InP/ZnS QDs对稀有鮈鲫子代软骨发育的影响

伍颖轶,陈 行,谢威威,金 丽

InP/ZnS QDs对稀有鮈鲫子代软骨发育的影响

伍颖轶,陈 行,谢威威,金 丽*

(西南大学生命科学学院,淡水鱼类资源与生殖发育教育部重点实验室,水产科学重庆市重点实验室,重庆 400715)

使用InP/ZnS QDs以雌性稀有鮈鲫()为实验动物,通过腹腔注射染毒,设计了(200, 400, 800nmol/L)3个实验组,在量子点暴露4和7d时取卵受精.以胚胎受精率、存活率、仔鱼体长、全长为指标,对仔鱼进行阿利新蓝染色和HE(Hematoxylin Eosin)染色,通过检测发育相关基因(,)的相对表达量,研究InP/ZnS QDs对稀有鮈鲫子代软骨发育的影响.结果表明:高浓度组仔鱼的体长减少6.2%、全长减少5.9%;颅面PQ-Meckel角增加24.8%,下颌骨长度减小14.6%和15.2%、宽度减小10.0%和10.7%;颅面软骨细胞肿大、数量减少.并发现QDs在不同发育时间对相关基因的相对表达量影响不同.总之InP/ZnS QDs会对稀有鮈鲫子代软骨发育产生不良影响.

InP/ZnS Quantum Dots；稀有鮈鲫；毒性；软骨发育

量子点(QDs)作为新型荧光纳米物质,具有独特光学性质,如光量子效率高、荧光发射强度高、稳定性好等[1-3],使其得到广泛的应用.然而,通过环境释放、职业暴露等途径,人们接触QDs的机会越来越多,其毒性效应也受到关注.研究表明,QDs可以穿透生物屏障对生物体的肝脏、脾脏、神经系统等造成影响,引起炎症反应,刺激ROS(Reactive Oxygen Species)产生[4].据有关报道,2nmol/kg体重的CdSe/ZnS QDs可以穿透血液-睾丸屏障,聚集在小鼠睾丸中,对后代的生长产生不利影响[5].无镉QDs因不含重金属元素,毒性较小.InP(Indium)QDs具有较大的吸收系数、广阔的颜色调节性和低毒性等特点[6].25μg/kg体重的InP/ZnS QDs注射进小鼠体内84d后,仍可在其脾脏、肾脏和肝脏中检测到,但对小鼠的生理指标无显著影响[7].在72h 内2.5μg/mLInP/ZnS QDs可使神经母细胞(SH-SY5Y)的活性显著降低[8],200～800nmol/L InP/ZnS QDs在稀有鮈鲫胚胎发育过程中均引起致畸效应和死亡[9],20mg/kg体重的 InP/ZnS QDs可以在小鼠体内激活巨噬细胞并引起炎症反应[10].不同表面基团修饰的2μg/mL InP/ZnS QDs均对人类肺癌细胞(HCC-15)和肺泡II型上皮细胞(RLE-6TN)产生细胞毒性,羧基和氨基修饰的QDs比羟基修饰的毒性更大[11].说明InP/ZnS QDs具有一定毒性,有必要进行深入研究.

骨形成蛋白2b()在控制骨外器官细胞形成及骨骼修复过程中起主导作用[12-13],(-)是最早被用来作为软骨形成的细胞指标.(Runt-related transcription factor 2b)是成骨细胞分化调节网络的重要位点之一[14],可作为胚胎发育后期的软骨细胞终末分化、成骨分化和骨形成的标志[15].本文重点研究InP/ZnS QDs处理稀有鮈鲫亲本对子一代胚胎与仔鱼骨骼生长发育的影响.利用软骨染色、HE染色探究仔鱼在不同时期软骨发育状况,利用实时荧光定量PCR技术探究InP/ZnS QDs对子代骨形成蛋白基因()软骨细胞转录因子() mRNA表达量变化,探讨InP/ZnS QDs对稀有鮈鲫子代骨骼发育的影响,为InP/ZnS QDs的广泛使用与研究提供基础科学依据.

1 材料与方法

1.1 实验准备

本实验采用QDs为羧基、水溶性聚乙二醇(PEG)修饰的红色的InP/ZnS QDs,浓度为10mg/mL,荧光最大发射波长为628nm,购于苏州星烁纳米科技有限公司.

实验用鱼来自西南大学淡水鱼类生殖与发育教育部重点实验室,在养殖体系中选取健康1龄亲鱼饲养于循环水养殖系统中,每缸水体1.5L.每个实验组选取18尾鱼,设置3个平行,每个平行组6尾鱼,雄:雌=1:1,雄性平均体重(1.05±0.21)g,平均体长(3.80±0.14)cm;雌性平均体重(1.38±0.29)g,平均体长(3.73±0.24)cm:每日早晚投喂1次饲料(四川正大集团S3号饲料),中午投喂1次丰年虫,水温控制(27±0.5)℃,光照时间为12h:12h.对其进行腹腔注射投毒,由于InP/ZnS QDs在现实环境中的浓度未见报道,所以本实验根据毒理浓度设置的一般原则:将胚胎半致死浓度的1/2即800nmol/L设置为最高浓度,并根据相等对数间距设置1个空白组和3个实验组:0,200, 400和800nmol/L.

投毒当天选取有成熟卵子的雌性个体,人工将其卵子完全排出体内后染毒,1个浓度组选取3尾雌性稀有鮈鲫,依据每尾鱼的体重(g),按2μL/g进行投毒,在每缸中放入3尾雄性稀有鮈鲫刺激其产卵.稀有鮈鲫的产卵周期为3～4d,稀有鮈鲫每尾雌鱼产卵量在96～655粒之间,因此选取排卵后第1,2个产卵周期取材,即投毒后的4与7d.

1.2 显微观察和数据收集

InP/ZnS QDs暴露后,在投毒后的4,7d进行取卵,与正常雄性稀有鮈鲫精子进行受精.在受精后4h统计受精率;在胚胎发育过程中每12h挑出死卵并统计个数,发育至72h统计存活率.96h在体式显微镜(SMZ25Nikon,日本)下对仔鱼进行拍照,并测量仔鱼体长、全长.

1.3 软骨染色

当胚胎发育至96h.各浓度选取6尾仔鱼进行软骨染色,观察其头部软骨发育情况.软骨染色方法为无酸双染色法[16].将仔鱼放入10%甲醛溶液中,4℃固定24h.用超纯水洗涤2～3次,50%酒精脱水10min.换无酸双染色溶液,由A、B两种溶液混合而成,现用现配,A液包括Alcian blue 8GX,B液包括茜素红S.在染色前,将含10mL B液和1mLA液的无酸双染色溶液混合.加入1mL染液于无酶管中,室温下在摇床放置过夜.用超纯水洗净,3%H2O2/2%KOH漂白,室温开盖3～4h.系列KOH和甘油(0.25%KOH/20%甘油,0.25%KOH/50%甘油,0.1%KOH/50%甘油)透明,4℃保存观察.

在体式显微镜下进行拍照,用Image J测量仔鱼下颌骨长度、宽度和PQ-Meckel角.每个浓度组测量6尾,用SPSS进行统计分析.

1.4 组织学观察

将96hpf(hours post-fertilization)的仔鱼放入4%的PFA中固定24h,从70%酒精开始至100%酒精脱水,各浓度处理15min.再将仔鱼放入50%二甲苯、100%二甲苯中各30min进行透明.随后用石蜡进行包埋,切片厚度为5μm.切片用苏木精-伊红(HE)染色,烘干后在荧光显微镜(Nikon eclipse 801,日本)下观察96hpf仔鱼的软骨细胞发育情况.

1.5 相关基因表达量测定

将36,48,72hpf的胚胎根据总RNA抽提试剂盒(上海生工生物工程有限公司)说明提取总RNA,并用PrimeScript®RT reagent Kit With gDNA Eraser(TaKaRa,大连,中国)试剂盒将各样品RNA进行逆转录.用稀有鮈鲫管家基因作为内参,参考FAN等[17]所设计的引物(表1).RT-PCR 反应体系如下(20μL):正、反向引物各0.8μL、TB Green Premix Ex Taq II(Tli RNaseH Plus)(2´)、cDNA模板2μL、ROX Reference Dye(50´)0.4μL,无菌水6μL.扩增条件参照说明书,各基因扩增效率范围在95%～ 97%,并分析结果.

表1 real-time PCR所用引物

2 结果与分析

2.1 胚胎早期发育影响

结果表明(表2),仅在QDs暴露7d的800nmol/L 浓度组出现受精率显著下降,其余浓度组的受精率和72hpf胚胎存活率无显著差异.而400nmol/L浓度组的4,7d仔鱼的体长减少6.2%、全长减少5.9%,其他各浓度组与对照组无显著差异.

表2 InP/ZnS QDs对稀有鮈鲫子代发育的影响

注:*表示与对照组差异显著(<0.05);**表示与对照组差异极显著(<0.01).

2.2 软骨染色结果

800nmol/L处理组QDs暴露4d后PQ-Meckel角(图1E,图2a)增加24.8%,其余处理组(图1C,D)与对照组(图1A,B)无显著性差异.在QDs暴露7d后,200和800nmol/L的下颌骨宽度(图1F、H,图2c) 减小10.0%和10.7%;400和800nmol/L的下颌骨长度(图1G、H,图2b)减小14.6%和15.2%.

图1 InP/ZnS QDs暴露后稀有鮈鲫子代颅面软骨的染色结果

A:对照组,PQ-Meckel角;B:对照组,ab:下颌骨宽度,cd:下颌骨长度;C:QDs暴露4d 200nmol/L;D:QDs暴露4d 400nmol/L;E:QDs暴露4d 800nmol/L;F:QDs暴露7d 200nmol/L;G:QDs暴露7d 400nmol/L;H:QDs暴露7d 800nmol/L.标尺=200μm.

图2 InP/ZnS QDs暴露后稀有鮈鲫子代颅面软骨染色测量结果

*表示与对照组差异显著(<0.05);**表示与对照组差异极显著(<0.01)

2.3 组织学观察结果

为探究InP/ZnS QDs对软骨细胞结构形态的影响,利用HE染色对仔鱼切片进行观察.结果显示,对照组软骨细胞排列密集、细胞核完整清晰(图3A).随着QDs浓度升高,实验组仔鱼的软骨细胞发生肿大、数量减少的现象(图3C～G).这种组织学结构改变的发生率见表3.

图3 InP/ZnS QDs对稀有鮈鲫子代咽软骨组织学影响

A:对照组;B:QDs暴露4d 200nmol/L;C:QDs暴露4d 400nmol/L;D:QDs暴露4d 800nmol/L;E:QDs暴露7d 200nmol/L;F:QDs暴露7d 400nmol/L;G:QDs暴露7d 800nmol/L.标尺=200μm

表3 咽软骨组织学结构改变的发生率

2.4 骨发育相关基因的相对表达量

在InP/ZnS QDs暴露至4d,在400和800nmol/L浓度组的72hpf mRNA表达水平下调均显著,,400nmol/L浓度组的36hpf、800nmol/L浓度组的48hpf上调显著.暴露后7d,400和800nmol/L浓度组的48hpf mRNA表达量均显著升高;在72hpf,200nmol/L浓度组显著上升、400和800nmol/L浓度组mRNA表达量显著下降,剩余浓度组mRNA表达量变化不显著(图4a,b).

在InP/ZnS QDs暴露到4d时,200和400nmol/L浓度组的48hpf mRNA表达量显著上调;而在72hpf 200nmol/L浓度组显著上调;400nmol/L浓度组mRNA表达量显著下调.在暴露后7d, 200nmol/L浓度组在48hpf mRNA表达显著上调,400nmol/L浓度组在36、48hpf mRNA表达量上调显著;而800nmol/L浓度组在36、48hpf mRNA表达量显著降低;在72hpf mRNA表达量显著上升.剩余浓度组mRNA表达量变化不显著(图4c,d).

在InP /ZnS QDs暴露到4d时,200nmol/L浓度组在48hpf mRNA表达显著上调;400nmol/L浓度组在36、48hpf mRNA表达量显著增加,72hpf表达量显著降低;800nmol/L浓度组在48、72hpf mRNA表达量显著下降.暴露后7d,3个浓度组在36、48hpf mRNA表达量多表现为显著增加;在72hpf各浓度组的mRNA表达量显著下降(图4e～f).

图4 InP/ZnS QDs对稀有鮈鲫子代bmp2b、sox9a、runx2b基因的影响

*表示与对照组差异显著(<0.05);**表示与对照组差异极显著(<0.01)

3 讨论

本研究将雌性稀有鮈鲫暴露于InP/ZnS QDs 4和7d,发现QDs会影响子一代胚胎骨骼发育,使其体长、全长短于对照组,颅骨形态结构发生变化;仔鱼软骨细胞形态结构发生变化、数量减少;骨发育相关基因的mRNA表达量也受到影响.

QDs的毒性与包被其的物质和核心金属离子等都有关系,其毒性会影响胚胎发育的多个方面.400和800nmol/LCdSe/ZnS QDs暴露于雌性稀有鮈鲫4d后,显著影响了子代的软骨发育,包括畸形率增加、颅面骨发育异常、骨骼发育相关基因表达水平显著下降[18].将稀有鮈鲫胚胎直接暴露于5～40nmol/LInP/ ZnS QDs中,会导致胚胎畸形率增加,并使其肝脏发育异常[19].50～800nmol/LCuInS2/ZnS QDs可使稀有鮈鲫胚胎畸形率显著升高[20],但InP/ZnS QDs对稀有鮈鲫子代骨骼发育的影响未有研究.

鱼类骨骼与人类骨骼的基本信号通路都高度保守[21],如斑马鱼()常被作为同时出现颅面和肢体缺陷的人类综合征的研究模式动物[22].母体稀有鮈鲫暴露于15和255μg/L的双酚A会使子代畸形率增加、抑制颅面软骨发育[17].本研究中,仅800nmol/L InP/ZnS QDs暴露7d时对稀有鮈鲫胚胎孵化率产生显著影响,400nmol/L浓度组的体长和全长显著变短.头部软骨作为鱼类软骨重要组成部分,其形态结构也作为软骨发育重要指标之一.颅面异常与许多疾病都有关联,颅面异常会影响摄食、呼吸等.本研究结果表明,高浓度组仔鱼的PQ-Meckel角显著增大,下颌骨宽度和长度显著减小.说明InP/ZnS QDs影响了稀有鮈鲫子代的下颌骨发育.颅骨畸形与软骨细胞形态、数量有直接关系[23].高浓度组仔鱼软骨细胞形态发生变化、细胞数量减少.

是家族的一员,可诱导间充质干细胞(MSC)定向分化为成软骨细胞或成骨细胞,对背侧正常发育十分重要[24],在骨愈合过程中发挥关键作用.可诱导ALP活性,促进矿化[25].可通过与基因家族相关产物结合来调节下游基因的表达,如[26-27].基因是软骨分化中必需的转录因子,对软骨缺损修复也有积极作用,在软骨细胞中持续表达[28].可使骨祖细胞分化为软骨细胞[29],在发育前期参与MSC的细胞凝结,细胞凝结对软骨细胞后续发育分化是必需的条件[30].是成骨细胞分化和软骨细胞成熟中必须的转录因子之一,对软骨细胞分化和骨吸收十分重要[31].过表达导致软骨细胞早熟,并使小鼠出现颅骨发育不良等骨骼畸形的现象[32]本研究中,在各时间点的表达趋势与较为相同,但因的主要功能为促使MSC向成软骨细胞分化,的主要功能为促使MSC向成骨细胞分化,并且信号通路在成骨分化过程中也存在着负反馈调节[33],所以推测基因间相互作用,导致基因表达趋势受到影响.各基因在仔鱼软骨发育阶段的表达量不同,如褐牙鲆()在胚胎和仔鱼时期几乎检测不到的表达,而在仔鱼时期表达量较高,在出膜后仔鱼中表达量先升高后递减[34].本研究中在仔鱼孵化前QDs多为促进这三个基因的表达,而在72hpf多为抑制其表达.说明QDs可能在子一代胚胎发育早期通过上调骨发育相关基因,将软骨细胞成熟和成骨细胞分化过程提前.72hpf为胚胎出膜时期,此时基因 mRNA表达量对仔鱼出膜可能产生影响,也会直接影响出膜后仔鱼骨骼发育.在仔鱼出膜后的36～48h,仔鱼开始开口摄食获取营养,这对于仔鱼后续生长发育十分重要.本文中,在72hpf QDs显著抑制骨骼发育相关的三个基因表达,这很有可能抑制下颌骨发育,与软骨染色、组织学观察结果一致.

QDs具有生殖毒性.之前研究表明,400 和800nmol/L ZnSe/ZnS QDs对雄性稀有鮈鲫造成生殖毒性,使其精子质量下降,并损伤子一代的胚胎DNA[35].800nmol/LInP/ZnS QDs可对稀有鮈鲫睾丸造成损伤,表现为线粒体肿胀、生殖细胞减少等[36]. CdSe/ZnS QDs使小鼠卵巢中的促卵泡激素受体()和黄体()mRNA的表达量显著下调,并减少成熟卵母细胞数量[37].本文通过腹腔注射的方式将雌性稀有鮈鲫暴露于InP/ZnS QDs,已有研究表明20nmol/LCdSe/ZnS QDs通过腹腔注射进入尼罗罗非鱼()体内7d内,各时间点均可观测QDs分布在卵母细胞周围的结缔组织内[38];大于2.0μg/mL InP/ZnS QDs可抑制体外小鼠卵母细胞的成熟[39].所以推测本实验中QDs可能进入卵巢对卵子发育过程产生影响.QDs对生殖和发育的影响机制包括:改变配子发生和胚胎发育基因的表达[40].本文InP/ZnS QDs通过改变胚胎时期骨骼发育相关基因表达,影响子一代颅骨发育.

Gottschalk 等[41]用模型预测出在自然界地表水(淡水)和海水的沉积物中浓度分别为0.2～452μg/ kg和0.04～2μg/kg.Wang等[42]通过模型预测2014年欧洲7个地区的地表水中QDs浓度范围为9.6～ 530fg/L(1fg/L=10-15g/L),在飞尘中浓度为环境中最高,范围在6.6～350μg/kg.研究表明,预估每年会有0.057～1.14t的QDs释放到环境中[43].QDs可以通过饮食暴露从浮游动物转移到斑马鱼,说明QDs可以通过潜在食物链转移[44].InP/ZnS QDs常被用于生物化学分析,如检测多巴胺[45]、腺苷[46]和辣根过氧化物酶[47],具有操作简便、灵明度高、选择性优异的优势.所以以稀有鮈鲫为实验对象探究InP/ZnS QDs的生物毒性是十分必要的.

4 结论

InP/ZnS QDs进入稀有鮈鲫母体后,400nmol/L实验组仔鱼的体长减少6.2%、全长减少5.9%;高浓度则颅面PQ-Meckel角增加24.8%,下颌骨长度减小14.6%和15.2%、宽度减小10.0%和10.7%. InP/ZnS QDs使子代颅面软骨细胞形态发生变化;并影响子代、和的mRNA表达量,推测在发育早期InP/ZnS QDs促进软骨细胞成熟、成骨细胞分化,而在出膜时抑制骨发育相关基因表达,影响仔鱼颅骨发育,从而影响其生长发育.

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Effects of InP/ZnS QDs on cartilage development in rare minnow () offspring.

WU Ying-yi, CHEN Hang, XIE Wei-wei, JIN Li*

(Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Key Laboratory of Aquatic Science of Chongqing, College of Life Sciences, Southwest University, Chongqing 400715,China)., 2023,43(12)：6732～6739

Quantum Dots (QDs) are a class of nanomaterials. With the wide application of QDs, its toxic affects on organisms are also concerned. In this experiment, InP/ZnS quantum dots were used, and female rare minnows () was used as the experimental animal. Three experimental groups (200, 400, 800nmol/L) were designed. Eggs were taken and fertilized at 4 and 7 days of quantum dots exposure. Using embryo fertilization rate, survival rate, body length and full length of larvae as indicators, the larvae were observed by Albion blue dye and Hematoxylin Eosin (HE) staining. The transcript expression levels of bone developmental related genes (,,) were detected to study the effect of quantum dots on the offspring cartilage development of rare minnows. The results showed that the body length and full length of larvae in high concentration group decreased by 6.2% and 5.9%. The PQ-Meckel’s angle increased by 24.8%, mandibular length decreased by 14.6% and 15.2%, and mandibular width decreased by 10.0% and 10.7%. Craniofacial chondrocytes were swelled and reduced in number. It was found that QDs had different affects on the transcript expression levels of related genes at different development time. In conclusion, InP/ZnS quantum dots can affect the skeletal development of offspring of rare minnows.

InP/ZnS quantum dots；；toxicity；skeletal development

X503.2

A

1000-6923(2023)12-6732-08

伍颖轶,陈 行,谢威威,等.InP/ZnS QDs对稀有鮈鲫子代软骨发育的影响 [J]. 中国环境科学, 2023,43(12):6732-6739.

Wu Y Y, Chen H, Xie W W, et al. Effects of InP/ZnS QDs on cartilage development in rare minnow () offspring [J]. China Environmental Science, 2023,43(12):6732-6739.

2023-04-14

* 责任作者, 副教授, jinll@swu.edu.cn

伍颖轶(1998-),女,新疆乌鲁木齐人,西南大学硕士研究生,主要从事从事动物形态发育与鱼类毒理学方面的研究.发表论文3篇. 970952060@qq.com.