纳米级炭黑颗粒对16HBE细胞毒性作用及影响因素的研究

发布时间：2018-07-05 10:10

  本文选题：炭黑纳米颗粒 + 氧化损伤　； 参考：《河北医科大学》2017年硕士论文

【摘要】：目的:炭黑(carbon black,CB)是一种超细碳粒子,是燃料不完全燃烧时产生的烟尘中的主要组成部分。炭黑纳米颗粒(carbon black nanoparticles,CBNPs)是通过控制碳氢化合物的气相裂解过程生产制造而成的纯的炭黑粉末,随着CBNPs的商业化及CB向大气的排放量持续升高,CBNPs的暴露与人类健康风险的评估备受关注。有研究证明处于不同周期的细胞会对纳米颗粒的摄取量存在差异,而细胞内纳米颗粒的含量与其所引起的细胞毒性密切相关。目前CBNPs对细胞产生的毒性损害以及不同细胞周期对于细胞的CBNPs摄取量是否产生影响尚不清楚,因此探究CBNPs产生的细胞损伤作用及不同周期对细胞摄取CBNPs量的影响具有十分重要的意义。本研究通过研究不同细胞周期对人支气管上皮细胞(16HBE)的CBNPs摄取量的影响为切入点,探究CBNPs对16HBE的细胞毒性效应及其影响因素。方法:1纳米炭黑颗粒表征观察通过扫描电子显微镜和透射电子显微镜观察纳米炭黑的结构特征。采用粒径分析仪分析纳米炭黑的粒径,BET比表面积测试法测量炭黑颗粒比表面积。2细胞培养和细胞模型建立16HBE(中国典型培养物保藏中心)用含10%热灭活胎牛血清的MEM培养基37℃,5%CO2饱和湿度下进行培养。选择对数生长期的细胞,用CBNPs处理24小时,剂量分别为50μg/mL、100μg/mL、200μg/mL;以MEM(含0.04%Tween80)为阴性对照。3 MTT法检测细胞存活率分别使用阴性对照组和1、10、50、100、200、300μg/mL CBNPs染毒组处理16HBE细胞3、6、12、24、36h。加入MTT孵育后使用酶标仪检测各孔在495nm波长处的吸光度值。每组设置六个复孔,计算各组吸光度并统计各染毒组细胞存活率。4细胞氧化应激水平的测定将cbnps染毒处理后的细胞制备成单细胞悬液,用2,7-二氢二氯荧光黄双乙酸钠(2,7-dichlorodihydrf-luoresceindiacetate,dcfh-da)荧光探针进行孵育,流式细胞仪检测不同剂量cbnps处理后16hbe细胞内活性氧(reactiveoxygenspecies,ros)的含量;16hbe细胞内抗氧化酶超氧化物歧化酶(superoxidedismutase,sod)、谷胱甘肽过氧化物酶(glutathioneperoxidase,gsh-px)活性以及脂质过氧化产物丙二醛(malonaldehyde,mda)含量采用生化试剂盒并按照说明书进行定量检测。5细胞dna损伤的测定应用单细胞凝胶电泳(singlecellgelectrophoresis,scge)实验,通过观测染毒前后olive尾踞的变化,检测cbnps对细胞dna的损伤。6annexinⅤ-pi双染检测细胞凋亡染毒24小时后将阴性对照和各染毒组细胞制备成单细胞悬液,采用annexinⅤ-fitc和pi双染标记,于流式细胞仪检测细胞凋亡率。7细胞周期检测将阴性对照和各染毒组制备成单细胞悬液,用-20℃预冷的80%的乙醇固定后,先后使用rnasea和pi处理细胞,之后使用流式细胞仪进行周期检测。8细胞周期的同步化使用害羞草碱使16hbe细胞同步于g0/g1期,胸苷双阻断法使16hbe细胞同步于s期,诺考达唑使细胞同步于g2/m期。9细胞摄取cbnps检测将阴性对照和各染毒组16hbe细胞制备成单细胞悬液,经pbs洗涤两次后,应用流式细胞仪进行检测,并通过ssc密度含量的改变来定量分析16hbe对cbnps的摄取量。另外采用imagestreamx单细胞成像流式细胞仪检测各组样本并获取各剂量组细胞的明场及暗场图像和数据,由此分析16hbe摄取cbnps的情况。结果:1纳米炭黑颗粒表征电子显微镜下观察,纳米炭黑为球状颗粒。透射电镜显示炭黑颗粒大体呈葡萄球状,分散良好,颗粒直径在30~50nm之间。扫描电镜显示炭黑颗粒近似呈球形,分散较均匀,团聚颗粒直径大约200~400nm之间。较大颗粒表面有很多球形突出物。利用bet比表面积测试法测量炭黑颗粒比表面积为74.85m2/g。炭黑颗粒难溶于水,在水溶剂中炭黑团聚颗粒直径为200~400nm,而在0.04%tween80中有较好的分散性,颗粒直径为30~50nm。2cbnps对16hbe细胞活性的影响当用100、200和300μg/mlcbnps处理16hbe细胞3h后,各剂量组细胞存活率逐渐降低(p0.05);当用50、100、200和300μg/mlcbnps处理16hbe细胞6h后,各剂量组细胞存活率逐渐降低(p0.05);当用1、10、50、100、200和300μg/mlcbnps处理细胞12、24、36h后,各剂量组细胞存活率逐渐降低(p0.05);随着染毒浓度和时间的增加,16hbe细胞活性降低更加明显。这些结果表明cbnps抑制16hbe细胞存活并且这种抑制效应存在剂量和时间依赖性。3cbnps诱导16hbe细胞凋亡用50、100和200μg/ml的cbnps处理16hbe细胞24小时。发现50μg/ml和100μg/mlcbnps处理的16hbe早期凋亡细胞数目略微升高(p0.05);200μg/mlcbnps处理可诱导16hbe细胞早期凋亡率显著增加(p0.05);随着cbnps染毒浓度增加,晚期凋亡细胞所占的百分率和细胞坏死细胞的百分率逐渐升高(p0.05)。这些结果表明cbnps对细胞增殖的抑制作用至少可以部分解释为cbnps诱导的凋亡和坏死。4cbnps对16hbe细胞氧化损伤的测定不同浓度cbnps处理16hbe细胞24小时后,随着染毒浓度的增加,ros水平逐渐升高(p0.05);mda含量逐渐增加(p0.05);sod和gsh-px活力随cbnps染毒浓度的增加而逐渐降低(p0.05)。说明cbnps可以诱导16hbe细胞氧化损伤。5cbnps对16hbe细胞dna损伤的诱导作用不同剂量的cbnps处理16hbe细胞24小时后,随着染毒浓度的增加,各剂量组olive尾距显著增加,与对照组相比,差异具有统计学意义(p0.05)。说明cbnps可以诱导16hbe细胞dna损伤。6cbnps对16hbe细胞周期的影响用100μg/mlcbnps对16hbe细胞进行连续处理,发现cbnps诱导S期和G2/M期细胞比率增加(P0.05)。S期细胞比率增加发生在6至24小时;G2/M期细胞比率增加发生在18至36小时。说明CBNPs诱导16HBE细胞周期阻滞,且阻滞发生于S期和G2/M期。7 16HBE细胞对CBNPs的摄取用50、100和200μg/mL的CBNPs处理16HBE细胞24小时,采用流式细胞仪和ImageStreamX成像流式细胞仪进行检测。发现细胞内CBNPs量逐渐升高(P0.05);将16HBE细胞分别同步化于G0/G1期,S期和G2/M期并进行CBNPs染毒处理。经6小时和24小时染毒后采用流式细胞仪检测观察到同步于S期和G2/M期的16HBE细胞比同步于G0/G1期的16HBE细胞有更高的CBNPs摄取量,其中同步于G2/M期的16HBE细胞对CBNPs摄取量最高,与对照组相比,差异有统计学意义(P0.05)。这表明不同细胞周期阶段可以影响16HBE细胞对CBNPs的摄取效果。不同细胞周期各阶段对CBNPs的摄取量由多到少排序为G2/M期S期G0/G1期。结论:1 CBNPs处理能够诱导16HBE细胞发生活性降低、凋亡率增加、活性氧水平升高、DNA损伤等急性毒性损伤,且这些损伤与16HBE摄取的CBNPs数量相关。2 S期及G2/M期是16HBE细胞摄取CBNPs的重要时期。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R994.6

【参考文献】

相关期刊论文 前1条

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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