【作者】 王萍；

【导师】 陈英华；

【作者基本信息】 南方医科大学 ， 人体解剖与组织胚胎学， 2020， 硕士

【摘要】 背景纳米氧化锌(ZnO nanoparticles,简称ZnO NPs)对紫外线具有广谱抵抗作用的同时还具有分散性好和透明性强等优点,因此作为防晒剂的主要添加成分被广泛应用于各类化妆品中。目前,关于ZnO NPs皮肤接触毒性越来越受到重视。完整的皮肤屏障能够抵御外界有害物和刺激物,而在皮肤屏障功能受损的情况下接触纳米颗粒,ZnO NPs可透过破损的皮肤屏障进入机体,然而透过破损的皮肤屏障渗入皮肤组织深层的ZnO NPs,其全身和皮肤毒性作用尚缺乏深入研究。研究表明表皮细胞Cdc42的缺失会导致皮肤屏障功能受损。本研究设想利用角质形成细胞Cdc42基因敲除小鼠,将其用于皮肤涂抹暴露ZnO NPs的毒性研究模型,检测ZnO NPs渗入对小鼠的全身和皮肤毒性作用,并进一步探讨ZnO NPs皮肤暴露毒性的作用机制。目标1.研究ZnO NPs渗入机体后在重要脏器的分布及毒性作用。2.研究ZnO NPs透过受损皮肤屏障对皮肤组织的毒性作用。3.探讨ZnO NPs渗入皮肤组织,对黑色素细胞的毒性作用及相关机制。方法本研究首先构建皮肤角质细胞Cdc42基因敲除(Knockout,KO)动物模型并于小鼠皮肤涂抹ZnO NPs。通过观察小鼠大体改变和记录体重改变、ICP-MS检测小鼠血液和各器官中锌元素的含量,血液生化检测研究ZnO NPs对小鼠全身毒性作用。体内实验通过小鼠皮肤组织含水量测定、H&E染色、TUNEL、Ki67免疫组化染色、氧化应激相关酶检测和8-OHdG免疫组织化学染色研究ZnO NPs对小鼠的皮肤毒性作用。通过全转录组基因测序研究小鼠皮肤相关信号改变并进行RT-PCR验证。通过免疫荧光检测DCT、TYR、FKBP51、BCL-2和NF-κB在表皮的表达。体外实验通过CCK-8检测ZnO NPs对黑色素细胞的活性影响,通过Edu和TUNEL观察ZnO NPs对黑色素细胞增殖和凋亡的影响,通过DCFH-DA检测ZnO NPs对细胞内ROS表达的影响,通过免疫荧光检测FKBP51、BCL-2和NF-κB在细胞的表达变化。进一步通过添加ROS抑制剂NAC,通过免疫印迹试验检测细胞内NF-κB的活化和BCL-2的蛋白表达变化。结果KO小鼠涂抹ZnO NPs组大体观察显示皮肤出现干燥和变黑,锌元素含量在血液、肝和脾明显高于其他各组,血清碱性磷酸酶含量较其余各组上升。KO小鼠涂抹ZnO NPs后皮肤含水量下降,病理显示表皮角质层不规则增生伴随明显过度角化和黑色素颗粒沉着,Ki67阳性表达上升以及TUNEL阳性表达下降,氧化应激损伤增加。全转录组基因测序结果显示KO小鼠涂抹ZnO NPs组皮肤组织FKBP51表达上调,免疫荧光检测DCT、TYR、FKBP51、BCL-2和NF-κB在此组小鼠表皮的表达增加。纳米氧化锌引起黑色素细胞内ROS表达增加,免疫荧光结果显示FKBP51、BCL-2和NF-κB在黑色素细胞的表达增加。ROS抑制剂NAC可抑制ZnO NPs引起黑色素细胞内NF-κB的活化和BCL-2的蛋白表达增加。结论ZnO NPs可透过破损的皮肤屏障,渗入深层组织,并随血液循环至肝脏和脾脏代谢,在一定程度上引起肝脏功能异常。渗入皮肤深层的ZnO NPs可导致明显的皮肤毒性,并引起皮肤表皮黑色素细胞过度增殖伴有黑色素瘤相关标志基因高表达等黑色素瘤样病变。该皮肤毒性可能由ZnO NPs通过ROS的积累刺激黑色素细胞NF-κB的活化并启动抗凋亡作用介导。更多还原

【Abstract】 BackgroundZinc oxide(ZnO)are abundantly produced nanomaterials and widely used in cosmetics products like sunscreens due to their opacifying,antimicrobial or UV protective properties.The skin barrier plays a critical role in preventing major environmental stresses.It can hinder the infiltration of external materials into the epidermis.Zhang et al.found that Cdc42 is essential for epidermal development and epidermal barrier formation and may be associated with skin barrier dysfunction and a variety of skin diseases.Recent reports suggest that nanoparticles(NPs)in sunscreens can penetrate through skin that was injured.In our study,the keratinocyte-restricted Cdc42 knockout mouse model was used to study the particle penetration.In this model,epidermal barrier dysfunction is caused by loss of Cdc42 in the epidermis in vivo.Although safety information on the dermal exposure to ZnO NPs is continuously increasing,there are deficiencies in the toxicological data,and therefore their effect cannot be confidently asserted.There is a lack of knowledge about the effects of these materials on epidermal barrier dysfunction skin.There is some concern about whether nanoparticles can cause health effects and skin change if the skin barrier integrity has been damaged.Objective1.To establish keratinocyte-restricted Cdc42 knockout(KO)mouse model and study the systemic toxic effects of ZnO NPs on KO mice after application.2.To study the toxic effects of ZnO NPs on skin tissue through the damaged skin barrier.3.To explore the toxic effects of ZnO NPs infiltrating into skin tissues on melanocytes and related mechanisms.MethodIn this study,KO mice were firstly established and used to study the toxicity of ZnO NPs exposed on the skin of mice.Observing the general changes and recording the weight changes of KO mice treated with 14-day ZnO NPs.Detecting the content of zinc in blood and organs of KO mice treated with 14-day ZnO NPs by ICP-MS and studying the systemic toxicity of KO mice treated with 14-day ZnO NPs by serum biochemical test.Studying the gene expression changes of KO skin treated with 14-day ZnO NPs by transcriptome sequencing,qRT-PCR and immunofluorescence staining.Detection of melanoma-like changes in KO mice treated with ZnO NPs by H&E staining pathological observation,electron microscopic observation,and TYR immunohistochemical staining of mouse skin tissue.TUNEL,Ki67 immunohistochemical staining,oxidative stress enzyme detection and 8-OHdG immunohistochemical staining were used to study the skin toxicity of ZnO NPs on KO mice.The mechanism of ZnO NPs-induced skin toxicity in KO mice was studied by changes in the expression of NF-κB and BCL-2 in melanocytes.The effect of gradient concentration of ZnO NPs on melanocytes cultured in vitro was detected by CCK-8;the effect of approporiate concentration of ZnO NPs on melanocytes proliferation and apoptosis was observed by Edu and TUNEL.The approporiate concentration of ZnO NPs was detected by immunofluorescence and qRT-PCR after treatment,the expression of FKBP51 was to determine the toxic effect with the treatment of ZnO NPs in vivo.The effect of 5 μg/ml ZnO NPs on intracellular ROS production was detected by DCFH-DA.The expression changes of NF-κB and BCL-2 were detected by immunofluorescence and western blot,and the mechanism was further detected by adding ROS inhibitor NAC to study NF-κB activation and BCL-2 protein expression.ResultsApplying ZnO NPs to KO mice can cause skin dryness and darkening.ICP-MS detection showed that the zinc content was higher in blood,liver and spleen in KO mice treated with 14-day ZnO NPs,and serum biochemical test showed the serum alkaline phosphatase content was higher than that in in KO mice treated with 14-day ZnO NPs.Transcriptome gene sequencing results showed that the expression of melanoma-related gene in skin tissue of KO mice treated with 14-day ZnO NPs changed.After applying ZnO NPs to KO mice,the skin water content decreased,and the epidermal hyperplasia was accompanied by obvious hyperkeratosis.After 14 days and 49 days ZnO NPs application,the number of melanocytes increased and even invaded into the dermal.With the increased days of ZnO NPs treatment,Ki67 positive cells significantly increased and TUNEL positive cells significantly reduced,oxidative stress damage increased as well.Immunofluorescence results showed that KO mice treated with ZnO NPs causing NF-κB activation and increasing BCL-2 expression in melanocytes.CCK-8 results showed that 2.5 μg/ml ZnO NPs can increase the viability of human epidermal melanocytes(HEM),5 μg/ml ZnO NPs can reduce cell viability at 24 h and 48 h after treatment,while increase viability at 72 h,but 7.5 μg/ml and 10μg/ml ZnO NPs can significantly inhibit cell viability.Edu and TUNEL staining results showed that 2.5 μg/ml ZnO NPs treatment led to proliferation without causing apoptosis.After treatment with 5 μg/ml ZnO NPs,HEM showed increased apoptotic cells at 24 h and 48 h,but showed decreased apoptotic cells accompanied by increased proliferative cells at 72 h.Treatment of melanocytes with 5μg/ml ZnO NPs for 72 h resulted in high expression of malignant transformation marker gene FKBP51.After 5μg/ml ZnO NPs treatment,ROS expression in melanocytes increased at 72 h,which resulted in the activation of NF-κB and BCL-2 expression.NAC,which is the ROS inhibitor,could inhibit the activation of NF-κB and BCL-2 expression in melanocytes.ConclusionZnO NPs can penetrate through the broken skin barrier and metabolize by blood and the liver and spleen,causing hepatic dysfunction to a certain extent.ZnO NPs that penetrate into the skin can cause obvious skin toxicity and cause excessive proliferation of melanocytes with melanoma-like lesions such as high expression of melanoma-related marker genes.This skin toxicity may be mediated by the accumulation of ROS caused ZnO NPs and stimulating the activation of NF-κB and initiating the anti-apoptotic effect in melanocytes.更多还原