基于暗场成像技术研究细胞膜外基质对金纳米颗粒内吞的影响

发布时间：2018-03-23 19:15

本文选题：细胞膜外基质　切入点：金纳米颗粒　出处：《湖南大学》2012年博士论文

【摘要】：前人已经证明了颗粒尺寸大小、形貌、表面化学、表面电荷等物理化学性质以及大颗粒的沉降、细胞周期等因素会影响纳米颗粒的内吞效率。但以往的研究中大家都普遍认为细胞膜是细胞内环境与外环境的唯一障碍。实际上，在大多真核细胞外都存在着一层细胞膜外基质。因膜外基质层透明不易被观察，因此在以往细胞内吞的研究中被认为是物质进入细胞的障碍或被忽略，更有研究工作通过降解膜外基质来提高小分子的内吞效率。在纳米颗粒的内吞研究进展中，膜外基质对纳米颗粒进入细胞的影响至今是空白。因膜外基质对细胞的贴壁、迁移、增殖、分化等生命活动起着很重要的作用，且存在于细胞膜外，是纳米颗粒接触细胞的第一层。因此，本论文在前人研究基础上，以金纳米颗粒作为探针，利用暗场显微镜研究了细胞膜外基质对纳米颗粒内吞过程及内吞效率的影响，，主要开展了以下工作：（1）第2章我们通过实时示踪单个金纳米颗粒的内吞过程，根据颗粒的运动受限程度，颗粒进入细胞可分为四个阶段，这与以往别人的研究结果不一致，新发现了内吞过程中膜外基质层对颗粒运动的影响。通过单颗粒扩散系数计算及暗场层层聚焦，发现颗粒在距离细胞膜几微米范围内运动受限，速度减慢，并且膜外基质层对颗粒扩散运动的影响与细胞系及颗粒的表面化学无关。（2）为了更加直接的证明膜外基质对金纳米颗粒内吞的影响，我们在第3章中用自身具有厚膜外基质的MG-63细胞作为厚膜外基质的细胞模型，降解膜外基质后的MG-63细胞作为薄膜外基质的细胞模型。利用暗场显微镜半定量的证明了在相同的颗粒浓度及培养条件下，厚膜外基质的细胞对颗粒具有更高的内吞效率，暗场结果显示厚膜外基质细胞的各个层面都聚集了大量的颗粒。接着，利用电感耦合等离子体发射光谱仪(ICP-AES)直接定量的证明了在相同条件下，厚膜外基质的细胞比薄膜外基质的细胞对金纳米颗粒具有更多的内吞量。初步说明了膜外基质促进了颗粒的内吞效率，相比之下，膜外基质变薄之后，颗粒内吞量大大降低，暗示膜外基质在颗粒内吞过程起着很重要的作用。（3）前面的工作已经证明了厚膜外基质细胞可以促进金纳米颗粒的内吞效率，暗场结果明显显示出颗粒在细胞膜外基质和细胞内都有大量的聚集。第4章主要研究膜外基质促进颗粒内吞效率的机制。将与颗粒共培养之后的厚膜外基质细胞和薄膜外基质细胞样品，用共聚焦显微镜对各个焦面上的颗粒进行分层聚焦，发现厚膜外基质细胞在膜外基质层聚集了大量的粒子，而薄膜外基质细胞中几乎没有聚集颗粒。再者，利用共聚焦显微镜实时示踪了颗粒在厚膜外基质细胞中的聚集过程，结果显示在较短的时间内有大量的颗粒在厚膜外基质细胞中聚集，提高了细胞膜外的颗粒浓度，从而促进了颗粒的内吞量。接着，我们通过单颗粒示踪发现颗粒在膜外基质中的扩散运动受限变慢，颗粒的扩散系数接近0.1m2/s，这个值与很多膜受体在细胞膜上的扩散系数接近，说明了颗粒扩散系数慢有利于颗粒与膜上受体的结合，从而促进了颗粒的内吞效率。而薄膜外基质情况下，颗粒的扩散系数偏大，不易于颗粒与膜上受体的结合。内吞较少。证明了膜外基质通过两方面促进颗粒内吞效率：一方面通过在膜外基质中捕获和聚集大量颗粒，提高了与细胞膜作用的颗粒浓度，高浓度可实现高的内吞效率；二是通过减慢颗粒的扩散系数，使其与膜上受体的扩散系数接近，便于受体与配体的结合，从而促进内吞量。通过能量实验和内吞抑制剂实验证明了细胞膜外基质是受体介导内吞过程的一部分，对纳米颗粒的聚集及促进内吞都是能量依赖的。（4）前人工作已证明细胞对纳米颗粒的内吞与颗粒的尺寸、形貌、表面化学都有很大的关系。第5章工作研究膜外基质促进颗粒内吞与颗粒自身的物理化学性质的关系。通过进行了50nm、60nm、90nm、120nm金颗粒的细胞内吞实验，证明了厚膜外基质促进颗粒的内吞效率与颗粒的尺寸无关。表面化学的结果说明了细胞膜外基质促进金纳米颗粒内吞与颗粒表面化学有很大的关系，膜外基质对不同表面化学的颗粒具有筛选的作用。（5）为了更进一步证明膜外基质促进金纳米颗粒的内吞不是只在MG-63细胞中，而是具有一定的普遍性。第6章根据别人报导的抗坏血酸(ascorbic acid,AA)处理细胞可以促进细胞膜外基质的成分增加。我们用HeLa细胞作为正常细胞的细胞模型，经AA处理之后的HeLa细胞作为厚膜外基质的细胞模型，降解膜外基质后的细胞作为薄膜外基质的细胞模型。利用暗场显微镜研究了不同处理的HeLa细胞对金纳米颗粒的内吞情况。与正常细胞的内吞结果相比，薄膜外基质细胞具有更少的内吞量，而厚膜外基质细胞具有更多的内吞量。（6）在研究细胞内吞的影响因素基础上，还考察了内吞不同尺寸的金纳米颗粒后细胞所产生的细胞反应(第7章)。通过研究20nm、50nm、137nm金颗粒对HeLa细胞贴壁、增殖、骨架排布等生命活动的影响。证明了宏观MTT毒性表征手段显示颗粒在较宽的浓度范围内是无毒的。但单细胞染色统计结果表明不同尺寸的颗粒内吞后对HeLa细胞的功能都产生了不同程度的影响。结果证明了以往利用MTT细胞活性表征方法研究纳米材料的毒性是不够的，纳米材料的进一步生物医学应用，需要更多的手段来表征纳米颗粒对细胞的毒性影响。并且以P19老鼠畸胎癌细胞为干细胞模型研究了内吞金纳米颗粒之后对干细胞神经向分化的影响，初步发现了金纳米颗粒能促进干细胞的神经向分化。
[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.

【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：O614.123;Q26

【共引文献】