本文選題：納米二氧化鈦　切入點(diǎn)：氣血屏障　出處：《浙江大學(xué)》2013年碩士論文

【摘要】：[研究背景] 納米顆粒具有獨(dú)特的物理、化學(xué)和生物特性,越來(lái)越廣泛地被應(yīng)用于生產(chǎn)生活的各個(gè)領(lǐng)域中。它們?cè)谶@些領(lǐng)域的應(yīng)用帶來(lái)了巨大的經(jīng)濟(jì)效益,但同時(shí)也產(chǎn)生潛在的生物安全性與環(huán)境安全性等問(wèn)題而引起世界范圍的廣泛關(guān)注。已有研究結(jié)果顯示納米顆�？梢鸺�(xì)胞代謝、凋亡、細(xì)胞周期、應(yīng)激反應(yīng)、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)和炎癥反應(yīng)等一系列的毒性效應(yīng)。納米顆粒進(jìn)入機(jī)體后可以轉(zhuǎn)運(yùn)到各器官,包括大腦和心臟等重要生命器官。轉(zhuǎn)運(yùn)可以是簡(jiǎn)單的物理性移位,還可以發(fā)生溶解(溶解大多數(shù)主要是物理過(guò)程,少數(shù)有化學(xué)變化)和蛋白結(jié)合等化學(xué)性過(guò)程。而多項(xiàng)研究結(jié)果表明納米二氧化鈦顆粒(nano-TiO2)易進(jìn)入上皮和間質(zhì)部位,nano-TiO2在肺灌洗液中只有20%的在巨噬細(xì)胞中可以被檢出,提示大多數(shù)nano-TiO2顆粒在上皮細(xì)胞或轉(zhuǎn)運(yùn)到間質(zhì)和循環(huán)系統(tǒng),然而nano-TiO2顆粒究竟是如何被轉(zhuǎn)運(yùn)的,現(xiàn)有的研究并沒(méi)有給出直接的證據(jù)。 [研究目的] 利用透射電子顯微鏡(TEM)觀察nano-TiO2顆粒是否可以直接穿越大鼠肺氣-血屏障,從而由肺進(jìn)入血循環(huán)。旨在為nano-TiO2的生物安全性評(píng)價(jià)、危險(xiǎn)度管理以及納米材料在生物醫(yī)學(xué)中的應(yīng)用提供有益的資料,進(jìn)一步促進(jìn)納米技術(shù)和納米產(chǎn)業(yè)的健康發(fā)展。 [研究方法] 本實(shí)驗(yàn)采用大鼠支氣管灌注實(shí)驗(yàn)探究nano-TiO2穿越肺氣血屏障的過(guò)程,具體觀察指標(biāo)包括：①肺組織病理切片常規(guī)HE染色：觀察肺組織結(jié)構(gòu)改變及nano-TiO2顆粒在肺泡的組織大體(一般指肉眼可見(jiàn)的)分布；②應(yīng)用透射電鏡技術(shù)觀察nano-TiO2顆粒在肺泡的微觀分布及對(duì)肺泡超微結(jié)構(gòu)的影響,特別是對(duì)肺氣血屏障的作用,并結(jié)合X射線(xiàn)能譜儀定性nano-TiO2顆粒的存在；③利用電感耦合等離子體質(zhì)譜法(ICP-MS)檢測(cè)灌注后大鼠靜脈血中nano-TiO2顆粒在不同時(shí)間點(diǎn)的含量變化。 [研究結(jié)果] 1.材料表征X-衍射和透射電鏡鑒定結(jié)果：試樣中銳鈦礦相和金紅石相的比例為80%：20%,nano-TiO2材料均呈圓形晶體顆粒,測(cè)得其平均粒徑約為25nm。 2.肺組織光鏡觀察結(jié)果：對(duì)照組,大鼠肺泡結(jié)構(gòu)正常,肺泡間隔較薄,肺泡腔干凈,無(wú)明顯炎癥變化。Nano-TiO2灌注后2h組,肺組織無(wú)明顯病理性改變,肺泡完整但呈現(xiàn)皺縮,少量納米顆粒以團(tuán)聚狀態(tài)沉積在肺泡內(nèi)表面,也有少量分布在肺泡隔。Nano-TiO2灌注后6h組,大量nano-TiO2顆粒團(tuán)聚物分布在大鼠肺部的呼吸性細(xì)支氣管、肺泡管、肺泡囊、肺泡和肺間質(zhì)；肺組織出現(xiàn)明顯充血、出血的病理性改變,肺泡腔內(nèi)有明顯的滲出和炎性細(xì)胞浸潤(rùn)。Nano-TiO2灌注后12h組,nano-TiO2顆粒團(tuán)聚物在肺組織中的分布比6h組減少,但病理性改變更加明顯,部分肺泡完整性遭到破壞,肺泡結(jié)構(gòu)排列紊亂或肺實(shí)變,整個(gè)肺泡結(jié)構(gòu)幾乎不存在。Nano-TiO2灌注后24h組,肺泡表面少見(jiàn)nano-TiO2顆粒沉積,間質(zhì)偶見(jiàn)小的團(tuán)聚顆粒分布,肺泡充血消失,水腫減輕,肺泡也有所修復(fù)。 3.透射電鏡觀察結(jié)果：在灌注后6h,肺泡腔內(nèi)出現(xiàn)大量nano-TiO2顆粒大團(tuán)聚物和鏈狀小團(tuán)聚物,肺泡腔內(nèi)游離巨噬細(xì)胞形態(tài)結(jié)構(gòu)完整,但胞質(zhì)中有大量無(wú)膜包被的nano-TiO2顆粒團(tuán)聚物。在氣血屏障處,有些nano-TiO2顆粒團(tuán)聚物粘附在肺泡上皮細(xì)胞上,有些nano-TiO2顆粒大團(tuán)聚物已破膜進(jìn)入氣血屏障的肺泡上皮細(xì)胞內(nèi),有nano-TiO2鏈狀小團(tuán)聚物跨越肺泡上皮細(xì)胞和基膜進(jìn)入毛細(xì)血管內(nèi)皮。在灌注后12h, nano-TiO2在肺組織中分布與6h相似,可見(jiàn)nano-TiO2顆粒以不同方式穿越氣血屏障,進(jìn)入毛細(xì)血管腔,鏈狀結(jié)構(gòu)團(tuán)聚物是直接穿越氣血屏障進(jìn)入的,塊狀團(tuán)聚物以破膜的形式穿越氣血屏障進(jìn)入。在灌注后24h, nano-TiO2顆粒大量進(jìn)入肺間質(zhì),肺泡隔明顯增厚,接觸nano-TiO2顆粒的細(xì)胞呈現(xiàn)細(xì)胞損傷,凋亡及壞死,細(xì)胞中出現(xiàn)大量空泡,細(xì)胞核縮小,線(xiàn)粒體腫大,脊散亂。在有nano-TiO2顆粒的Ⅱ型肺泡上皮細(xì)胞中生成大量板層小體,細(xì)胞表面微絨毛消失。 4. ICP-MS結(jié)果：與對(duì)照組相比,nano-TiO2顆粒氣管灌注組在各時(shí)間點(diǎn)(0、2、6、12、24和48h)全血Ti元素均高于對(duì)照組,但灌注組不同時(shí)間點(diǎn)間比較并沒(méi)有發(fā)現(xiàn)明顯差異。 [結(jié)論] 1)本研究實(shí)驗(yàn)條件下,25nm粒徑的nano-TiO2顆粒支氣管灌注后能夠跨越氣-血屏障進(jìn)入血液循環(huán),其中小鏈狀結(jié)構(gòu)團(tuán)聚物是以直接穿越氣血屏障進(jìn)入毛細(xì)血管腔的,大塊狀團(tuán)聚物則是以破壞氣血屏障的形式進(jìn)入毛細(xì)血管腔的。 2)本研究實(shí)驗(yàn)條件下,25nm粒徑的nano-TiO2顆粒支氣管灌注后可造成肺充血、肺水腫、肺泡破壞和細(xì)胞凋亡等肺損傷。
[Abstract]:[research background]
Nano particles have unique physical, chemical and biological characteristics of various fields more and more widely used in production and life. They are applied in these areas has brought enormous economic benefits, but also produce biological safety and environmental safety of potential problems and attracted worldwide attention. The existing research results showed that nano particles can cause cell metabolism, apoptosis, cell cycle, stress response, signal transduction and cell toxicity of inflammatory reaction. A series of nano particles into the body can be transported to various organs, including the brain and heart and other vital organs. Transport can be physical simple shift, can also be dissolved (most dissolved is the main physical process, there are a few chemical changes) and protein binding chemical process. And a number of research results show that the nano TiO2 particles (nano-TiO2) Easy to enter the epithelial and stromal parts, nano-TiO2 was detected in lung lavage fluid in only 20% in macrophages, suggesting that most of the nano-TiO2 particles in epithelial cells or transported to the interstitial and circulatory system, but how was nano-TiO2 particle transport, the existing research does not give direct evidence.
[research purposes]
Using transmission electron microscopy (TEM) to observe whether the nano-TiO2 particles can be directly through the rat lung qi - blood barrier and enter blood circulation by the lung. Aimed at the biological safety evaluation of nano-TiO2, provide useful information to application of risk management as well as the nano materials in the biomedical field, to further promote the healthy development of nanotechnology and nano industry.
[research methods]
This experiment adopts the process of experimental inquiry nano-TiO2 through pulmonary bronchial perfusion blood barrier in rats, including specific outcome measures: lung tissue was stained by HE, observe the lung tissue structure changes and nano-TiO2 particles in the alveolar tissue specimens (usually visible) distribution; II nano-TiO2 particles in the micro distribution of alveoli and influence on the alveoli ultrastructure was observed by TEM, especially on pulmonary blood barrier function, and combined with X ray spectrometer qualitative nano-TiO2 particles; using inductively coupled plasma mass spectrometry (ICP-MS) detection of nano-TiO2 particles after reperfusion in rat blood content changes at different time points.
[results]
1. material characterization X- diffraction and transmission electron microscopy identification results: the ratio of anatase and rutile in the samples is 80%:20%, and nano-TiO2 materials are round crystal particles, and the average particle size is about 25nm..
Results 2. lung tissue under light microscope: the control group, the rats with normal alveolar structure, alveolar septum is thinner, the alveolar cavity clean, no obvious inflammatory changes in.Nano-TiO2 after reperfusion in 2H group, the lung tissue had no obvious pathological changes, but showed complete alveolar collapse, a small amount of nano particles to agglomerate state of deposition in the alveolar surface, also a small amount of the distribution in the alveolar septum.Nano-TiO2 after reperfusion in 6h group, a large number of nano-TiO2 aggregates distribution in rat lung respiratory bronchioles, alveolar ducts, alveolar sac, alveolar and interstitial lung; lung tissue pathological changes appeared obvious hyperemia, hemorrhage, exudation and inflammatory cell infiltration was.Nano-TiO2 after reperfusion in 12h group the alveolar cavity nano-TiO2 particles distribution in lung tissues than in the 6h group decreased, but the pathological changes were more obvious, some alveolar integrity destruction of alveolar structure disorder or consolidation of the lung, the alveoli There was almost no.Nano-TiO2 perfusion in the 24h group. There was little nano-TiO2 particle deposition on the alveolar surface. Occasionally, there were small aggregation particles in the interstitium, alveolar hyperemia disappeared, edema reduced and alveoli repaired.
3. transmission electron microscope: in 6h after reperfusion, the alveolar cavities appear a large number of large nano-TiO2 particles agglomerate and a chain of small agglomerates, alveolar macrophage free intact structure, but there are a lot of cytoplasmic membrane coated nano-TiO2 particles were agglomerated. In the blood barrier, some nano-TiO2 particles agglomerate adhesion in alveolar epithelium nano-TiO2 cells, some large particles agglomerate has broken blood barrier membrane into the alveolar epithelial cells, nano-TiO2 chain small agglomerates across the alveolar epithelial cells and capillary endothelial basement membrane into. In 12h after reperfusion, nano-TiO2 in lung tissue distribution is similar with 6h, visible nano-TiO2 particles in different ways through the blood barrier, into the capillaries cavity chain structure is agglomerate directly through the barrier into blood, massive agglomerates to break the membrane form through blood perfusion in the barrier to enter the 24h, A large number of nano-TiO2 particles into the interstitial lung, alveolar septum thickening, contact nano-TiO2 granule cells showed cell damage, apoptosis and necrosis, vacuolization, cell nucleus reduced, mitochondrial swelling, ridge scattered. Generate a large number of lamellar bodies in nano-TiO2 particles of type II alveolar epithelial cells, cell surface microvilli disappeared.
4. ICP-MS results: compared with the control group, the Ti elements in the whole blood of nano-TiO2 granule tracheal perfusion group at each time point (0,2,6,12,24 and 48h) were all higher than those in the control group, but no significant difference was found in the perfusion group at different time points.
[Conclusion]
1) the experimental conditions, 25nm nano-TiO2 particle size after bronchial perfusion can cross the blood air barrier into blood circulation, the chain like structure is to agglomerate directly through the blood capillary barrier into the cavity, the bulk agglomerate is into the capillary lumen to destroy blood barrier form.
2) under the condition of this study, the 25nm particle size of nano-TiO2 particles can cause pulmonary hyperemia, pulmonary edema, alveolar destruction and cell apoptosis.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R114

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