卷烟烟气暴露下AA549和BEAS-22B细胞促炎性因子的释放变化

为比较不同类型细胞对卷烟烟气诱导的促炎症反应的应答差异,采用中性红细胞毒性试验测试了卷烟烟气总粒相物(TPM)和全烟气(WS)对人肺腺癌细胞A549和人支气管上皮细胞BEAS-2B的细胞毒性效应;采用酶联免疫法(ELISA)分析了TPM和WS暴露后,A549和BEAS-2B促炎性细胞因子白细胞介素-6(IL-6)和白细胞介素-8(IL-8)的释放水平。结果表明:BEAS-2B细胞较A549细胞对卷烟烟气的细胞毒性效应敏感。卷烟烟气暴露下A549细胞没有增加促炎性细胞因子IL-6和IL-8的释放;而BEAS-2B细胞在TPM和WS暴露下其IL-6和IL-8的释放显著增加。卷烟烟气诱导的促炎症反应存在细胞类型的差异。     

三维细胞支架的制备及在卷烟烟气细胞毒性评价中的应用

为开发用于卷烟烟气体外毒理学评价的三维细胞支架,采用高内相乳液法制备材料,以其为载体进行人肺腺癌细胞A549的三维培养,评价3R4F和CM8两种参比卷烟烟气总粒相物(TPM)的细胞毒性,并与二维平板培养细胞的实验结果进行了对比。结果表明:(1)A549细胞在三维细胞支架上可以吸附、增殖并融合形成细胞层,且在亲水改性支架上增殖更快;(2)培养21d后,三维细胞支架上A549细胞的吸光度(OD值)为3.601,高于二维平板上细胞长满(培养7 d)时的OD值(3.055);(3)3R4F和CM8卷烟烟气TPM浓度与二维平板和三维细胞支架培养的A549细胞存活率均具有良好的剂量-反应关系,且在高TPM浓度时,三维细胞体系下的细胞存活率高于二维细胞体系。该研究为卷烟烟气体外毒理学评价提供了一种新的三维细胞培养方法。     

基于两种细胞的卷烟烟气体外免疫毒性评价

为评价卷烟烟气总粒相物(TPM)的体外免疫毒性,选择小鼠淋巴瘤细胞(EL-4细胞)和小鼠巨噬细胞(Ana-1细胞)两种细胞,采用CCK-8法检测肯塔基3R4F参比卷烟烟气TPM染毒后细胞的存活率,采用Luminex液相芯片技术检测细胞上清液中20种细胞因子(FGF-basic、GM-CSF、IFN-γ、IL-1α、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、IL-13、IL-17、KC、IP-10、MCP-1、MIP-1α、VEGF、MIG、TNF-α)的分泌量。结果表明:两种细胞经烟气TPM染毒后,随着染毒剂量的增加,EL-4细胞中GM-CSF、IL-10和VEGF的分泌量均下调;Ana-1细胞中MCP-1、MIP-1α和TNF-α的分泌量均上调;两种细胞中其余17种细胞因子的分泌量无明显变化。不同类型/来源的细胞对烟气的毒性效应有不同的反应,因此采用两种细胞有助于更好地评价卷烟烟气的体外免疫毒性。该方法具有快速、灵敏、高通量等特点,适用于卷烟烟气体外免疫毒性评价。     

食品污染物苯并[a]芘致细胞损伤作用的研究

目的:对食品污染物苯并[a]芘所致细胞损伤作用进行研究。方法:用不同浓度的苯并[a]芘(1、5、10、50、100μmol/L)对肺腺癌细胞A549染毒24h,MTT法测定细胞存活率、单细胞琼脂糖凝胶电泳检测细胞DNA损伤,用溶剂二甲基亚砜处理细胞组作为未染毒细胞组。结果:苯并[a]芘染毒作用时,随染毒浓度的增加,A549细胞存活率呈下降趋势;A549细胞的DNA损伤程度也随染毒浓度增加而增加,与未染毒A549细胞相比,染毒A549细胞的Olive尾矩值均有明显增加(p<0.05)。结论:食品污染物苯并[a]芘致A549细胞明显的损伤作用。     

卷烟添加木犀草素与黄芩苷混合物对细胞损伤的减害作用研究

将木犀草素、黄芩苷混合物加入卷烟制成样品卷烟,研究其在卷烟烟气中对人肺泡上皮细胞损伤的影响。用不同浓度卷烟烟气提取物(cigarette smoke extract,CSE)刺激人肺泡上皮细胞A549,采用MTT实验检测细胞存活率,彗星实验观察DNA损伤,荧光法测定细胞内活性氧(reactive oxygen species,ROS)含量。结果表明,与对照卷烟比较,样品卷烟组细胞存活率升高,细胞内活性氧(ROS)彗星尾长、尾部DNA含量、尾距、Olive尾距均小于对照卷烟组。因此,添加木犀草素与黄芩苷混合物的特制卷烟烟气对细胞的损伤较少,产生较少ROS,减少一定的DNA损伤,对细胞毒性有一定的减害作用。     

枸杞多糖联合顺铂对人肺腺癌细胞A549氧化损伤及凋亡的影响

目的:探讨枸杞多糖(Lycium barbarum polysaccharides,LBP)联合顺铂(cisplatin,DDP)对人肺腺癌细胞A549氧化损伤、细胞凋亡及凋亡相关蛋白表达的影响。方法:细胞分为对照组、DDP组(6 mg/L)、LBP组(8 mg/L)和DDP(6 mg/L)+LBP(8 mg/L)组,CCK-8法检测DDP、LBP单独及联合用药对A549细胞存活率的影响;黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(superoxide dismutase,SOD)活力;二硫代二硝基苯甲酸法测定谷胱甘肽(glutathione,GSH)含量;硫代巴比妥酸法测定丙二醛(malondialdehyde,MDA)含量;流式细胞术检测活性氧(reaction oxygen species,ROS)含量和细胞凋亡率;Western blot检测细胞凋亡相关蛋白Bcl-2、Bax和Caspase-3的表达。结果:LBP能极显著增强DDP对A549细胞存活率的抑制作用(P0.01)。DDP导致A549细胞SOD活力和GSH含量极显著降低(P0.01),MDA含量和ROS含量极显著升高(P0.01)。而LBP联合DDP组与DDP组比较,细胞内SOD活力、GSH含量、MDA含量无显著变化(P0.05),而ROS含量明显降低(P0.01)。一定质量浓度DDP和LBP单独及联合处理均能极显著促进细胞凋亡(P0.01),并且能极显著降低Bcl-2、提高Bax、Caspase-3表达量及Bax/Bcl-2值(P0.01)。结论:LBP与DDP联合作用可明显促进A549细胞凋亡,其机制主要与两者对细胞凋亡相关蛋白表达的调节有关。     

γ-萜品烯的体内外抗氧化性研究

本文通过细胞荧光强度、3种抗氧化酶活性(超氧化物岐化酶,SOD;谷胱甘肽过氧化物酶,GSH-Px;过氧化氢酶,CAT)、谷胱甘肽(GSH)和丙二醛(MDA)含量的测定,在细胞模型和动物实验中研究γ-萜品烯的体内外抗氧化能力。结果表明,Hep-G2细胞内的荧光强度随着γ-萜品烯质量浓度的增加而下降,二者呈现良好的剂量-作用关系,说明γ-萜品烯可以有效结合细胞膜内外的自由基或活性氧,从而减少细胞内带有荧光的氧化产物的积累;过氧化氢诱导建立的氧化应激细胞模型和动物实验结果表明,γ-萜品烯可以显著提高超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的活性及谷胱甘肽的含量(p<0.05),并显著降低丙二醛的含量(p<0.05)。以上研究结果表明,γ-萜品烯在体内外均表现出一定的抗氧化作用。     

鲑鱼皮明胶水解物的抗氧化活性及其对细胞氧化损伤的保护作用

采用碱性蛋白酶、木瓜蛋白酶和中性蛋白酶水解鲑鱼皮明胶,得到水解度为4.7％到13.5％的7个明胶水解物,分别评价明胶水解物对自由基的清除能力.明胶水解物(0.5、1、2mg/mL)预先作用BRL大鼠肝细胞2h后,通过H2O2(5mmol/L)诱导氧化损伤,分别测定细胞存活率、乳酸脱氢酶(LDH)、丙二醛(MDA)和谷胱甘肽(GSH)等细胞内抗氧化产物的含量变化.结果表明,明胶水解物的抗氧化活性随着水解度的增大呈增强趋势.明胶水解物对细胞具有保护作用,可显著提高细胞存活率,降低LDH渗出量和MDA生成量,且存在一定的剂量关系,但细胞中GSH含量变化不显著;7个明胶水解物对H2O2诱导肝细胞损伤保护时,细胞存活率与水解物的体外抗氧化活性大小显著正相关,而LDH渗出量和MDA生成量与水解物的体外抗氧化活性大小负相关.     

韩国产芝麻酱油对AAPH诱发LLC-PK1细胞氧化应激的保护作用

以芝麻酿造酱油乙醇提取物(ethanol extract sesame sauce,SSE)为研究对象,探讨其对1 mmol/L 2,2’-偶氮二异丁基脒二盐酸盐(2,2’-azobis(2-methylpropionamidine)dihydrochloride,AAPH)引发的LLC-PK1猪肾近曲小管上皮细胞氧化应激损伤的保护作用。方法:LLC-PK1细胞与不同质量浓度(10~100μg/m L)的SSE预先共同培养24 h后,用含AAPH的DMEM培养液继续培养4 h建立细胞损伤模型。细胞存活率用四甲基偶氮唑蓝法检测,细胞内丙二醛(malondialdehyde,MDA)含量和总活性氧簇(reactive oxygen species,ROS)水平分别以硫代巴比妥酸比色法和2’,7’-二氢二氯荧光黄双乙酸钠探针法测定。细胞内过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化酶(glutathione peroxidase,GSH-Px)、谷胱甘肽巯基转移酶(glutathione S-transferase,GST)、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-GCS)活力和谷胱甘肽(glutathione,GSH)含量按试剂盒说明书以比色法测定。结果表明,SSE预处理可以提高受损细胞存活率,降低细胞内总ROS水平和MDA含量。同时,SSE还能提高受损细胞内抗氧化酶(CAT、SOD、GSH-Px)及γ-GCS活力并提高细胞内GSH含量。实时荧光定量聚合酶链式反应分析也提示SSE能上调细胞内抗氧化物酶(CAT、SOD和GSH-Px)的m RNA表达量。此外,SSE可以通过提高细胞内源性抗氧化系统能力来降低细胞内MDA和ROS水平,并由此缓解AAPH对LLC-PK1细胞所造成的氧化应激损伤。     

小麦蛋白水解物对肠道上皮细胞抵御氧化应激损伤的作用

采用双酶和三酶对小麦蛋白复合水解,获得富含谷氨酰胺(Gln)的双酶酶解产物(PWGH)和三酶酶解产物(AWGH)。比较小麦蛋白及其2种酶解产物经体外模拟消化前、后,抵御肠上皮细胞(Caco-2)氧化应激损伤能力的变化。结果表明:2种水解产物均能在一定程度上缓解细胞因氧化应激损伤导致的存活率降低现象,减少细胞氧化损伤的程度。二者经体外模拟消化降解后仍有此作用,而小麦蛋白的体外模拟消化产物对损伤细胞存活率无显著影响。2种水解产物均能在一定程度上提高氧化应激损伤细胞的抗氧化能力,提高细胞内还原型谷胱甘肽(GSH)水平,减少损伤细胞的脂质氧化,然而,经体外模拟消化降解后,PWGH仍有上述作用,而AWGH对损伤细胞的抗氧化能力和GSH水平无显著影响。研究表明:体外模拟消化对AWGH的活性肽段有破坏,而PWGH活性肽段经消化后仍有保留。值得注意的是,小麦蛋白经体外模拟消化,其产物(WGH)虽能减少损伤细胞的脂质氧化,但对损伤细胞的抗氧化能力和GSH水平均无显著影响。     

不同抽吸模式下烟气冷凝物对人口腔细胞体外生长增殖的影响

为考察卷烟烟气暴露对人口腔细胞体外生长增殖的影响,以肯塔基参比卷烟3R4F 为试验卷烟,人口腔表皮样癌细胞为实验模型,分别在ISO 和加拿大深度抽吸(HCI)模式下制备烟气冷凝物并对细胞进行染毒暴露,评估了两种抽吸模式下烟气暴露对人口腔细胞代谢活性及核酸复制活性的影响,并结合主流烟气总粒相物(TPM)中主要有害成分的释放水平进行了分析。结果表明:①烟气冷凝物暴露对人口腔细胞体外增殖有抑制作用,对人口腔细胞代谢活性以及核酸的复制活性有一定的毒性作用。②HCI 抽吸模式下3R4F 卷烟单位TPM 和单位烟碱中有害成分的释放量低于ISO 模式,HCI 抽吸模式下制备的烟气冷凝物含水率高于ISO 模式,这两种因素是导致3R4F 卷烟HCI 模式下烟气冷凝物细胞毒性弱于ISO模式的主要原因。      

采用全烟气暴露系统测试卷烟主流烟气细胞毒性

基于VITROCELL系统结合中性红摄取试验建立了全烟气暴露细胞毒性测试方法,采用建立的方法测试了加拿大深度(HCI)抽吸条件下国内外29个牌号市售卷烟主流烟气的细胞毒性。结果表明:①建立的全烟气暴露细胞毒性测试方法可以获得良好的烟气细胞毒性剂量-效应关系;②测试结果的变异系数(CV)在25%以内,方法稳定性较好。建立的全烟气暴露实验方法适合于卷烟主流烟气细胞毒性测试。     

卷烟烟气pH与烟气总粒相物中游离烟碱的关系

对烟气总粒相物pH的测定方法进行了实验研究,确定了准确测定烟气总粒相物pH的方法。对烟气总粒相物中游离烟碱含量的测定方法进行了实验研究,确定了用配备FID的气相色谱仪准确测定烟气总粒相物中的游离烟碱含量的方法。按照国标方法测定烟丝中总植物碱、还原糖、总氮、挥发碱的含量;按照国标方法测定卷烟的焦油、烟碱、水分。请评吸专家对卷烟进行评吸,针对烟气劲头和刺激性进行打分。对测定结果进行分析比较,研究结果表明:烟气中游离烟碱占总烟碱的比例与烟气总粒相物pH呈现显著的线性相关关系;烟丝总氮含量与烟气总粒相物pH呈显著线性相关;烟气烟碱、烟丝总烟碱、还原糖对烟气劲头有较大影响,而对烟气刺激性影响较大的因素有烟气总粒相物pH、烟叶pH、总氮等。      

Toxicological evaluation of aspartame against Madin–Darby canine kidney cells

Aspartame is most widely used as artificial sweeteners in more than 6000 food varieties. Aspartame digested into aspartic acid, phenylalanine, and methanol, and several peroxides, superoxide molecules also generated together. The kidney is the secondary site for the cellular metabolism. The present study examined whether the treatment of aspartame induces oxidative stress in the Madin–Darby kidney cells (MDCK). The effects of aspartame on MDCK cell viability were investigated by the sulphorhodamine-B assay and flow cytometry. Morphology of MDCK cells following aspartame exposure was observed. Mitochondria-derived reactive oxygen species (ROS) was also determined using 2′,7′-dichlorodihydrofluorescein diacetate. Lipid peroxidation (LPO), glutathione reduced (GSH) levels and activities of superoxide dismutase (SOD), and catalase enzymes were also determined. Cell viability was significantly altered following aspartame exposure. Morphology of MDCK cells did not change significantly. However, there was a marginal morphological change, including rounding, sporadic distribution and loss of adherence were observed at higher doses of aspartame exposure. Mitochondria-derived ROS was increased in a dose-dependent manner following aspartame exposure. A significant increase in LPO levels, whereas GSH level was reduced after 48 and 72 h of aspartame exposure. SOD and catalase enzyme activities were significantly reduced in a dose- and time-dependent manner. Taking all these data together, it is concluded that aspartame may induce oxidative stress in the MDCK cells.     

不同类型流式细胞仪检测卷烟烟气体外微核比对研究

目的:为了评估流式细胞仪（FCM）检测卷烟烟气体外微核的广泛适用性和稳定性。方法:采用4种不同类型的FCM,对国内5种品牌的卷烟烟气总粒相物（TPM）处理的中国仓鼠卵巢细胞（CHO）和人支气管上皮细胞（BEAS-2B）微核进行检测并比较检测数据。结果:不同类型FCM检测参数差异较大,但经过调整优化之后均可达到卷烟烟气体外微核检测的技术要求。不同类型FCM测定的微核率之间统计学分析没有显著差异（P>0.05）。结论:不同类型的FCM均适用于卷烟烟气体外微核的检测,且检测结果具有一致性和稳定性。      

不同形态硒与VE联用对H_2O_2诱导人脐静脉血管内皮细胞氧化应激损伤的保护作用

目的:研究不同形态硒化合物(有机硒:L-硒甲基硒代半胱氨酸;无机硒:亚硒酸钠)单独使用及其与VE联合使用后对H_2O_2诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)产生氧化应激损伤的保护作用。方法:采用体外细胞培养方法,将HUVECs随机分为7组:对照组、H_2O_2组、L-硒甲基硒代半胱氨酸(L-Se-methylselenocysteine,L-Se MSC)组、亚硒酸钠(sodium selenite,SS)组、VE组、L-Se MSC+VE联用组、SS+VE联用组。经不同样品干预处理后,采用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测HUVECs存活率;检测各组细胞内脂质氧化终产物丙二醛(malondialdehyde,MDA)含量以及超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性的变化。结果:各处理组HUVECs存活率之间没有显著差异;与H_2O_2组比较,0.1 mg/L亚硒酸钠、10 mg/L VE处理能使HUVECs内MDA生成量显著减少,SOD和GSH-Px活性显著升高,并且亚硒酸钠和VE联合使用在细胞水平上具有显著的协同作用,而L-Se MSC组与对照组无显著差异。结论:亚硒酸钠和VE均可保护由H_2O_2诱导引起的HUVECs氧化损伤,提高HUVECs抗氧化能力,并且亚硒酸钠和VE联用具有明显的协同抗氧化作用。     

P20添加剂的设计及其对烟气TPM含量的影响

采用均匀设计理论安排了１４组不同配比的Ｐ２０添加剂（主要成分为苹果酸和碳酸钾）,添加到卷烟烟支中,检测其ＴＰＭ递送量。将有关数据在计算机上进行回归分析,得到苹果酸和钾量与ＴＰＭ递送量的回归方程,分析了不同配比的Ｐ２０添加剂与ＴＰＭ递送量的关系,并将该方程在约束条件下用复形调优法求极值,得出最优条件。在此条件下,ＴＰＭ递送量比对照烟支降低了３．６７ｍｇ／支。     

The effect of modified starches on the adsorption of cigarette mainstream smoke composition

In this work modified starches such as porous starch (PS) and cross‐linked phosphorylated porous starch (PPS) were used as tobacco filter to investigate their adsorption efficiency on the tar, ammonia (NH₃), hydrocyanic acid (HCN), crotonaldehyde, and total particulate matter (TPM) in mainstream cigarette smoke. The pore properties and structural stability of the modified starches have been evaluated and the adsorption relationship between the modified starches and the main toxicants in tobacco smoke has also been established. Analytes of mainstream smoke from experimental cigarettes show reductions in yields of some measured constituents in mainstream cigarette smoke, such as tar, HCN, NH₃, crotonaldehyde, and TPM. The largest reductions are for NH₃ levels, up to 35.93% in one case, observed from the tobacco filter containing PPS. The largest reductions in mainstream yields of HCN (13.04%) and crotonaldehyde (31.06%) smoke toxicants were also observed. These results indicate that reducing exposure to some smoke toxicants is possible using the modified starches such as PS and PPS as tobacco filter.     

基于遗传算法的卷烟主流烟气粒相物pH值影响因素研究

为研究影响烟气pH值的关键性烟丝化学成分,以国内代表性卷烟为研究对象,分别对样品的烟丝生物碱、糖、氨基酸、有机酸等化学成分含量及主流烟气粒相物pH值进行了测定。采用遗传算法结合偏最小二乘法,筛选出8个对烟气总粒相物pH值影响最大的化学指标,建立了烟气pH值与烟丝化学成分之间的定量关系模型。结果表明,烟丝丁酸、异戊酸、乙酰丙酸、苹果酸、葡萄糖和精氨酸可以降低卷烟烟气pH值,烟碱和异亮氨酸可以增加卷烟烟气pH值。本研究可为卷烟产品研发提供科学依据。      

Oxidative effects of cigarette smoke on the human skin

The oxidative effects of cigarette smoke on the human skin were investigated. A remarkable increase in the conversion ratio of squalene (SQ) to squalene monohydroperoxide (SQHPO) due to exposure to cigarette smoke was observed using a CL-HPLC (high performance liquid chromatography with a chemiluminescence detector) system. The results showed that cigarette smoke caused lipid peroxidation. We also found that the addition of chain-breaking-type antioxidants, such as oolong tea extract, inhibited the peroxidation. When cultured human skin fibroblasts were exposed to cigarette smoke, this increased the intensity of ultraweak chemiluminescence (CL), leading us to assume that cigarette smoke caused oxidation in cultured human skin fibroblasts. When the cultured human skin fibroblasts were treated with antioxidants such as glutathione, thiotaurine, hypotaurine and ascorbic acid there was little increase in CL, meaning that oxidation had been prevented in the human skin fibroblasts. We also exposed the human forearm to cigarette smoke and obtained sebum using cotton immersed in acetone in order to measure hydroperoxide levels by means of a CL-HPLC system. The exposure of skin to the smoke caused a dose-dependent increase in hydroperoxides derived from cigarette smoke. Further exposure of the forearm to cigarette smoke increased the intensity of CL, but pretreating the skin with antioxidants such as glutathione, thiotaurine and hypotaurine inhibited this increase. From these results, we concluded that cigarette smoke had an oxidative effect on SQ, cultured human skin fibroblasts and the surface of the human skin. The application of antioxidants prevented the cigarette smoke-induced oxidation. We consider that these oxidative effects on the skin could be a cause of skin disorders and skin aging.