞效率：一方面通過在膜外基質(zhì)中捕獲和聚集大量顆粒，提高了與細(xì)胞膜作用的顆粒濃度，高濃度可實現(xiàn)高的內(nèi)吞效率；二是通過減慢顆粒的擴(kuò)散系數(shù)，使其與膜上受體的擴(kuò)散系數(shù)接近，便于受體與配體的結(jié)合，從而促進(jìn)內(nèi)吞量。通過能量實驗和內(nèi)吞抑制劑實驗證明了細(xì)胞膜外基質(zhì)是受體介導(dǎo)內(nèi)吞過程的一部分，對納米顆粒的聚集及促進(jìn)內(nèi)吞都是能量依賴的。 （4）前人工作已證明細(xì)胞對納米顆粒的內(nèi)吞與顆粒的尺寸、形貌、表面化學(xué)都有很大的關(guān)系。第5章工作研究膜外基質(zhì)促進(jìn)顆粒內(nèi)吞與顆粒自身的物理化學(xué)性質(zhì)的關(guān)系。通過進(jìn)行了50nm、60nm、90nm、120nm金顆粒的細(xì)胞內(nèi)吞實驗，證明了厚膜外基質(zhì)促進(jìn)顆粒的內(nèi)吞效率與顆粒的尺寸無關(guān)。表面化學(xué)的結(jié)果說明了細(xì)胞膜外基質(zhì)促進(jìn)金納米顆粒內(nèi)吞與顆粒表面化學(xué)有很大的關(guān)系，膜外基質(zhì)對不同表面化學(xué)的顆粒具有篩選的作用。 （5）為了更進(jìn)一步證明膜外基質(zhì)促進(jìn)金納米顆粒的內(nèi)吞不是只在MG-63細(xì)胞中，而是具有一定的普遍性。第6章根據(jù)別人報導(dǎo)的抗壞血酸(ascorbic acid,AA)處理細(xì)胞可以促進(jìn)細(xì)胞膜外基質(zhì)的成分增加。我們用HeLa細(xì)胞作為正常細(xì)胞的細(xì)胞模型，經(jīng)AA處理之后的HeLa細(xì)胞作為厚膜外基質(zhì)的細(xì)胞模型，降解膜外基質(zhì)后的細(xì)胞作為薄膜外基質(zhì)的細(xì)胞模型。利用暗場顯微鏡研究了不同處理的HeLa細(xì)胞對金納米顆粒的內(nèi)吞情況。與正常細(xì)胞的內(nèi)吞結(jié)果相比，薄膜外基質(zhì)細(xì)胞具有更少的內(nèi)吞量，而厚膜外基質(zhì)細(xì)胞具有更多的內(nèi)吞量。 （6）在研究細(xì)胞內(nèi)吞的影響因素基礎(chǔ)上，還考察了內(nèi)吞不同尺寸的金納米顆粒后細(xì)胞所產(chǎn)生的細(xì)胞反應(yīng)(第7章)。通過研究20nm、50nm、137nm金顆粒對HeLa細(xì)胞貼壁、增殖、骨架排布等生命活動的影響。證明了宏觀MTT毒性表征手段顯示顆粒在較寬的濃度范圍內(nèi)是無毒的。但單細(xì)胞染色統(tǒng)計結(jié)果表明不同尺寸的顆粒內(nèi)吞后對HeLa細(xì)胞的功能都產(chǎn)生了不同程度的影響。結(jié)果證明了以往利用MTT細(xì)胞活性表征方法研究納米材料的毒性是不夠的，納米材料的進(jìn)一步生物醫(yī)學(xué)應(yīng)用，需要更多的手段來表征納米顆粒對細(xì)胞的毒性影響。并且以P19老鼠畸胎癌細(xì)胞為干細(xì)胞模型研究了內(nèi)吞金納米顆粒之后對干細(xì)胞神經(jīng)向分化的影響，初步發(fā)現(xiàn)了金納米顆粒能促進(jìn)干細(xì)胞的神經(jīng)向分化。
[Abstract]:It has been proved that the particle size, morphology, surface chemistry, settlement of physical and chemical properties of surface charge and large particles, cell cycle and other factors will affect the nanoparticle endocytosis efficiency. But previous studies have generally agreed that the cell membrane is the only obstacle to cell internal environment and external environment. In fact, there are a layer of extracellular matrix in most eukaryotic cells. The extracellular matrix layer is not easy to be observed, so in previous studies of endocytosis is considered the material into the cell barrier or ignored, more research work to improve the efficiency of small molecule endocytosis by degrading the extracellular matrix. Progress in research on endocytosis of nanoparticles, effects of extracellular matrix on nano particles into the cells is still blank. Because the extracellular matrix of cell adhesion, migration, proliferation, differentiation and other life activities play a very important role, And in cell membrane, cell contact is the first layer of nanoparticles. Therefore, this paper on the basis of previous studies, using gold nanoparticles as probes, the effect of extracellular matrix on the cell membrane internalization and endocytosis efficiency in the use of nano particle darkfield microscopy, mainly carried out the following work:
(1) the second chapter we through endocytosis process real-time tracing single gold nanoparticles, according to the degree of limitation of movement of particles, particles into the cells can be divided into four stages, the previous research and the results of others are not consistent, newly discovered membrane matrix layer movement effect on particles through. Single particle diffusion coefficient calculation and dark layers of focus, found particle motion at a distance of a few microns within the cell membrane is limited, slow down, and the film surface chemical matrix layer diffusion effect and cell lines and particles of particles.
(2) in order to prove the film more direct matrix on gold nanoparticle endocytosis effect, we use their own in the third chapter, with a thick film matrix MG-63 cell as a model of the thick film matrix, MG-63 cells to degrade the extracellular matrix as the cell model of thin film matrix. Proved by means of semi quantitative darkfield microscopy the particle concentration in the same culture and under the condition of thick film matrix cells have higher endocytosis efficiency of particles, the dark field showed that each level of thick film extracellular matrix cells are gathered a large number of particles. Then, using inductively coupled plasma emission spectrometer (ICP-AES) direct quantitative proved that under the same conditions thick film, the extracellular matrix of cells than the film matrix cells with endocytosis more of gold nanoparticles. The preliminary research showed that the extracellular matrix promotes particle endocytosis efficiency, by contrast, film When the outer matrix is thinner, the endocytosis of the particles is greatly reduced, suggesting that the extracellular matrix plays an important role in the process of endocytosis.
(3) the previous work has proved that extracellular matrix can promote cell thick film gold nanoparticles endocytosis efficiency, dark field results clearly show that particles in the cell membrane and extracellular matrix cells have large aggregation. The fourth chapter mainly studies the extracellular matrix to promote the mechanism of particle endocytosis efficiency. The thick film cells and extracellular matrix film samples after coculture with stromal cell particles, using confocal microscopy for each focal plane of particles are found outside the matrix of thick film layer focus, stromal cells in the outer membrane layer gathered a large number of particles, while the film matrix cells almost no aggregation of particles. Furthermore, using confocal microscopy in real time tracing aggregation the process of particles in thick film extracellular matrix cells, results in a relatively short period of time has gathered a large number of particles in the film matrix cells, increase the concentration of particles outside the cell membrane, from promoting The amount of endocytosis of particles. Then, we found by single particle tracer diffusion limited particle matrix in the outer membrane of slow diffusion coefficient of particles is close to 0.1m2/s, and this value is close to many membrane receptors on the cell membrane diffusion coefficient, the diffusion coefficient of particles to slow particles and membrane receptor the combination, so as to promote the particle endocytosis efficiency. The matrix film, the diffusion coefficient of particles is too large, not easy to particles and membrane receptor endocytosis. Less extracellular matrix. It is proved that through the two aspects of promoting particle endocytosis efficiency: on the one hand through the extracellular matrix in the capture and gathered a large number of particles, the particle concentration and improve the function of cell membrane, high concentration can achieve high endocytic efficiency; two is through the slow diffusion coefficient of particles, and the diffusion coefficient of membrane receptors to facilitate the receptor and the ligand. In conclusion, the energy and endocytosis inhibitors show that extracellular matrix is a part of receptor mediated endocytosis process, and it is energy dependent on aggregation and promoting endocytosis of nanoparticles.
(4) the previous work has shown that endocytosis and granule cells of nano particle size, morphology, surface chemistry have a great relationship. The fifth chapter research work of extracellular matrix to promote the relationship between the physical and chemical properties of particles and particle endocytosis itself. Through the 50nm, 60NM, 90nm, 120nm gold particles cells endocytosis experiments proved that extracellular matrix promotes endocytosis of thick film efficiency and particle size. The results show that the surface chemistry of extracellular matrix promotes gold nanoparticles in surface chemistry and swallow particles have a great relationship, particles of extracellular matrix of different surface chemistry has a role in screening.
(5) in order to further prove that extracellular matrix promotes endocytosis of gold nanoparticles not only in MG-63 cells, but has a certain universality. In the sixth chapter, according to the reports of others (ascorbic acid ascorbic acid, AA) treating cells extracellular matrix components increase. We used HeLa cell as a model of normal the cell, after the AA treatment of HeLa cells as a cell model of thick film matrix cells, degradation of extracellular matrix as the cell model of thin film matrix. Endocytosis study of different treatment by darkfield microscopy of HeLa cells on the gold nanometer particles. Compared with normal cell endocytosis results, endocytosis amount the film has less stromal cells and extracellular matrix, thick film cells with endocytosis more volume.
(6) the basic factors in the study of endocytosis, cell reaction was also investigated with different sizes of gold nanoparticles endocytosis generated after the cell (the seventh chapter). Through the study of 20nm, 50nm, 137nm gold particles on HeLa cell adhesion, proliferation, cytoskeleton arrangement and other life activities. The influence of proof the macro MTT toxicity characterization showed that the particles are non-toxic in a wide concentration range. But the single cell staining on HeLa cell function statistical results show that the particles of different sizes after endocytosis are affected in different degree. Results show that the previous use of MTT cell activity characterization methods of nanomaterials toxicity is not enough further, biomedical applications of nano materials, need more means to characterize the toxicity of nanoparticles on cell. The effects of P19 and mouse teratocarcinoma cells for stem cell model to study the way in nanoparticles after the dry The effect of cell nerve to differentiation has been found that gold nanoparticles can promote the neural differentiation of stem cells.

【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：O614.123;Q26

【共引文獻(xiàn)】

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