本文選題：炭黑納米顆粒 + 氧化損傷��； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:炭黑(carbon black,CB)是一種超細碳粒子,是燃料不完全燃燒時產(chǎn)生的煙塵中的主要組成部分。炭黑納米顆粒(carbon black nanoparticles,CBNPs)是通過控制碳氫化合物的氣相裂解過程生產(chǎn)制造而成的純的炭黑粉末,隨著CBNPs的商業(yè)化及CB向大氣的排放量持續(xù)升高,CBNPs的暴露與人類健康風險的評估備受關注。有研究證明處于不同周期的細胞會對納米顆粒的攝取量存在差異,而細胞內(nèi)納米顆粒的含量與其所引起的細胞毒性密切相關。目前CBNPs對細胞產(chǎn)生的毒性損害以及不同細胞周期對于細胞的CBNPs攝取量是否產(chǎn)生影響尚不清楚,因此探究CBNPs產(chǎn)生的細胞損傷作用及不同周期對細胞攝取CBNPs量的影響具有十分重要的意義。本研究通過研究不同細胞周期對人支氣管上皮細胞(16HBE)的CBNPs攝取量的影響為切入點,探究CBNPs對16HBE的細胞毒性效應及其影響因素。方法:1納米炭黑顆粒表征觀察通過掃描電子顯微鏡和透射電子顯微鏡觀察納米炭黑的結構特征。采用粒徑分析儀分析納米炭黑的粒徑,BET比表面積測試法測量炭黑顆粒比表面積。2細胞培養(yǎng)和細胞模型建立16HBE(中國典型培養(yǎng)物保藏中心)用含10%熱滅活胎牛血清的MEM培養(yǎng)基37℃,5%CO2飽和濕度下進行培養(yǎng)。選擇對數(shù)生長期的細胞,用CBNPs處理24小時,劑量分別為50μg/mL、100μg/mL、200μg/mL;以MEM(含0.04%Tween80)為陰性對照。3 MTT法檢測細胞存活率分別使用陰性對照組和1、10、50、100、200、300μg/mL CBNPs染毒組處理16HBE細胞3、6、12、24、36h。加入MTT孵育后使用酶標儀檢測各孔在495nm波長處的吸光度值。每組設置六個復孔,計算各組吸光度并統(tǒng)計各染毒組細胞存活率。4細胞氧化應激水平的測定將cbnps染毒處理后的細胞制備成單細胞懸液,用2,7-二氫二氯熒光黃雙乙酸鈉(2,7-dichlorodihydrf-luoresceindiacetate,dcfh-da)熒光探針進行孵育,流式細胞儀檢測不同劑量cbnps處理后16hbe細胞內(nèi)活性氧(reactiveoxygenspecies,ros)的含量;16hbe細胞內(nèi)抗氧化酶超氧化物歧化酶(superoxidedismutase,sod)、谷胱甘肽過氧化物酶(glutathioneperoxidase,gsh-px)活性以及脂質(zhì)過氧化產(chǎn)物丙二醛(malonaldehyde,mda)含量采用生化試劑盒并按照說明書進行定量檢測。5細胞dna損傷的測定應用單細胞凝膠電泳(singlecellgelectrophoresis,scge)實驗,通過觀測染毒前后olive尾踞的變化,檢測cbnps對細胞dna的損傷。6annexinⅤ-pi雙染檢測細胞凋亡染毒24小時后將陰性對照和各染毒組細胞制備成單細胞懸液,采用annexinⅤ-fitc和pi雙染標記,于流式細胞儀檢測細胞凋亡率。7細胞周期檢測將陰性對照和各染毒組制備成單細胞懸液,用-20℃預冷的80%的乙醇固定后,先后使用rnasea和pi處理細胞,之后使用流式細胞儀進行周期檢測。8細胞周期的同步化使用害羞草堿使16hbe細胞同步于g0/g1期,胸苷雙阻斷法使16hbe細胞同步于s期,諾考達唑使細胞同步于g2/m期。9細胞攝取cbnps檢測將陰性對照和各染毒組16hbe細胞制備成單細胞懸液,經(jīng)pbs洗滌兩次后,應用流式細胞儀進行檢測,并通過ssc密度含量的改變來定量分析16hbe對cbnps的攝取量。另外采用imagestreamx單細胞成像流式細胞儀檢測各組樣本并獲取各劑量組細胞的明場及暗場圖像和數(shù)據(jù),由此分析16hbe攝取cbnps的情況。結果:1納米炭黑顆粒表征電子顯微鏡下觀察,納米炭黑為球狀顆粒。透射電鏡顯示炭黑顆粒大體呈葡萄球狀,分散良好,顆粒直徑在30~50nm之間。掃描電鏡顯示炭黑顆粒近似呈球形,分散較均勻,團聚顆粒直徑大約200~400nm之間。較大顆粒表面有很多球形突出物。利用bet比表面積測試法測量炭黑顆粒比表面積為74.85m2/g。炭黑顆粒難溶于水,在水溶劑中炭黑團聚顆粒直徑為200~400nm,而在0.04%tween80中有較好的分散性,顆粒直徑為30~50nm。2cbnps對16hbe細胞活性的影響當用100、200和300μg/mlcbnps處理16hbe細胞3h后,各劑量組細胞存活率逐漸降低(p0.05);當用50、100、200和300μg/mlcbnps處理16hbe細胞6h后,各劑量組細胞存活率逐漸降低(p0.05);當用1、10、50、100、200和300μg/mlcbnps處理細胞12、24、36h后,各劑量組細胞存活率逐漸降低(p0.05);隨著染毒濃度和時間的增加,16hbe細胞活性降低更加明顯。這些結果表明cbnps抑制16hbe細胞存活并且這種抑制效應存在劑量和時間依賴性。3cbnps誘導16hbe細胞凋亡用50、100和200μg/ml的cbnps處理16hbe細胞24小時。發(fā)現(xiàn)50μg/ml和100μg/mlcbnps處理的16hbe早期凋亡細胞數(shù)目略微升高(p0.05);200μg/mlcbnps處理可誘導16hbe細胞早期凋亡率顯著增加(p0.05);隨著cbnps染毒濃度增加,晚期凋亡細胞所占的百分率和細胞壞死細胞的百分率逐漸升高(p0.05)。這些結果表明cbnps對細胞增殖的抑制作用至少可以部分解釋為cbnps誘導的凋亡和壞死。4cbnps對16hbe細胞氧化損傷的測定不同濃度cbnps處理16hbe細胞24小時后,隨著染毒濃度的增加,ros水平逐漸升高(p0.05);mda含量逐漸增加(p0.05);sod和gsh-px活力隨cbnps染毒濃度的增加而逐漸降低(p0.05)。說明cbnps可以誘導16hbe細胞氧化損傷。5cbnps對16hbe細胞dna損傷的誘導作用不同劑量的cbnps處理16hbe細胞24小時后,隨著染毒濃度的增加,各劑量組olive尾距顯著增加,與對照組相比,差異具有統(tǒng)計學意義(p0.05)。說明cbnps可以誘導16hbe細胞dna損傷。6cbnps對16hbe細胞周期的影響用100μg/mlcbnps對16hbe細胞進行連續(xù)處理,發(fā)現(xiàn)cbnps誘導S期和G2/M期細胞比率增加(P0.05)。S期細胞比率增加發(fā)生在6至24小時;G2/M期細胞比率增加發(fā)生在18至36小時。說明CBNPs誘導16HBE細胞周期阻滯,且阻滯發(fā)生于S期和G2/M期。7 16HBE細胞對CBNPs的攝取用50、100和200μg/mL的CBNPs處理16HBE細胞24小時,采用流式細胞儀和ImageStreamX成像流式細胞儀進行檢測。發(fā)現(xiàn)細胞內(nèi)CBNPs量逐漸升高(P0.05);將16HBE細胞分別同步化于G0/G1期,S期和G2/M期并進行CBNPs染毒處理。經(jīng)6小時和24小時染毒后采用流式細胞儀檢測觀察到同步于S期和G2/M期的16HBE細胞比同步于G0/G1期的16HBE細胞有更高的CBNPs攝取量,其中同步于G2/M期的16HBE細胞對CBNPs攝取量最高,與對照組相比,差異有統(tǒng)計學意義(P0.05)。這表明不同細胞周期階段可以影響16HBE細胞對CBNPs的攝取效果。不同細胞周期各階段對CBNPs的攝取量由多到少排序為G2/M期S期G0/G1期。結論:1 CBNPs處理能夠誘導16HBE細胞發(fā)生活性降低、凋亡率增加、活性氧水平升高、DNA損傷等急性毒性損傷,且這些損傷與16HBE攝取的CBNPs數(shù)量相關。2 S期及G2/M期是16HBE細胞攝取CBNPs的重要時期。
[Abstract]:Objective: carbon black (carbon black, CB) is a kind of superfine carbon particles, the main component of the smoke produced in the incomplete combustion of fuel. The carbon black nanoparticles (carbon black nanoparticles, CBNPs) are pure carbon black powders produced by controlling the gas phase cracking process of the hydrocarbons, with the commercialization of CBNPs and the CB to the large. The exposure of CBNPs and the assessment of human health risk have attracted much attention. Studies have shown that there are differences in the uptake of nanoparticles in cells in different cycles, and the content of nanoparticles in cells is closely related to the cytotoxicity caused by the CBNPs. It is not clear whether the cell cycle has an effect on the CBNPs uptake of cells. Therefore, it is of great significance to explore the effect of CBNPs's cell damage and the effect of different cycles on the uptake of CBNPs. This study is to explore the effect of different cell cycles on the CBNPs uptake of human bronchial epithelial cells (16HBE). Point, explore the cytotoxic effect of CBNPs on 16HBE and its influencing factors. Methods: 1 nano carbon black particles were observed by scanning electron microscope and transmission electron microscope to observe the structural characteristics of nano carbon black. The particle size analyzer was used to analyze the particle size of nano carbon black, and the specific surface area.2 cells were measured by BET surface product test. The culture and cell model established 16HBE (Chinese typical culture preservation Center) with the MEM medium containing 10% heat inactivated fetal bovine serum at 37 and 5%CO2 saturated humidity. The logarithmic growth period cells were selected for 24 hours with CBNPs, 100 mu g/mL, 200 g/mL, respectively. MEM (containing 0.04%Tween80) was negative control.3 MTT method. The survival rate of the cell measured by the negative control group and the 1,10,50100200300 mu g/mL CBNPs infected group were treated with the 16HBE cell 3,6,12,24,36h. to be incubated with MTT, and the absorbance value of the pores in the 495nm wave length was detected by the enzyme labeling instrument. Each group was set up with six complex holes, and the absorbance of each group was calculated and the cell survival rate of.4 cells in each group was calculated to be oxidative stress. The cell suspension of cbnps infected cells was prepared by the level determination, and the 2,7- two hydrogen two chloride fluorescein sodium acetate (2,7-dichlorodihydrf-luoresceindiacetate, DCFH-DA) fluorescence probe was incubated. The content of reactive oxygen species (reactiveoxygenspecies, ROS) in 16HBE cells after cbnps treatment at different doses was detected by flow cytometry; 16HBE Superoxidedismutase (SOD), glutathione peroxidase (glutathioneperoxidase, GSH-Px) activity and the content of malonaldehyde (MDA) of lipid peroxidation products (malonaldehyde, MDA) were used in biochemical reagent box and quantitative determination of DNA damage in.5 cells according to the specification Singlecellgelectrophoresis (SCGE) experiment, by observing the change of the entrenched olive tail before and after exposure to the virus, detecting the damage of cbnps to the cell DNA by.6annexin V -pi, after 24 hours of apoptosis, the negative control and the infected cells were prepared into single cell suspension, and annexin V -fitc and PI double staining markers were used to test the flow cytometry. The cell cycle detection of cell apoptosis rate.7 cell cycle tests the negative control and the infected groups to form a single cell suspension. After 80% ethanol fixed at -20 centigrade, the cells were treated with RNaseA and PI successively. Then the synchronization of.8 cell cycle detection by flow cytometry was used to synchronize 16HBE cells to g0/g1 phase and thymidine double. The 16HBE cells were synchronized with the S phase, and the cells were synchronized with the.9 cells in g2/m phase for cbnps detection, and the negative control and the 16HBE cells were prepared to form a single cell suspension. After two times of PBS washing, a flow cytometer was used to detect the cells, and the amount of 16HBE to cbnps was quantitatively analyzed by the change of SSC density. Imagestreamx single cell imaging flow cytometry was used to detect each group of samples and obtain the clear field and dark field images and data of each dose group. Thus the situation of 16HBE uptake of cbnps was analyzed. Results: 1 nano carbon black particles were observed under electron microscope and nano carbon black was spherical granules. Transmission electron microscopy showed that the carbon black particles were in general grape The spherical shape is well dispersed and the particle diameter is between 30~50nm. The scanning electron microscope shows that the carbon black particles are approximately spherical, the dispersion is more uniform and the diameter of the aggregate particles is about 200~400nm. The surface of the larger particles has a lot of spherical projecting. The specific surface area of the carbon black particles is difficult to dissolve in water with the surface area of the carbon black particles by the BET specific surface area test method. The diameter of carbon black agglomerate in the solvent was 200~400nm, but there was better dispersion in 0.04%tween80. The effect of particle diameter was 30~50nm.2cbnps on the activity of 16HBE cells. When 16HBE cell 3H was treated with 100200 and 300 mu g/mlcbnps, the cell survival rate of each dose group decreased gradually (P0.05); 16HBE cell 6H was treated with 50100200 and 300 micron. The cell survival rate in each dose group decreased gradually (P0.05). After 1,10,50100200 and 300 micron g/mlcbnps were used to treat the cell 12,24,36h, the cell survival rate in each dose group decreased gradually (P0.05). As the concentration and time increased, the activity of 16HBE cells decreased more obviously. These results suggest that cbnps inhibits the survival of 16HBE cells and the inhibition of this inhibition. The effect of dose and time dependent.3cbnps induced 16HBE cell apoptosis to treat 16HBE cells with 50100 and 200 mu g/ml cbnps for 24 hours. It was found that the number of apoptotic cells in early 16HBE was slightly higher (P0.05) by 50 g/ml and 100 micron g/mlcbnps. 200 micron g/mlcbnps treatment could induce significant increase of apoptosis rate in the early stage of 16HBE cells (P0.05). The increase of toxic concentration, the percentage of the late apoptotic cells and the percentage of cell necrotic cells increased gradually (P0.05). These results suggest that the inhibitory effect of cbnps on cell proliferation is at least partly explained by cbnps induced apoptosis and necrotic.4cbnps for the oxidative damage of 16HBE cells in 16HBE cells with different concentrations of cbnps for the treatment of 16HBE cells for 24 hours With the increase of concentration, the level of ROS increased gradually (P0.05), the content of MDA increased gradually (P0.05), and the activity of SOD and GSH-Px decreased gradually with the increase of the concentration of cbnps (P0.05). It indicated that cbnps could induce the oxidative damage of 16HBE cells to induce the injury of 16HBE cells at different doses for 24 hours. With the increase of concentration, the tail distance of olive increased significantly in each dose group. Compared with the control group, the difference was statistically significant (P0.05). It indicated that cbnps could induce the DNA damage of 16HBE cells to the 16HBE cell cycle by 100 mu g/mlcbnps to the 16HBE cell continuous treatment, and found that cbnps induced S phase and G2/M phase ratio increased. 0.05) the increase of cell ratio in.S period occurred between 6 and 24 hours, and the increase in G2/M cell ratio occurred at 18 to 36 hours. It indicated that CBNPs induced 16HBE cell cycle arrest, and the inhibition of.7 16HBE cells in S and G2/M stages of CBNPs uptake by 50100 and 200 micron g/mL for 16HBE cells for 24 hours, using flow cytometry and imaging The flow cytometry was used to detect the increase of CBNPs in the cells (P0.05), and the 16HBE cells were synchronized at G0/G1, S and G2/M stages and treated with CBNPs. After 6 hours and 24 hours the flow cytometry was used to detect 16HBE cells that synchronize at S and G2/M than those in the G0/G1 phase. The uptake of CBNPs, of which the CBNPs uptake of 16HBE cells in phase G2/M was the highest, compared with the control group, the difference was statistically significant (P0.05). This indicated that the different cell cycle stages could affect the effect of 16HBE cells on the uptake of CBNPs. The uptake of CBNPs in various stages of different cell cycles was from G2/M to G0/G1 phase of S phase. Conclusion: 1 CBNPs treatment can induce 16HBE cells to decrease in life, increase the rate of apoptosis, increase the level of reactive oxygen species, DNA damage and other acute toxicity, and these injuries are associated with the CBNPs number of 16HBE uptake in.2 S phase and G2/M phase is an important period for 16HBE cell uptake of CBNPs.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R994.6

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相關期刊論文 前1條

1 Raymond Bujdoso;Matthias Landgraf;Walker S Jackson;Alana M Thackray;;Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response[J];World Journal of Virology;2015年03期

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本文編號：2099862