纳米氧化锌对人肝肾细胞的毒性作用研究

发布时间：2018-04-15 14:53

本文选题：纳米氧化锌 + HL-7702肝细胞　；参考：《中国计量学院》2012年硕士论文

【摘要】：纳米氧化锌具有极强的抗氧化性、抗腐蚀性、光催化性和独特的抗紫外线性，使其在光电学、橡胶工业、日用化工、食品添加剂、生物医学等方面应用广泛。纳米氧化锌可通过皮肤、呼吸道或消化道进入生物体并在体内积蓄，但机体很难排除这类微小物质，因此潜在一定的生物毒性效应，对其生物安全性的研究已成为目前的研究热点。为了探讨纳米氧化锌是否具有肝肾细胞毒性，本研究以人正常肝细胞HL-7702和人胚肾细胞HEK293为研究对象，首先通过形态学观察、MTT(噻唑盐)比色法来检测纳米氧化锌对细胞活性的影响，然后通过单细胞凝胶电泳和微核试验检测其诱导的单链DNA断裂程度和染色体损伤程度。检测细胞内活性氧水平(包括GSH、MDA和SOD水平)，考马斯亮蓝法测定蛋白质羰基含量来探讨纳米氧化锌对细胞的氧化损伤作用及可能机制。同时，利用DNA-laddering法来研究纳米氧化锌诱导的细胞凋亡作用。一、纳米氧化锌对HL-7702及HEK293细胞的毒性作用研究通过透射电镜确定纳米氧化锌粒子粒径分布，制备成不同浓度的含氧化锌培养液与人正常肝细胞HL-7702及人胚肾细胞HEK293接触培养，，通过倒置显微镜观察其形态，采取MTT法检测细胞毒性，计算相对增值率(RGR)，并进行毒性评价。形态学观察结果显示，高剂量纳米氧化锌粒子作用细胞对细胞形态的影响更为明显，可导致细胞形态变化，细胞凋亡或坏死。MTT法进行细胞毒性检测结果表明，对于HL-7702和HEK293细胞，当纳米氧化锌粒子浓度分别达到10μg/mL和25μg/mL时，纳米氧化锌实验组与对照组组相比出现显著性差异(P0.05)，并表现出浓度-效应关系。二、纳米氧化锌对HL-7702及HEK293细胞的遗传毒性探讨纳米氧化锌对人肝肾细胞的遗传毒性效应，采用不同浓度纳米氧化锌10、25、50、75和100μg/mL对分别对HL-7702和HEK293细胞进行染毒12h，24h和48h后，用单细胞凝胶电泳技术检测细胞的DNA的损伤程度。并采用微核试验检测染毒后双核细胞的微核发生率，以期得出纳米氧化锌体外遗传毒性。结果显示，纳米氧化锌染毒肝肾细胞后，随着培养基中纳米氧化锌浓度的增加，与对照组相比，高剂量染毒组细胞头部DNA含量(Head DNA%)显著降低，尾部DNA含量(Tail DNA%)、尾矩(Tail Moment)、Olive尾矩(Olive Tail Moment)数值显著升高(P＜0.05)；微核试验发现染毒组的微核率显著升高。研究结果提示，高浓度的纳米氧化锌可引起人肝胚肾细胞DNA和染色体水平损伤，表现出遗传毒性效应，认为其对细胞DNA的损伤个的纳米氧化锌致体外细胞毒性的主要机制之一。三、纳米氧化锌致HL-7702及HEK293细胞氧化损伤作用的研究验证纳米氧化锌是否致使HL-7702及HEK293细胞产生氧化损伤作用，采用不同浓度的纳米氧化锌10、25、50、75和100μg/mL对细胞进行染毒处理，24h后收集细胞，检测其谷胱甘肽GSH、丙二醛MDA和超氧化物歧化酶SOD水平，探讨细胞内氧化应激水平，并通过检测蛋白质羰基含量来反映纳米材料对蛋白质的氧化损伤作用。结果表明，在纳米氧化锌处于一定剂量范围内(≤100μg/mL)，纳米氧化锌处理组总SOD和GSH酶活力降低，而MDA含量增加，且呈现一定的剂量-效应关系，在高浓度纳米氧化锌组中蛋白质羰基含量与对照组相比有显著性的升高(P0.05)。进一步证实纳米氧化锌可对人肝肾细胞产生明显氧化损伤作用，并认为氧化损伤是纳米氧化锌导致细胞毒性的主要机制之一。四、纳米氧化锌致HL-7702及HEK293细胞凋亡作用的研究采用DNA-ladder法探讨纳米氧化锌对人肝肾细胞凋亡作用的影响，结果表明：纳米氧化锌处理细胞后进行的DNA电泳图显示，DNA在100-200bp发生不同程度的特异性断裂，出现较明显的DNA ladder片段，提示纳米氧化锌能诱导人肝肾细胞发生凋亡作用。纳米氧化锌诱发的细胞凋亡可能是发生细胞毒性的机制之一，其有关机理还有待进一步研究。
[Abstract]:Nano Zinc Oxide has highly resistant to oxidation, corrosion resistance, photocatalysis and unique UV resistance, the photoelectricity, rubber industry, daily chemical, food additives, biomedical and other aspects of a wide range of applications. Nano Zinc Oxide through the skin, respiratory or digestive tract into organisms and accumulated in the body, but it is difficult for the body to eliminate this kind of small matter, therefore some potential biological toxicity effects on the biological safety has become a hot research topic at present. In order to investigate whether Zinc Oxide has nano liver and kidney cells toxicity, this study in normal human liver cell HL-7702 and human embryo kidney cell HEK293 as the research object, firstly by morphological observation MTT, (MTT) colorimetric method to detect the effect of nano Zinc Oxide on cell activity, followed by single cell gel electrophoresis and micronucleus test to detect the induction of single strand DNA fragmentation and dyeing Body injury. The level of intracellular reactive oxygen species detection (including GSH, MDA and SOD), to explore the effect of nano Zinc Oxide oxidative damage to cells and the possible mechanism of the determination of protein carbonyl content Kaumas Bradford method. At the same time, to study the cell apoptosis induced by nano Zinc Oxide DNA-laddering method.
Study on the toxic effect of nano Zinc Oxide on HL-7702 and HEK293 cells
By transmission electron microscopy to determine the particle size distribution of nano Zinc Oxide, prepared by different concentration of HL-7702 and liquid containing Zinc Oxide culture and human normal liver cell embryo kidney cell HEK293 contact culture, were observed by inverted microscope, cell toxicity test MTT method, calculation of the value-added rate (RGR), and toxicity evaluation.
Morphological observation showed that the effect of high dose of nano particle effects on cell morphology of Zinc Oxide cells is more obvious, can lead to changes in cell morphology, cell apoptosis or necrosis of.MTT cell toxicity test showed that the HL-7702 and HEK293 cells, when the nano particle concentration Zinc Oxide reached 10 g/mL and 25 g/mL, nano Zinc Oxide the experimental group and the control group was significantly difference (P0.05), and showed dose effect relationship.
Two, the genetic toxicity of nanoscale Zinc Oxide to HL-7702 and HEK293 cells
To investigate the genetic toxic effect on liver and kidney cells of nano Zinc Oxide, with different concentrations of nano Zinc Oxide 10,25,50,75 and 100 g/mL respectively for 12h exposure on HL-7702 and HEK293 cells, 24h and 48h after injury were detected by single cell gel electrophoresis DNA and micronucleus test were detected. The incidence after binucleate cells. Micronucleus, in order to obtain nano Zinc Oxide in vitro genotoxicity.
The results showed that liver and kidney cells after exposure to nano Zinc Oxide, with the increase of nano Zinc Oxide culture medium concentrations, compared with the control group, high dose group cells (Head DNA%) head DNA content decreased significantly, the tail DNA content (Tail DNA%), tail moment (Tail Moment), Olive (Olive Tail Moment) tail moment value increased significantly (P < 0.05); micronucleus test found that the micronuclear rates increased. The results suggest that high concentrations of nano DNA Zinc Oxide can cause renal cell and embryo liver injury showed chromosome level, genetic toxicity effect, that the main mechanism of in vitro cytotoxicity induced on DNA damage induced by a nano Zinc Oxide one.
Three, study on the oxidative damage of HL-7702 and HEK293 cells induced by nano Zinc Oxide
Verify whether Zinc Oxide led HL-7702 and nano HEK293 cells to produce oxidative damage, with different concentrations of nano 10,25,50,75 and Zinc Oxide 100 g/mL cells were treated with 24h, were collected after the detection of glutathione GSH and malondialdehyde MDA superoxide dismutase SOD levels of oxidative stress in cells, and the protein carbonyl content test to reflect the effects of nanomaterials on oxidative damage of proteins. The results showed that the nano in Zinc Oxide in a certain dose range (less than 100 g/mL), Zinc Oxide group SOD and nano GSH enzyme activity decreased, while the content of MDA increased, and showed a dose effect, in the high concentration of nano protein in Zinc Oxide group the carbonyl content increased as compared with the control group there was significant (P0.05). Further confirmed that Zinc Oxide can produce obvious nano oxygen on human liver and kidney cells of injury, It is also considered that oxidative damage is one of the main mechanisms of the cytotoxicity of nanoscale Zinc Oxide.
Four, study on the effect of nano Zinc Oxide on the apoptosis of HL-7702 and HEK293 cells
To investigate the effect of Zinc Oxide, nano effect on human liver and kidney cells apoptosis by DNA-ladder method showed that DNA electrophoresis for processing nano Zinc Oxide after the cells showed that DNA specific fracture at different level of 100-200bp, DNA fragment of ladder obviously, suggesting that Zinc Oxide nano induced apoptosis of human liver cells. The apoptosis of nano Zinc Oxide may be one of the mechanisms of induced cell toxicity occurred, its mechanism remains to be further studied.

【学位授予单位】：中国计量学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114;TB383.1

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