本文關(guān)鍵詞： 納米二氧化鈦 C17.2細(xì)胞 胞吞 胞排 生物相容性　出處：《上海大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：隨著納米技術(shù)的迅速發(fā)展，納米材料的廣泛應(yīng)用和納米材料產(chǎn)量的迅速增加，人們開(kāi)始關(guān)注納米材料的安全性。例如，在生物醫(yī)學(xué)領(lǐng)域，納米材料作為藥物傳輸?shù)妮d體對(duì)病理組織和細(xì)胞有很好的靶向性并且能夠起到安全有效的藥物療法的作用。其中，納米二氧化鈦(nano-TiO_2)因其獨(dú)特的物理和化學(xué)性質(zhì)在諸多領(lǐng)域得到廣泛的應(yīng)用。納米二氧化鈦對(duì)于生物體的安全性研究變得尤為重要，特別是納米材料在細(xì)胞內(nèi)的基本代謝過(guò)程，如細(xì)胞的胞吞作用和胞排作用。神經(jīng)干細(xì)胞(NSCs)是神經(jīng)系統(tǒng)中各類神經(jīng)細(xì)胞的原組細(xì)胞，它既具有增值特征又擁有分化發(fā)育的潛能。目前，對(duì)于納米TiO_2的生物毒性、環(huán)境效應(yīng)、與生物組織的相互作用還沒(méi)有系統(tǒng)的研究結(jié)果。針對(duì)目前的研究現(xiàn)狀，本論文擬在細(xì)胞水平上，以神經(jīng)干細(xì)胞為細(xì)胞模型，對(duì)三種不同形貌和粒徑的納米TiO_2的胞吞和胞排作用進(jìn)行定量的研究。 納米顆粒的物理化學(xué)性質(zhì)及表面性能決定其生物學(xué)作用，論文首先對(duì)三種不同粒徑不同形貌的納米TiO_2的純度、粒徑、形貌、晶型、表面電勢(shì)等進(jìn)行了充分的表征。隨后采用CCK-8和活性氧檢測(cè)等手段評(píng)估納米TiO_2對(duì)神經(jīng)干細(xì)胞的細(xì)胞活力和細(xì)胞內(nèi)ROS水平的影響。毒性檢測(cè)結(jié)果顯示，納米TiO_2在低濃度下對(duì)細(xì)胞無(wú)毒。納米TiO_2對(duì)細(xì)胞的毒性存在著時(shí)間效應(yīng)和濃度效應(yīng)，當(dāng)納米顆粒濃度達(dá)到150mg/L以上時(shí)，細(xì)胞活力開(kāi)始下降，細(xì)胞內(nèi)的活性氧顯著增加，但與納米材料的尺寸和形貌沒(méi)有相關(guān)性。 納米TiO_2的胞吞和胞排過(guò)程研究：胞吞作用隨著時(shí)間的增加，攝取量也明顯增加，在48h后攝取量幾乎達(dá)到了飽和狀態(tài)。粒徑越小的納米顆粒胞吞速度越快，更容易進(jìn)入細(xì)胞，管狀的納米顆粒比球狀的更難進(jìn)入細(xì)胞。對(duì)于不同濃度的納米TiO_2來(lái)說(shuō)，納米材料的濃度增加，細(xì)胞攝取量也成倍增加，但對(duì)攝取率沒(méi)有影響。胞吞作用受納米材料的尺寸、形貌和濃度的影響。細(xì)胞對(duì)納米管的攝取速度比對(duì)另外兩種納米顆粒的攝取速度快。根據(jù)胞吞量隨時(shí)間的變化曲線，選擇48h為起點(diǎn)，采用細(xì)胞多步傳代的方法，通過(guò)激光共聚焦顯微鏡對(duì)細(xì)胞的胞排過(guò)程做了動(dòng)態(tài)的追蹤實(shí)驗(yàn)。在移除細(xì)胞外的納米顆粒后，隨著時(shí)間的變化，圍繞在細(xì)胞核周圍的納米顆粒由團(tuán)聚慢慢分散，最后被細(xì)胞排出。ICP-AES的分析結(jié)果顯示隨著時(shí)間的增加，細(xì)胞內(nèi)的Ti元素的濃度顯著減少。濃度為50mg/L的三種納米TiO_2在24h的胞排率分別為35.0%(NP1)，34.6%(NP2)和41.7%(NT)。當(dāng)納米TiO_2的濃度增加為100mg/L時(shí)，胞吞量增加一倍而胞排量卻沒(méi)有同樣增加一倍。納米材料的胞排作用受到細(xì)胞胞吞的納米材料的數(shù)量的影響。例如，就NT而言，隨著納米TiO_2濃度的不同，胞排率從41.7%降低至26.7%。研究結(jié)果顯示，細(xì)胞的胞排作用受到加入細(xì)胞的納米材料的濃度、細(xì)胞胞吞的納米材料的濃度和納米材料的形貌的影響，但是與納米材料的尺寸無(wú)關(guān)。 胞吞和胞排作用的機(jī)理研究：與37°C條件相比，在4°C下，納米TiO_2的胞吞和胞排量明顯減少了，說(shuō)明細(xì)胞對(duì)于納米材料的胞吞作用和胞吞作用都存在著能量依賴，并且其胞吞過(guò)程是通過(guò)受體介導(dǎo)來(lái)完成的。隨后，我們研究了血清對(duì)納米TiO_2的胞吞和胞排作用的影響。結(jié)果顯示，沒(méi)有血清的條件下，對(duì)于三種不同的納米TiO_2，胞吞量和胞排量都被抑制了，其中胞吞量分別被抑制了24.9%(NP1)，18.6%(NP2)和30.4%(NT)，同時(shí)，胞排量被抑制了23.0%(NP1)，，19.5%(NP2)和58.2%(NT)。在透射電鏡下觀察到，經(jīng)過(guò)血清處理的納米材料表面吸附了一層膠狀的血清蛋白，吸附了血清蛋白的納米顆粒更容易進(jìn)入細(xì)胞。納米顆粒-蛋白的復(fù)合體減少了納米粒子的團(tuán)聚，促進(jìn)了細(xì)胞的胞吞和胞排作用。在胞排過(guò)程中，細(xì)胞形貌發(fā)生了變化，其原因是細(xì)胞微管發(fā)生了變化，經(jīng)納米材料處理過(guò)的細(xì)胞微管不規(guī)則排列。微管起著細(xì)胞內(nèi)物質(zhì)運(yùn)輸?shù)穆奋壍淖饔�，破壞微管�?huì)抑制細(xì)胞內(nèi)的物質(zhì)運(yùn)輸，這也可能是引起胞排過(guò)程中細(xì)胞內(nèi)活性氧略微升高的原因。
[Abstract]:With the rapid development of nanotechnology, rapidly increasing widely the application of nano materials and nano materials production, people began to pay more attention to the safety of nano materials. For example, in the field of biomedicine, nano materials as drug delivery carrier has good targeting and to safe and effective medication effect on pathological tissue and cells. Among them, nano titanium dioxide (nano-TiO_2) because of its unique physical and chemical properties have been widely used in many fields. Nano titanium dioxide is particularly important for the safety of the organism becomes, especially the basic metabolism in the cells of nano materials, such as cell endocytosis and exocytosis of neural stem cells. (NSCs) is a group of cells of various neural cells in the nervous system, it not only has the characteristics of value-added have differentiation potential. At present, the biological toxicity of TiO_2 nanoparticles Of environmental effects, the interaction with the biological tissue has no systematic research results. According to the current situation of the research, this paper aims at the cellular level, the neural stem cells as a cell model to study on the three kinds of different morphology and particle size of nano TiO_2 endocytosis and exocytosis effect quantitatively.
The physical and chemical properties and surface properties of nanoparticles determines its biological function, purity, firstly, three different sizes of different morphologies of TiO_2 nano particle size, morphology, crystal type and surface potential were fully characterized. Then using CCK-8 and ROS detection in the assessment of the impact of nano TiO_2 on the level of ROS neural stem cells, cell viability and cell toxicity. Test results showed that nano TiO_2 with low concentration of cell toxicity. Toxicity of nano TiO_2 on cell has time effect and concentration effect, when the concentration of the nano particles can reach more than 150mg/L, the cell viability decreased, intracellular reactive oxygen species increased significantly, but the size of and there was no correlation between morphology and nano materials.
Study on nano TiO_2 endocytosis and exocytosis, endocytosis as time increases, consumption also increased significantly, after 48h intake almost reached saturation. Particles size of the endocytosis speed faster, more easily into the cells, tubular nano particles is more difficult than the ball into the cells. For different concentrations of nano TiO_2, increasing the concentration of nano materials, cell uptake also increased exponentially, but has no effect on the uptake rate of endocytosis by nano materials. The size, morphology and impact velocity on the nanotube concentration. The uptake of cells than to the other two kinds of nanoparticles uptake speed according to the. Endocytosis volume versus time curves, choose 48h as the starting point, using the method of multi step cell passage, by laser scanning confocal microscopy of cell line process to make the tracking experiment dynamic. Nanoparticles in the removal of extracellular Later, with the change of time, around the nanoparticles from agglomeration slowly dispersed around the nucleus, was finally discharged.ICP-AES analysis results showed that the cells increased with time, concentrations of Ti in cells was significantly reduced. The concentration of three kinds of nano TiO_2 50mg/L in 24h cell line rates were 35% (NP1). 34.6% (NP2) and 41.7% (NT). When the concentration of nano TiO_2 increased to 100mg/L, doubling the amount of endocytosis and cell displacement is no doubling. Nano materials are affected by the cell exocytosis endocytosis the number of nano material effects. For example, NT, with nano TiO_2 different concentration of exocytosis rate decreased from 41.7% to 26.7%. the results of the study showed that the cell line is affected by the concentration of nano material into cells, cell morphology influence endocytosis of nanomaterials and the concentration of nano materials and nano materials, but the size of Irrelevant.
Study on the mechanism of endocytosis and exocytosis effect: compared with 37 DEG C, under the temperature of 4 C nano TiO_2 endocytosis and cell volume significantly reduced, indicating that cells for nanomaterials endocytosis and endocytosis are energy dependent, and its endocytosis is mediated by the receptor to complete. Then, we study the effect of serum on nano TiO_2 endocytosis and exocytosis effect. The results showed that no serum conditions for three different kinds of nano TiO_2, endocytosis and cell displacement are suppressed, which respectively endocytosis was inhibited by 24.9% (NP1). 18.6% (NP2) and 30.4% (NT), at the same time, the displacement of cells was inhibited by 23% (NP1), 19.5% (NP2) and 58.2% (NT). Observed under transmission electron microscope, the surface of the nano material after serum treatment serum protein adsorption layer of colloid, adsorption of serum albumin nanoparticles more easy access to the nanoparticle cells. Protein complex reduced the agglomeration of nanoparticles, promote cell endocytosis and exocytosis. In the exocytosis process, changes in cell morphology, which is due to the changes of cell microtubules, nanometer material treated cells arranged irregularly. Microtubule plays an intracellular material transport track effect of disruption of microtubules can inhibit the intracellular material transportation, which may be caused by exocytosis of intracellular reactive oxygen species slightly higher.

【學(xué)位授予單位】：上海大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08;TB383.1

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