InP/ZnS QDs對(duì)稀有鮈鯽子代軟骨發(fā)育的影響

伍穎軼,陳 行,謝威威,金 麗

InP/ZnS QDs對(duì)稀有鮈鯽子代軟骨發(fā)育的影響

伍穎軼,陳 行,謝威威,金 麗*

(西南大學(xué)生命科學(xué)學(xué)院,淡水魚類資源與生殖發(fā)育教育部重點(diǎn)實(shí)驗(yàn)室,水產(chǎn)科學(xué)重慶市重點(diǎn)實(shí)驗(yàn)室,重慶 400715)

使用InP/ZnS QDs以雌性稀有鮈鯽()為實(shí)驗(yàn)動(dòng)物,通過(guò)腹腔注射染毒,設(shè)計(jì)了(200, 400, 800nmol/L)3個(gè)實(shí)驗(yàn)組,在量子點(diǎn)暴露4和7d時(shí)取卵受精.以胚胎受精率、存活率、仔魚體長(zhǎng)、全長(zhǎng)為指標(biāo),對(duì)仔魚進(jìn)行阿利新藍(lán)染色和HE(Hematoxylin Eosin)染色,通過(guò)檢測(cè)發(fā)育相關(guān)基因(,)的相對(duì)表達(dá)量,研究InP/ZnS QDs對(duì)稀有鮈鯽子代軟骨發(fā)育的影響.結(jié)果表明:高濃度組仔魚的體長(zhǎng)減少6.2%、全長(zhǎng)減少5.9%;顱面PQ-Meckel角增加24.8%,下頜骨長(zhǎng)度減小14.6%和15.2%、寬度減小10.0%和10.7%;顱面軟骨細(xì)胞腫大、數(shù)量減少.并發(fā)現(xiàn)QDs在不同發(fā)育時(shí)間對(duì)相關(guān)基因的相對(duì)表達(dá)量影響不同.總之InP/ZnS QDs會(huì)對(duì)稀有鮈鯽子代軟骨發(fā)育產(chǎn)生不良影響.

InP/ZnS Quantum Dots；稀有鮈鯽；毒性；軟骨發(fā)育

量子點(diǎn)(QDs)作為新型熒光納米物質(zhì),具有獨(dú)特光學(xué)性質(zhì),如光量子效率高、熒光發(fā)射強(qiáng)度高、穩(wěn)定性好等[1-3],使其得到廣泛的應(yīng)用.然而,通過(guò)環(huán)境釋放、職業(yè)暴露等途徑,人們接觸QDs的機(jī)會(huì)越來(lái)越多,其毒性效應(yīng)也受到關(guān)注.研究表明,QDs可以穿透生物屏障對(duì)生物體的肝臟、脾臟、神經(jīng)系統(tǒng)等造成影響,引起炎癥反應(yīng),刺激ROS(Reactive Oxygen Species)產(chǎn)生[4].據(jù)有關(guān)報(bào)道,2nmol/kg體重的CdSe/ZnS QDs可以穿透血液-睪丸屏障,聚集在小鼠睪丸中,對(duì)后代的生長(zhǎng)產(chǎn)生不利影響[5].無(wú)鎘QDs因不含重金屬元素,毒性較小.InP(Indium)QDs具有較大的吸收系數(shù)、廣闊的顏色調(diào)節(jié)性和低毒性等特點(diǎn)[6].25μg/kg體重的InP/ZnS QDs注射進(jìn)小鼠體內(nèi)84d后,仍可在其脾臟、腎臟和肝臟中檢測(cè)到,但對(duì)小鼠的生理指標(biāo)無(wú)顯著影響[7].在72h 內(nèi)2.5μg/mLInP/ZnS QDs可使神經(jīng)母細(xì)胞(SH-SY5Y)的活性顯著降低[8],200～800nmol/L InP/ZnS QDs在稀有鮈鯽胚胎發(fā)育過(guò)程中均引起致畸效應(yīng)和死亡[9],20mg/kg體重的 InP/ZnS QDs可以在小鼠體內(nèi)激活巨噬細(xì)胞并引起炎癥反應(yīng)[10].不同表面基團(tuán)修飾的2μg/mL InP/ZnS QDs均對(duì)人類肺癌細(xì)胞(HCC-15)和肺泡II型上皮細(xì)胞(RLE-6TN)產(chǎn)生細(xì)胞毒性,羧基和氨基修飾的QDs比羥基修飾的毒性更大[11].說(shuō)明InP/ZnS QDs具有一定毒性,有必要進(jìn)行深入研究.

骨形成蛋白2b()在控制骨外器官細(xì)胞形成及骨骼修復(fù)過(guò)程中起主導(dǎo)作用[12-13],(-)是最早被用來(lái)作為軟骨形成的細(xì)胞指標(biāo).(Runt-related transcription factor 2b)是成骨細(xì)胞分化調(diào)節(jié)網(wǎng)絡(luò)的重要位點(diǎn)之一[14],可作為胚胎發(fā)育后期的軟骨細(xì)胞終末分化、成骨分化和骨形成的標(biāo)志[15].本文重點(diǎn)研究InP/ZnS QDs處理稀有鮈鯽親本對(duì)子一代胚胎與仔魚骨骼生長(zhǎng)發(fā)育的影響.利用軟骨染色、HE染色探究仔魚在不同時(shí)期軟骨發(fā)育狀況,利用實(shí)時(shí)熒光定量PCR技術(shù)探究InP/ZnS QDs對(duì)子代骨形成蛋白基因()軟骨細(xì)胞轉(zhuǎn)錄因子() mRNA表達(dá)量變化,探討InP/ZnS QDs對(duì)稀有鮈鯽子代骨骼發(fā)育的影響,為InP/ZnS QDs的廣泛使用與研究提供基礎(chǔ)科學(xué)依據(jù).

1 材料與方法

1.1 實(shí)驗(yàn)準(zhǔn)備

本實(shí)驗(yàn)采用QDs為羧基、水溶性聚乙二醇(PEG)修飾的紅色的InP/ZnS QDs,濃度為10mg/mL,熒光最大發(fā)射波長(zhǎng)為628nm,購(gòu)于蘇州星爍納米科技有限公司.

實(shí)驗(yàn)用魚來(lái)自西南大學(xué)淡水魚類生殖與發(fā)育教育部重點(diǎn)實(shí)驗(yàn)室,在養(yǎng)殖體系中選取健康1齡親魚飼養(yǎng)于循環(huán)水養(yǎng)殖系統(tǒng)中,每缸水體1.5L.每個(gè)實(shí)驗(yàn)組選取18尾魚,設(shè)置3個(gè)平行,每個(gè)平行組6尾魚,雄:雌=1:1,雄性平均體重(1.05±0.21)g,平均體長(zhǎng)(3.80±0.14)cm;雌性平均體重(1.38±0.29)g,平均體長(zhǎng)(3.73±0.24)cm:每日早晚投喂1次飼料(四川正大集團(tuán)S3號(hào)飼料),中午投喂1次豐年蟲,水溫控制(27±0.5)℃,光照時(shí)間為12h:12h.對(duì)其進(jìn)行腹腔注射投毒,由于InP/ZnS QDs在現(xiàn)實(shí)環(huán)境中的濃度未見報(bào)道,所以本實(shí)驗(yàn)根據(jù)毒理濃度設(shè)置的一般原則:將胚胎半致死濃度的1/2即800nmol/L設(shè)置為最高濃度,并根據(jù)相等對(duì)數(shù)間距設(shè)置1個(gè)空白組和3個(gè)實(shí)驗(yàn)組:0,200, 400和800nmol/L.

投毒當(dāng)天選取有成熟卵子的雌性個(gè)體,人工將其卵子完全排出體內(nèi)后染毒,1個(gè)濃度組選取3尾雌性稀有鮈鯽,依據(jù)每尾魚的體重(g),按2μL/g進(jìn)行投毒,在每缸中放入3尾雄性稀有鮈鯽刺激其產(chǎn)卵.稀有鮈鯽的產(chǎn)卵周期為3～4d,稀有鮈鯽每尾雌魚產(chǎn)卵量在96～655粒之間,因此選取排卵后第1,2個(gè)產(chǎn)卵周期取材,即投毒后的4與7d.

1.2 顯微觀察和數(shù)據(jù)收集

InP/ZnS QDs暴露后,在投毒后的4,7d進(jìn)行取卵,與正常雄性稀有鮈鯽精子進(jìn)行受精.在受精后4h統(tǒng)計(jì)受精率;在胚胎發(fā)育過(guò)程中每12h挑出死卵并統(tǒng)計(jì)個(gè)數(shù),發(fā)育至72h統(tǒng)計(jì)存活率.96h在體式顯微鏡(SMZ25Nikon,日本)下對(duì)仔魚進(jìn)行拍照,并測(cè)量仔魚體長(zhǎng)、全長(zhǎng).

1.3 軟骨染色

當(dāng)胚胎發(fā)育至96h.各濃度選取6尾仔魚進(jìn)行軟骨染色,觀察其頭部軟骨發(fā)育情況.軟骨染色方法為無(wú)酸雙染色法[16].將仔魚放入10%甲醛溶液中,4℃固定24h.用超純水洗滌2～3次,50%酒精脫水10min.換無(wú)酸雙染色溶液,由A、B兩種溶液混合而成,現(xiàn)用現(xiàn)配,A液包括Alcian blue 8GX,B液包括茜素紅S.在染色前,將含10mL B液和1mLA液的無(wú)酸雙染色溶液混合.加入1mL染液于無(wú)酶管中,室溫下在搖床放置過(guò)夜.用超純水洗凈,3%H2O2/2%KOH漂白,室溫開蓋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℃保存觀察.

在體式顯微鏡下進(jìn)行拍照,用Image J測(cè)量仔魚下頜骨長(zhǎng)度、寬度和PQ-Meckel角.每個(gè)濃度組測(cè)量6尾,用SPSS進(jìn)行統(tǒng)計(jì)分析.

1.4 組織學(xué)觀察

將96hpf(hours post-fertilization)的仔魚放入4%的PFA中固定24h,從70%酒精開始至100%酒精脫水,各濃度處理15min.再將仔魚放入50%二甲苯、100%二甲苯中各30min進(jìn)行透明.隨后用石蠟進(jìn)行包埋,切片厚度為5μm.切片用蘇木精-伊紅(HE)染色,烘干后在熒光顯微鏡(Nikon eclipse 801,日本)下觀察96hpf仔魚的軟骨細(xì)胞發(fā)育情況.

1.5 相關(guān)基因表達(dá)量測(cè)定

將36,48,72hpf的胚胎根據(jù)總RNA抽提試劑盒(上海生工生物工程有限公司)說(shuō)明提取總RNA,并用PrimeScript?RT reagent Kit With gDNA Eraser(TaKaRa,大連,中國(guó))試劑盒將各樣品RNA進(jìn)行逆轉(zhuǎn)錄.用稀有鮈鯽管家基因作為內(nèi)參,參考FAN等[17]所設(shè)計(jì)的引物(表1).RT-PCR 反應(yīng)體系如下(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,無(wú)菌水6μL.擴(kuò)增條件參照說(shuō)明書,各基因擴(kuò)增效率范圍在95%～ 97%,并分析結(jié)果.

表1 real-time PCR所用引物

2 結(jié)果與分析

2.1 胚胎早期發(fā)育影響

結(jié)果表明(表2),僅在QDs暴露7d的800nmol/L 濃度組出現(xiàn)受精率顯著下降,其余濃度組的受精率和72hpf胚胎存活率無(wú)顯著差異.而400nmol/L濃度組的4,7d仔魚的體長(zhǎng)減少6.2%、全長(zhǎng)減少5.9%,其他各濃度組與對(duì)照組無(wú)顯著差異.

表2 InP/ZnS QDs對(duì)稀有鮈鯽子代發(fā)育的影響

注:*表示與對(duì)照組差異顯著(<0.05);**表示與對(duì)照組差異極顯著(<0.01).

2.2 軟骨染色結(jié)果

800nmol/L處理組QDs暴露4d后PQ-Meckel角(圖1E,圖2a)增加24.8%,其余處理組(圖1C,D)與對(duì)照組(圖1A,B)無(wú)顯著性差異.在QDs暴露7d后,200和800nmol/L的下頜骨寬度(圖1F、H,圖2c) 減小10.0%和10.7%;400和800nmol/L的下頜骨長(zhǎng)度(圖1G、H,圖2b)減小14.6%和15.2%.

圖1 InP/ZnS QDs暴露后稀有鮈鯽子代顱面軟骨的染色結(jié)果

A:對(duì)照組,PQ-Meckel角;B:對(duì)照組,ab:下頜骨寬度,cd:下頜骨長(zhǎng)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.標(biāo)尺=200μm.

圖2 InP/ZnS QDs暴露后稀有鮈鯽子代顱面軟骨染色測(cè)量結(jié)果

*表示與對(duì)照組差異顯著(<0.05);**表示與對(duì)照組差異極顯著(<0.01)

2.3 組織學(xué)觀察結(jié)果

為探究InP/ZnS QDs對(duì)軟骨細(xì)胞結(jié)構(gòu)形態(tài)的影響,利用HE染色對(duì)仔魚切片進(jìn)行觀察.結(jié)果顯示,對(duì)照組軟骨細(xì)胞排列密集、細(xì)胞核完整清晰(圖3A).隨著QDs濃度升高,實(shí)驗(yàn)組仔魚的軟骨細(xì)胞發(fā)生腫大、數(shù)量減少的現(xiàn)象(圖3C～G).這種組織學(xué)結(jié)構(gòu)改變的發(fā)生率見表3.

圖3 InP/ZnS QDs對(duì)稀有鮈鯽子代咽軟骨組織學(xué)影響

A:對(duì)照組;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.標(biāo)尺=200μm

表3 咽軟骨組織學(xué)結(jié)構(gòu)改變的發(fā)生率

2.4 骨發(fā)育相關(guān)基因的相對(duì)表達(dá)量

在InP/ZnS QDs暴露至4d,在400和800nmol/L濃度組的72hpf mRNA表達(dá)水平下調(diào)均顯著,,400nmol/L濃度組的36hpf、800nmol/L濃度組的48hpf上調(diào)顯著.暴露后7d,400和800nmol/L濃度組的48hpf mRNA表達(dá)量均顯著升高;在72hpf,200nmol/L濃度組顯著上升、400和800nmol/L濃度組mRNA表達(dá)量顯著下降,剩余濃度組mRNA表達(dá)量變化不顯著(圖4a,b).

在InP/ZnS QDs暴露到4d時(shí),200和400nmol/L濃度組的48hpf mRNA表達(dá)量顯著上調(diào);而在72hpf 200nmol/L濃度組顯著上調(diào);400nmol/L濃度組mRNA表達(dá)量顯著下調(diào).在暴露后7d, 200nmol/L濃度組在48hpf mRNA表達(dá)顯著上調(diào),400nmol/L濃度組在36、48hpf mRNA表達(dá)量上調(diào)顯著;而800nmol/L濃度組在36、48hpf mRNA表達(dá)量顯著降低;在72hpf mRNA表達(dá)量顯著上升.剩余濃度組mRNA表達(dá)量變化不顯著(圖4c,d).

在InP /ZnS QDs暴露到4d時(shí),200nmol/L濃度組在48hpf mRNA表達(dá)顯著上調(diào);400nmol/L濃度組在36、48hpf mRNA表達(dá)量顯著增加,72hpf表達(dá)量顯著降低;800nmol/L濃度組在48、72hpf mRNA表達(dá)量顯著下降.暴露后7d,3個(gè)濃度組在36、48hpf mRNA表達(dá)量多表現(xiàn)為顯著增加;在72hpf各濃度組的mRNA表達(dá)量顯著下降(圖4e～f).

圖4 InP/ZnS QDs對(duì)稀有鮈鯽子代bmp2b、sox9a、runx2b基因的影響

*表示與對(duì)照組差異顯著(<0.05);**表示與對(duì)照組差異極顯著(<0.01)

3 討論

本研究將雌性稀有鮈鯽暴露于InP/ZnS QDs 4和7d,發(fā)現(xiàn)QDs會(huì)影響子一代胚胎骨骼發(fā)育,使其體長(zhǎng)、全長(zhǎng)短于對(duì)照組,顱骨形態(tài)結(jié)構(gòu)發(fā)生變化;仔魚軟骨細(xì)胞形態(tài)結(jié)構(gòu)發(fā)生變化、數(shù)量減少;骨發(fā)育相關(guān)基因的mRNA表達(dá)量也受到影響.

QDs的毒性與包被其的物質(zhì)和核心金屬離子等都有關(guān)系,其毒性會(huì)影響胚胎發(fā)育的多個(gè)方面.400和800nmol/LCdSe/ZnS QDs暴露于雌性稀有鮈鯽4d后,顯著影響了子代的軟骨發(fā)育,包括畸形率增加、顱面骨發(fā)育異常、骨骼發(fā)育相關(guān)基因表達(dá)水平顯著下降[18].將稀有鮈鯽胚胎直接暴露于5～40nmol/LInP/ ZnS QDs中,會(huì)導(dǎo)致胚胎畸形率增加,并使其肝臟發(fā)育異常[19].50～800nmol/LCuInS2/ZnS QDs可使稀有鮈鯽胚胎畸形率顯著升高[20],但I(xiàn)nP/ZnS QDs對(duì)稀有鮈鯽子代骨骼發(fā)育的影響未有研究.

魚類骨骼與人類骨骼的基本信號(hào)通路都高度保守[21],如斑馬魚()常被作為同時(shí)出現(xiàn)顱面和肢體缺陷的人類綜合征的研究模式動(dòng)物[22].母體稀有鮈鯽暴露于15和255μg/L的雙酚A會(huì)使子代畸形率增加、抑制顱面軟骨發(fā)育[17].本研究中,僅800nmol/L InP/ZnS QDs暴露7d時(shí)對(duì)稀有鮈鯽胚胎孵化率產(chǎn)生顯著影響,400nmol/L濃度組的體長(zhǎng)和全長(zhǎng)顯著變短.頭部軟骨作為魚類軟骨重要組成部分,其形態(tài)結(jié)構(gòu)也作為軟骨發(fā)育重要指標(biāo)之一.顱面異常與許多疾病都有關(guān)聯(lián),顱面異常會(huì)影響攝食、呼吸等.本研究結(jié)果表明,高濃度組仔魚的PQ-Meckel角顯著增大,下頜骨寬度和長(zhǎng)度顯著減小.說(shuō)明InP/ZnS QDs影響了稀有鮈鯽子代的下頜骨發(fā)育.顱骨畸形與軟骨細(xì)胞形態(tài)、數(shù)量有直接關(guān)系[23].高濃度組仔魚軟骨細(xì)胞形態(tài)發(fā)生變化、細(xì)胞數(shù)量減少.

是家族的一員,可誘導(dǎo)間充質(zhì)干細(xì)胞(MSC)定向分化為成軟骨細(xì)胞或成骨細(xì)胞,對(duì)背側(cè)正常發(fā)育十分重要[24],在骨愈合過(guò)程中發(fā)揮關(guān)鍵作用.可誘導(dǎo)ALP活性,促進(jìn)礦化[25].可通過(guò)與基因家族相關(guān)產(chǎn)物結(jié)合來(lái)調(diào)節(jié)下游基因的表達(dá),如[26-27].基因是軟骨分化中必需的轉(zhuǎn)錄因子,對(duì)軟骨缺損修復(fù)也有積極作用,在軟骨細(xì)胞中持續(xù)表達(dá)[28].可使骨祖細(xì)胞分化為軟骨細(xì)胞[29],在發(fā)育前期參與MSC的細(xì)胞凝結(jié),細(xì)胞凝結(jié)對(duì)軟骨細(xì)胞后續(xù)發(fā)育分化是必需的條件[30].是成骨細(xì)胞分化和軟骨細(xì)胞成熟中必須的轉(zhuǎn)錄因子之一,對(duì)軟骨細(xì)胞分化和骨吸收十分重要[31].過(guò)表達(dá)導(dǎo)致軟骨細(xì)胞早熟,并使小鼠出現(xiàn)顱骨發(fā)育不良等骨骼畸形的現(xiàn)象[32]本研究中,在各時(shí)間點(diǎn)的表達(dá)趨勢(shì)與較為相同,但因的主要功能為促使MSC向成軟骨細(xì)胞分化,的主要功能為促使MSC向成骨細(xì)胞分化,并且信號(hào)通路在成骨分化過(guò)程中也存在著負(fù)反饋調(diào)節(jié)[33],所以推測(cè)基因間相互作用,導(dǎo)致基因表達(dá)趨勢(shì)受到影響.各基因在仔魚軟骨發(fā)育階段的表達(dá)量不同,如褐牙鲆()在胚胎和仔魚時(shí)期幾乎檢測(cè)不到的表達(dá),而在仔魚時(shí)期表達(dá)量較高,在出膜后仔魚中表達(dá)量先升高后遞減[34].本研究中在仔魚孵化前QDs多為促進(jìn)這三個(gè)基因的表達(dá),而在72hpf多為抑制其表達(dá).說(shuō)明QDs可能在子一代胚胎發(fā)育早期通過(guò)上調(diào)骨發(fā)育相關(guān)基因,將軟骨細(xì)胞成熟和成骨細(xì)胞分化過(guò)程提前.72hpf為胚胎出膜時(shí)期,此時(shí)基因 mRNA表達(dá)量對(duì)仔魚出膜可能產(chǎn)生影響,也會(huì)直接影響出膜后仔魚骨骼發(fā)育.在仔魚出膜后的36～48h,仔魚開始開口攝食獲取營(yíng)養(yǎng),這對(duì)于仔魚后續(xù)生長(zhǎng)發(fā)育十分重要.本文中,在72hpf QDs顯著抑制骨骼發(fā)育相關(guān)的三個(gè)基因表達(dá),這很有可能抑制下頜骨發(fā)育,與軟骨染色、組織學(xué)觀察結(jié)果一致.

QDs具有生殖毒性.之前研究表明,400 和800nmol/L ZnSe/ZnS QDs對(duì)雄性稀有鮈鯽造成生殖毒性,使其精子質(zhì)量下降,并損傷子一代的胚胎DNA[35].800nmol/LInP/ZnS QDs可對(duì)稀有鮈鯽睪丸造成損傷,表現(xiàn)為線粒體腫脹、生殖細(xì)胞減少等[36]. CdSe/ZnS QDs使小鼠卵巢中的促卵泡激素受體()和黃體()mRNA的表達(dá)量顯著下調(diào),并減少成熟卵母細(xì)胞數(shù)量[37].本文通過(guò)腹腔注射的方式將雌性稀有鮈鯽暴露于InP/ZnS QDs,已有研究表明20nmol/LCdSe/ZnS QDs通過(guò)腹腔注射進(jìn)入尼羅羅非魚()體內(nèi)7d內(nèi),各時(shí)間點(diǎn)均可觀測(cè)QDs分布在卵母細(xì)胞周圍的結(jié)締組織內(nèi)[38];大于2.0μg/mL InP/ZnS QDs可抑制體外小鼠卵母細(xì)胞的成熟[39].所以推測(cè)本實(shí)驗(yàn)中QDs可能進(jìn)入卵巢對(duì)卵子發(fā)育過(guò)程產(chǎn)生影響.QDs對(duì)生殖和發(fā)育的影響機(jī)制包括:改變配子發(fā)生和胚胎發(fā)育基因的表達(dá)[40].本文InP/ZnS QDs通過(guò)改變胚胎時(shí)期骨骼發(fā)育相關(guān)基因表達(dá),影響子一代顱骨發(fā)育.

Gottschalk 等[41]用模型預(yù)測(cè)出在自然界地表水(淡水)和海水的沉積物中濃度分別為0.2～452μg/ kg和0.04～2μg/kg.Wang等[42]通過(guò)模型預(yù)測(cè)2014年歐洲7個(gè)地區(qū)的地表水中QDs濃度范圍為9.6～ 530fg/L(1fg/L=10-15g/L),在飛塵中濃度為環(huán)境中最高,范圍在6.6～350μg/kg.研究表明,預(yù)估每年會(huì)有0.057～1.14t的QDs釋放到環(huán)境中[43].QDs可以通過(guò)飲食暴露從浮游動(dòng)物轉(zhuǎn)移到斑馬魚,說(shuō)明QDs可以通過(guò)潛在食物鏈轉(zhuǎn)移[44].InP/ZnS QDs常被用于生物化學(xué)分析,如檢測(cè)多巴胺[45]、腺苷[46]和辣根過(guò)氧化物酶[47],具有操作簡(jiǎn)便、靈明度高、選擇性優(yōu)異的優(yōu)勢(shì).所以以稀有鮈鯽為實(shí)驗(yàn)對(duì)象探究InP/ZnS QDs的生物毒性是十分必要的.

4 結(jié)論

InP/ZnS QDs進(jìn)入稀有鮈鯽母體后,400nmol/L實(shí)驗(yàn)組仔魚的體長(zhǎng)減少6.2%、全長(zhǎng)減少5.9%;高濃度則顱面PQ-Meckel角增加24.8%,下頜骨長(zhǎng)度減小14.6%和15.2%、寬度減小10.0%和10.7%. InP/ZnS QDs使子代顱面軟骨細(xì)胞形態(tài)發(fā)生變化;并影響子代、和的mRNA表達(dá)量,推測(cè)在發(fā)育早期InP/ZnS QDs促進(jìn)軟骨細(xì)胞成熟、成骨細(xì)胞分化,而在出膜時(shí)抑制骨發(fā)育相關(guān)基因表達(dá),影響仔魚顱骨發(fā)育,從而影響其生長(zhǎng)發(fā)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

伍穎軼,陳 行,謝威威,等.InP/ZnS QDs對(duì)稀有鮈鯽子代軟骨發(fā)育的影響 [J]. 中國(guó)環(huán)境科學(xué), 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

* 責(zé)任作者, 副教授, jinll@swu.edu.cn

伍穎軼(1998-),女,新疆烏魯木齊人,西南大學(xué)碩士研究生,主要從事從事動(dòng)物形態(tài)發(fā)育與魚類毒理學(xué)方面的研究.發(fā)表論文3篇. 970952060@qq.com.