本文選題：氧化鋯 + 納米顆粒　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景納米材料是由納米顆粒所構(gòu)成的材料。納米顆粒的定義是單個(gè)顆粒的三維尺寸中至少有一個(gè)維度的尺寸在1～100 nm的材料。由于顆粒直徑小,納米材料具有了大塊固體材料所不具備的多種理化特性,也因此被廣泛用于工業(yè)生產(chǎn)的各個(gè)領(lǐng)域。但納米材料本身并非完全無害的。隨著納米材料應(yīng)用范圍的擴(kuò)展,相關(guān)領(lǐng)域的學(xué)者也開始關(guān)注其生物安全性問題。大量的實(shí)驗(yàn)研究顯示納米材料在研發(fā)、生產(chǎn)以及應(yīng)用的過程中,納米顆粒可進(jìn)入水體、土壤等自然環(huán)境,再通過多種途徑進(jìn)入人體,經(jīng)血液循環(huán)進(jìn)入組織器官和細(xì)胞中,進(jìn)而產(chǎn)生一系列的損傷效應(yīng)[1]。納米氧化鋯材料在口腔醫(yī)學(xué)領(lǐng)域也有廣闊的應(yīng)用空間。通過使用納米氧化鋯對(duì)傳統(tǒng)口腔材料進(jìn)行改性,可極大的改善傳統(tǒng)材料的性能[2-4]。但作為納米材料,納米氧化鋯的生物安全性問題同樣值得關(guān)注。然而,現(xiàn)有文獻(xiàn)關(guān)于納米氧化鋯的毒性研究十分有限。本研究擬建立納米氧化鋯顆粒的體內(nèi)染毒模型,通過電感耦合等離子體質(zhì)譜技術(shù),分析納米氧化鋯顆粒在心、脾、肺、腎中的分布及代謝規(guī)律;通過血清中炎癥因子指標(biāo)的變化分析顆粒對(duì)脾臟的影響;通過組織病理技術(shù)、生物化學(xué)技術(shù)進(jìn)一步分析其對(duì)脾臟的毒性作用,為納米氧化鋯的毒性研究提供新思路。研究目的:(1)建立納米氧化鋯顆粒急性染毒模型,觀察顆粒在大鼠體內(nèi)的分布情況。(2)通過血清中炎癥因子指標(biāo)的變化分析納米氧化鋯對(duì)脾臟的影響。(3)研究納米氧化鋯對(duì)大鼠脾組織的損傷作用。(4)探究納米氧化鋯對(duì)脾臟毒性作用的機(jī)制。材料與方法:第一部分:納米氧化鋯顆粒的表征(1)透射電子顯微鏡檢測(cè)納米原始粒徑及形態(tài)。(2)電位測(cè)定儀檢測(cè)納米氧化鋯顆粒在混懸液中的電位值及水合粒徑。(3)能譜分析儀檢測(cè)納米顆粒的化學(xué)元素構(gòu)成。(4)氮?dú)馕綄?shí)驗(yàn)檢測(cè)納米顆粒的比表面積。第二部分:鋯元素的體內(nèi)分布及血清中炎癥因子含量的分析(1)構(gòu)建動(dòng)物模型模型構(gòu)建前選用0.9%的無菌生理鹽水將納米氧化鋯顆粒分散均勻并配置顆�；鞈乙�,然后對(duì)6周齡的雄性wistar大鼠進(jìn)行混懸液的單次尾靜脈注射處理(20 mg/kg b.w.)。(2)鋯元素的生物學(xué)分布在給藥后的第1 d、7 d、14 d,分別收集對(duì)照組和實(shí)驗(yàn)組大鼠的心、脾、肺、腎組織,經(jīng)濃硝酸與H202消解后,采用電感耦合等離子體質(zhì)譜分析檢測(cè)各樣品中鋯元素含量。(3)血清中炎癥因子含量的分析在給藥后的第1d、7d、14d,分別收集對(duì)照組和實(shí)驗(yàn)組大鼠的血清,參照液相蛋白分析試劑盒說明準(zhǔn)備樣品及標(biāo)準(zhǔn)品,采用液相蛋白分析儀測(cè)定血清中炎癥因子的含量。第三部分:納米氧化鋯對(duì)脾組織的損傷研究(1)脾組織的病理學(xué)觀察收集對(duì)照組7d與實(shí)驗(yàn)組7d、14d大鼠的新鮮脾組織(n=3),浸泡于4%的中性甲醛溶液完全固定后,制作石蠟切片。采用常規(guī)的蘇木精伊紅染色觀察脾組織的結(jié)構(gòu)變化。(2)脾組織的免疫組織化學(xué)染色觀察進(jìn)行常規(guī)蘇木素伊紅染色的同時(shí),分別采用Ki-67與末端標(biāo)記法凋亡染色技術(shù)觀察脾組織的增殖與凋亡情況。第四部分:納米氧化鋯毒性作用的機(jī)制研究(1)組織勻漿的制備與總蛋白含量的測(cè)定分別收集實(shí)驗(yàn)組(1d、7 d、14 d)和對(duì)照組(1d、7 d、14 d)大鼠的脾組織,用組織勻漿機(jī)制備組織勻漿,離心收集后稀釋至所需濃度。根據(jù)總蛋白定量試劑盒的說明,檢測(cè)勻漿中的蛋白含量。(2)氧化應(yīng)激相關(guān)酶活性的檢測(cè)同樣使用實(shí)驗(yàn)組(1d、7 d、14 d)和對(duì)照組(1d、7 d、14 d)大鼠脾組織所制備的勻漿。按超氧化物歧化酶試劑盒之操作說明完成檢測(cè)樣品制備。采用熒光酶標(biāo)儀測(cè)定樣品OD值,并計(jì)算出組織中超氧化物歧化酶的活性。(3)脂質(zhì)過氧化產(chǎn)物的含量檢測(cè)使用適當(dāng)濃度的組織勻漿,按照丙二醛試劑盒之操作說明制備檢測(cè)樣品。樣品經(jīng)水浴加熱后反應(yīng)完全,測(cè)定樣品OD值,并計(jì)算丙二醛含量。結(jié)果:第一部分:透射電子顯微鏡觀察結(jié)果顯示納米氧化鋯顆粒近似球形,原始粒徑平均約為38 nm。電位測(cè)定儀顯示納米顆粒在水溶液中的電動(dòng)電位為30.6 mV,水合粒徑約為181.2 nm。能譜儀結(jié)果示納米顆粒的元素組成符合氧化鋯的特點(diǎn),無其他雜質(zhì)。氮?dú)馕綄?shí)驗(yàn)結(jié)果示納米氧化鋯顆粒的比表面積為33.06 m2/g。第二部分:生物學(xué)分布檢測(cè)結(jié)果顯示,鋯元素在對(duì)照組大鼠的心、脾、肺、腎組織中未檢出,在實(shí)驗(yàn)組大鼠的心、腎組織中未檢出,實(shí)驗(yàn)組大鼠脾、肺組織中的含量顯著高于對(duì)照組。分析實(shí)驗(yàn)組脾、肺組織中鋯元素的含量變化,脾組織中鋯元素的含量14 d7d1d,提示鋯元素含量隨時(shí)間累積而逐漸升高。肺組織中鋯元素的含量7 d1 d=14 d,鋯元素含量先升后降。血清中IL-1α的含量,實(shí)驗(yàn)組7 d顯著高于對(duì)照組(1d、7 d、14 d)與實(shí)驗(yàn)組(1d、14 d);血清中IL-1β含量,實(shí)驗(yàn)組1d顯著高于對(duì)照組(1d、7 d、14 d)與實(shí)驗(yàn)組(7d、14d);血清中IL-2的含量各組無統(tǒng)計(jì)學(xué)差異;血清中IL-6的含量各組無統(tǒng)計(jì)學(xué)差異;血清中IL-12含量,實(shí)驗(yàn)組1 d顯著低于對(duì)照組(1 d、7 d、14 d)與實(shí)驗(yàn)組(7 d、14 d);實(shí)驗(yàn)組7 d顯著高于對(duì)照組(1d、7 d、14 d)與實(shí)驗(yàn)組(1d、14 d);對(duì)照組(1d、7 d、14 d)與實(shí)驗(yàn)組14 d間無統(tǒng)計(jì)學(xué)差異;血清中TNF-α的含量,實(shí)驗(yàn)組7 d顯著高于對(duì)照組(1d、7 d、14 d)與實(shí)驗(yàn)組(1d、14d);實(shí)驗(yàn)組14d低于對(duì)照組(1d、7d、14d)與實(shí)驗(yàn)組(7d);對(duì)照組(1d、7 d、14 d)與實(shí)驗(yàn)組1d間無統(tǒng)計(jì)學(xué)差異。第三部分:蘇木素伊紅染色結(jié)果顯示,實(shí)驗(yàn)組7 d、14 d動(dòng)物脾組織相較對(duì)照組7 d無明顯組織病理學(xué)改變,對(duì)照組7 d、實(shí)驗(yàn)組(7 d、14 d)的脾組織切片中均清晰可見白髓、紅髓及脾小梁等正常結(jié)構(gòu)。Ki-67染色結(jié)果,實(shí)驗(yàn)組7 d脾組織中細(xì)胞陽(yáng)性率與對(duì)照組7 d無明顯差異,但對(duì)照組7 d脾組織的紅髓與白髓中可見陽(yáng)性細(xì)胞分布規(guī)律,而實(shí)驗(yàn)組7 d的脾組織中陽(yáng)性細(xì)胞主要集中于白髓中,紅髓中少見陽(yáng)性細(xì)胞分布,實(shí)驗(yàn)組14 d的細(xì)胞陽(yáng)性率低于對(duì)照組7 d和實(shí)驗(yàn)組7 d。末端標(biāo)記法凋亡染色結(jié)果示,對(duì)照組7 d脾組織中可見少量陽(yáng)性胞,實(shí)驗(yàn)組7 d脾組織中的部分區(qū)域陽(yáng)性細(xì)胞數(shù)多于對(duì)照組7 d,實(shí)驗(yàn)組14 d的陽(yáng)性細(xì)胞比率與對(duì)照組7 d無差異。提示實(shí)驗(yàn)組7 d脾組織中部分區(qū)域凋亡細(xì)胞數(shù)目增加。第四部分:氧化應(yīng)激相關(guān)酶活性檢測(cè)結(jié)果顯示,實(shí)驗(yàn)組1d、14 d的脾組織中超氧化物歧化酶活力較對(duì)照組顯著下降(P0.01)。表明給藥后實(shí)驗(yàn)組動(dòng)物脾組織中該酶的活性受到了損害;脂質(zhì)過氧化產(chǎn)物的含量檢測(cè)表明,丙二醛的含量在給藥后的實(shí)驗(yàn)組動(dòng)物中脾組織中未有明顯改變。結(jié)論:1.納米氧化鋯顆粒可沉積于脾、肺組織中,并引起脾組織炎癥反應(yīng)和氧化應(yīng)激。2.納米氧化鋯顆粒沉積可引起脾紅髓細(xì)胞增殖減少、誘導(dǎo)脾組織細(xì)胞凋亡,引起功能損傷。3.納米氧化鋯顆粒在脾臟中隨時(shí)間而逐漸沉積,并至少可維持14 d。4.納米氧化鋯顆粒對(duì)脾臟損傷的機(jī)制與氧化應(yīng)激密切相關(guān)。
[Abstract]:Background nanomaterials are made of nanoparticles. The nanoparticles are defined as a material with at least one dimension in the dimension of a single particle in a dimension of 1~100 nm. Due to the small diameter of the particles, the nanomaterials have many physical and chemical properties that the bulk solid materials do not have, and are widely used in industrial production. Nanomaterials themselves are not completely harmless. With the expansion of the application scope of nanomaterials, scholars in the related fields have also begun to pay attention to their biological safety problems. A large number of experimental studies show that nanomaterials can enter the water, soil and other natural environments in the process of development, production and application of nanomaterials. A variety of ways to enter the human body and circulate through the blood into tissues and organs and cells, and then produce a series of damage effects [1]. nano zirconia materials have wide application space in the field of Stomatology. By using nano zirconium oxide to modify traditional oral materials, it can greatly improve the performance of traditional materials [2-4]. but as a Na The biosafety of rice materials and nano zirconia is also worthy of concern. However, the current literature about the toxicity of nano zirconia is very limited. This study is to establish an in vivo toxicity model of nano zirconia particles. The distribution of nano zirconia particles in the heart, spleen, lung and kidney is analyzed by inductively coupled plasma mass spectrometry. And the regulation of metabolism; the effect of granule on spleen was analyzed through the changes of inflammatory factors in serum. Through histopathology, biochemical technology was used to further analyze its toxic effect on the spleen and provide new ideas for the study of the toxicity of nano zirconia. (1) to establish an acute poisoning model of nano zirconia particles and observe the particles. Distribution in rats. (2) analysis of the effect of nano zirconia on spleen by changes of inflammatory factors in serum. (3) study the damage effect of nano zirconia on spleen tissue of rats. (4) explore the mechanism of the toxicity of nano zirconia to spleen. Material and method: the first part: characterization of nano zirconia particles (1) transmission The original particle size and morphology of nanoscale nanoparticles were detected by electron microscopy. (2) potential measurement instrument was used to detect the potential value and hydrous particle size of nano zirconia particles in suspension. (3) the chemical element composition of nanoparticles was detected by the energy spectrum analyzer. (4) the specific surface area of nano particles was detected by nitrogen adsorption experiment. The second part: in vivo distribution of zirconium elements and in serum Analysis of the content of inflammatory factors (1) before the construction of animal model model, 0.9% of the aseptic saline was selected to disperse the nano zirconia particles evenly and to configure the granular suspension. Then, the male Wistar rats of 6 weeks old were treated with a single tail vein injection (20 mg/kg b.w.). (2) the biological distribution of zirconium elements was given after the drug delivery. First D, 7 d, 14 d, the heart, spleen, lung and kidney tissue of the control group and the experimental group were collected respectively. The content of zirconium elements in the samples was detected by inductively coupled plasma mass spectrometry after the digestion of concentrated nitric acid and H202. (3) the serum levels of inflammatory factors were analyzed at 1D, 7d, and 14d after administration, and the blood of the rats in the control group and the experimental group were collected respectively. The content of inflammatory factors in serum was determined by liquid phase protein analyzer. The third part: Study on the damage of the spleen tissue by nano zirconium oxide (1) the pathological observation of spleen tissue (1) the control group 7d and experimental group 7d, the fresh splenic tissue of 14d rats (n=3) were soaked in 4%. After the sex Formaldehyde Solution was completely fixed, the paraffin section was made. The structural changes of spleen tissue were observed with routine hematoxylin eosin staining. (2) immuno histochemical staining of spleen tissue was used to observe the routine hematoxylin staining of routine hematoxylin, and the proliferation and apoptosis of spleen tissue were observed by Ki-67 and terminal labeling method. The fourth part: Study on the mechanism of the toxicity of nano zirconia (1) the preparation of tissue homogenate and the determination of the total protein content of the experimental group (1D, 7 d, 14 d) and the control group (1D, 7 d, 14 d) of the spleen tissue of the rats, the tissue homogenate was prepared by the homogenate mechanism, and then diluted to the required concentration after centrifugation. According to the description of the total protein quantitative reagent box, Detection of protein content in homogenate. (2) detection of oxidative stress related enzyme activity was also used in the homogenate prepared by the experimental group (1D, 7 d, 14 d) and the control group (1D, 7 d, 14 d). The sample was prepared by the operation instructions of the superoxide dismutase Kit. Activity of oxide dismutase. (3) the content of lipid peroxidation products was detected by proper concentration of tissue homogenate, and the test samples were prepared according to the operation instructions of the malondialdehyde kit. The sample was heated by water bath and reacted completely, measured the sample o value and calculated the content of malondialdehyde. Results: the first part: transmission electron microscope observation results show that The nano zirconia particles are approximately spherical. The average size of the original particle size is about 38 nm. potential meter. The electrokinetic potential of nano particles in aqueous solution is 30.6 mV, and the hydrous particle size is about 181.2 nm.. The results show that the element composition of the nanoparticles conforms to the characteristics of zirconia and has no other impurities. The experimental results of nitrogen adsorption show nano zirconia particles. The specific surface area was 33.06 m2/g. second parts: the results of biological distribution detection showed that the zirconium element was not detected in the heart, spleen, lung and kidney tissue of the control group, and was not detected in the heart and kidney tissue of the experimental group. The content of the spleen and lung tissue in the experimental group was significantly higher than that in the control group. The content of zirconium elements in the spleen was 14 d7d1d, suggesting that the content of zirconium elements increased gradually with time. The content of zirconium elements in the lung tissue was 7 D1 d=14 D, the content of zirconium elements increased first and then decreased. The content of IL-1 alpha in the serum, 7 d in the experimental group was significantly higher than that of the control group (1D, 7 d, 14 d) and the experimental group (1D, 14 d), and the content of IL-1 beta in the serum. Compared with the control group (1D, 7 d, 14 d) and the experimental group (7d, 14d), there was no statistical difference in serum IL-2 content, and there was no statistical difference in the content of IL-6 in serum. The content of IL-12 in the serum was significantly lower than that of the control group (1 D, 7 d, 14 d) and the experimental group (7, 14), and the experimental group 7 was significantly higher than that of the control group (7, 14 concerned) and experimental group. 14 d); there was no statistical difference between the control group (1D, 7 d, 14 d) and the experimental group 14 d; the content of TNF- alpha in the serum and the experimental group 7 d were significantly higher than that of the control group (1D, 7 d, 14 d) and the experimental group (1D, 14d); the experimental group was lower than the control group, and the control group was not statistically different from the experimental group. Third: Su Mu Su Hong The staining results showed that the experimental group 7 d, 14 d animal spleen tissue compared with the control group 7 d no obvious histopathological changes, 7 d in the control group, the experimental group (7 d, 14 d) of the spleen tissue sections clearly visible white pulp, red pulp and Pi Xiaoliang and other normal structure.Ki-67 staining results, the experimental group 7 d spleen tissue positive rate and the control group 7 d no significant difference, But the distribution of positive cells in the red pulp and white pulp of the spleen tissue of the control group was 7 d, while the positive cells in the spleen tissue of the experimental group were mainly concentrated in the white pulp and the rare positive cells in the red pulp were mainly concentrated in the 7 d of the experimental group. The positive rate of the 14 d in the experimental group was lower than that of the control group 7 d and the experimental group 7 d. terminal labeling method apoptosis staining results, and the control group 7 d spleen group. The number of positive cells in the 7 d splenic tissues in the experimental group was more than 7 d in the control group. The ratio of positive cells to 14 d in the experimental group was not different from that of the control group 7 d. It suggested that the number of apoptotic cells in the part of the spleen tissue of the experimental group increased. Fourth: the test results of the oxidative stress related enzyme activity showed that the experimental group was 1D, 1, 1. The activity of superoxide dismutase in the spleen tissue of 4 D was significantly lower than that in the control group (P0.01). It showed that the activity of the enzyme in the spleen tissues of the experimental group was damaged after the administration, and the content of lipid peroxidation products showed that the content of malondialdehyde was not significantly changed in the spleen tissue of the experimental group after the administration. Conclusion: 1. nano zirconia. The particles can be deposited in the spleen, lung tissue, and the inflammatory response of the spleen tissue and the oxidative stress of the spleen.2. nanoparticles can cause the proliferation and decrease of the splenic red marrow cells and induce the apoptosis of the spleen tissue cells. The.3. nano zirconia particles are gradually deposited in the spleen with time, and at least 14 d.4. nanoscale zirconia nanoparticles can be maintained. The mechanism of granule damage to spleen is closely related to oxidative stress.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R783.1;TB383.1

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本文編號(hào)：1781166