本文選題：納米二氧化鈦 + 普通粒徑二氧化鈦�。� 參考：《寧波大學(xué)》2013年碩士論文

【摘要】：目的 通過(guò)體內(nèi)動(dòng)物實(shí)驗(yàn)和體外細(xì)胞實(shí)驗(yàn)，系統(tǒng)研究納米粒徑二氧化鈦粉塵（TiO_2NPs）與普通粒徑二氧化鈦粉塵（TiO_2FPs）對(duì)動(dòng)物和培養(yǎng)細(xì)胞的毒性；在此基礎(chǔ)上探索了N-乙酰半胱氨酸（NAC）對(duì)TiO_2NPs產(chǎn)生細(xì)胞毒性的拮抗作用及機(jī)制，為TiO_2NPs的職業(yè)與環(huán)境毒性評(píng)價(jià)和安全使用提供科學(xué)依據(jù)。 方法 體內(nèi)動(dòng)物實(shí)驗(yàn)：通過(guò)采用尾靜脈注射法對(duì)小鼠進(jìn)行不同劑量染毒，Horn’s法計(jì)算TiO_2NPs與TiO_2FPs的半數(shù)致死劑量（LD50）；臟器稱重計(jì)算臟器系數(shù)；全血檢測(cè)血常規(guī)指標(biāo)；分離血清，檢測(cè)血生化指標(biāo)；HE染色法觀察組織的病理?yè)p傷情況；骨髓細(xì)胞微核實(shí)驗(yàn)法檢測(cè)TiO_2NPs是否具有遺傳毒性。體外細(xì)胞實(shí)驗(yàn)：采用FITC標(biāo)記過(guò)的TiO_2NPs染毒小鼠JB6上皮細(xì)胞，研究TiO_2NPs在染毒細(xì)胞中的熒光定位；采用熒光染料H2DCFDA和DHE染色觀察細(xì)胞內(nèi)ROS的產(chǎn)生情況，比較TiO_2NPs與TiO_2FPs對(duì)細(xì)胞的氧化應(yīng)激毒性；Hoechst法染色細(xì)胞核，比較TiO_2NPs與TiO_2FPs對(duì)細(xì)胞核的毒性；Luciferase法比較TiO_2NPs與TiO_2FPs對(duì)AP-1細(xì)胞熒光素酶表達(dá)的影響；蛋白免疫印跡法比較TiO_2NPs與TiO_2FPs染毒后對(duì)細(xì)胞C-jun和P53蛋白表達(dá)的影響，探索TiO_2NPs與TiO_2FPs致腫瘤作用的分子機(jī)制。抗氧化劑NAC對(duì)TiO_2NPs氧化應(yīng)激毒性的抑制作用：通過(guò)在細(xì)胞染毒前加入20nM抗氧化劑NAC，檢測(cè)細(xì)胞活性氧自由基的產(chǎn)生變化情況，觀察NAC是否可以對(duì)TiO_2NPs產(chǎn)生的細(xì)胞毒性起到一定的拮抗作用。 結(jié)果體內(nèi)實(shí)驗(yàn)： LD50結(jié)果表明雌性小鼠（948mg/kg）對(duì)TiO_2NPs毒性的敏感性大于雄性小鼠（1392mg/kg）。TiO_2NPs染毒后，可不同程度的損傷小鼠的肝、肺和腎，呈一定的劑量反應(yīng)關(guān)系。但是，并未發(fā)現(xiàn)TiO_2NPs具有遺傳毒性；TiO_2FPs LD50為114.9mg/kg。與TiO_2NPs毒性相似，，也可引起小鼠的肝、肺和腎的毒性損傷。 體外實(shí)驗(yàn)：通過(guò)FITC熒光探針定位，發(fā)現(xiàn)TiO_2NPs可以透過(guò)細(xì)胞膜，未見(jiàn)其侵入細(xì)胞核內(nèi)；Hoechst染色法發(fā)現(xiàn)TiO_2NPs造成的細(xì)胞凋亡明顯大于TiO_2FPs；H2DCFDA，DHA，Hoechst共同染色結(jié)果顯示，在相同的染毒劑量下，TiO_2NPs所產(chǎn)生的活性氧自由基（ROS）明顯大于TiO_2FPs，且存在一定的劑量反應(yīng)關(guān)系；TiO_2NPs與TiO_2FPs均可引起細(xì)胞AP-1熒光素酶的表達(dá)升高，并且，TiO_2NPs組的升高程度明顯高于TiO_2FPs組。此外，蛋白免疫印跡實(shí)驗(yàn)結(jié)果顯示，25μg/cm2的TiO_2NPs可引起C-jun蛋白表達(dá)量升高和P53水平下降�？寡趸瘎㎞AC對(duì)TiO_2NPs產(chǎn)生毒性的抑制作用：加入抗氧化劑NAC后，不僅細(xì)胞產(chǎn)生的ROS受到了抑制，同時(shí)細(xì)胞AP-1熒光素酶的表達(dá)也有所降低。說(shuō)明NAC對(duì)TiO_2NPs產(chǎn)生的細(xì)胞氧化應(yīng)激有一定的拮抗作用。 結(jié)論 體內(nèi)實(shí)驗(yàn)結(jié)果表明：TiO_2NPs與TiO_2FPs經(jīng)過(guò)尾靜脈染毒可以引起小鼠臟器不同程度的損傷，提示經(jīng)血液接觸一定量的TiO_2NPs與TiO_2FPs均可能對(duì)機(jī)體造成潛在危害。體外實(shí)驗(yàn)結(jié)果表明：TiO_2NPs可以通過(guò)細(xì)胞膜進(jìn)入細(xì)胞漿內(nèi)，引起促癌基因表達(dá)上調(diào)，其毒性主要表現(xiàn)為細(xì)胞內(nèi)氧化應(yīng)激損傷，且毒性大于TiO_2FPs。此外，抗氧化劑NAC可以考慮作為一種新型的藥物用來(lái)拮抗TiO_2NPs對(duì)細(xì)胞產(chǎn)生的氧化應(yīng)激損傷。
[Abstract]:objective
Through in vivo animal experiments and in vitro cell experiments, the toxicity of nanoparticle titanium dioxide dust (TiO_2NPs) and ordinary particle size titanium dioxide dust (TiO_2FPs) on animals and cultured cells was systematically studied. On this basis, the antagonism and mechanism of N- acetylcysteine (NAC) on the toxicity of TiO_2NPs producing cells were explored, which was a TiO_2NPs job. It provides scientific basis for environmental toxicity assessment and safe use.
Method
In vivo animal experiments: the mice were poisoned by the tail vein injection, the Horn 's method was used to calculate the half lethal dose of TiO_2NPs and TiO_2FPs (LD50); the organ weight was weighed to calculate the organ coefficient; the whole blood was used to detect the blood routine index; the serum was separated and the blood biochemical index was detected; the pathological damage of the tissue was observed by HE staining. Bone marrow cell micronucleus test was used to detect whether TiO_2NPs had genetic toxicity. In vitro cell experiment: using FITC labeled TiO_2NPs to poison the JB6 epithelial cells of mice, the fluorescence localization of TiO_2NPs in the infected cells was studied. The production of ROS in the cells was observed by the fluorescent dye H2DCFDA and DHE staining, and the TiO_2NPs and TiO_2FPs were compared. Cytotoxicity of cellular oxidative stress; Hoechst staining of nuclei to compare the toxicity of TiO_2NPs and TiO_2FPs to the nucleus; Luciferase method compared the effect of TiO_2NPs and TiO_2FPs on the expression of luciferase in AP-1 cells; protein immunoblotting was used to compare the effect of TiO_2NPs and TiO_2FPs on the expression of C-jun and P53 proteins after TiO_2NPs and TiO_2FPs. The molecular mechanism of O_2FPs induced tumor action. The inhibitory effect of antioxidant NAC on the oxidative stress toxicity of TiO_2NPs: by adding 20nM antioxidant NAC before the cells were infected, the changes in the production of free radicals of reactive oxygen species were detected, and the effect of NAC on the cytotoxicity of TiO_2NPs produced by TiO_2NPs was observed.
Results in the experiment in vivo: LD50 results showed that the sensitivity of female mice (948mg/kg) to TiO_2NPs toxicity was greater than that of male mice (1392mg/kg).TiO_2NPs, and there was a certain dose response relationship between the liver, lung and kidney of mice in varying degrees. However, TiO_2NPs was not found to have genetic toxicity; TiO_2FPs LD50 was 114.9mg/kg. and TiO_2N. Ps is similar in toxicity and can cause liver, lung and kidney toxicity in mice.
In vitro experiment: it was found that TiO_2NPs could penetrate cell membrane and not intruder into the nucleus by FITC fluorescence probe. The apoptosis of TiO_2NPs caused by Hoechst staining was obviously greater than that of TiO_2FPs; H2DCFDA, DHA, Hoechst CO staining showed that the reactive oxygen free radical (ROS) produced by TiO_2NPs under the same amount of poison. It was obviously greater than TiO_2FPs, and there was a certain dose response relationship. Both TiO_2NPs and TiO_2FPs could cause the increase of AP-1 luciferase expression in cell, and the increase of TiO_2NPs group was significantly higher than that of TiO_2FPs group. In addition, the results of protein immunoblot test showed that the 25 u g/cm2 TiO_2NPs could cause the increase of C-jun protein expression and P53 level. Decrease. Anti oxidant NAC inhibits the toxicity of TiO_2NPs: after adding antioxidant NAC, not only the ROS of the cells is inhibited, but also the expression of AP-1 luciferase is also reduced. It shows that NAC has certain antagonism to the oxidative stress produced by TiO_2NPs.
conclusion
The results of the experiment in vivo show that TiO_2NPs and TiO_2FPs can cause different degrees of damage to the organs of mice through the tail vein, suggesting that the certain amount of TiO_2NPs and TiO_2FPs may cause potential harm to the body. In vitro experimental results show that TiO_2NPs can enter the cytoplasm through the cell membrane and cause the cancer promoting gene table. Up regulation, its toxicity is mainly manifested in intracellular oxidative stress damage, and the toxicity is greater than TiO_2FPs.. Antioxidant NAC can be considered as a new drug used to antagonize oxidative stress damage caused by TiO_2NPs to cells.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R114

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