InP/ZnS QDs對稀有鮈鯽子代軟骨發育的影響

伍穎軼,陳 行,謝威威,金 麗

InP/ZnS QDs對稀有鮈鯽子代軟骨發育的影響

伍穎軼,陳 行,謝威威,金 麗*

(西南大學生命科學學院,淡水魚類資源與生殖發育教育部重點實驗室,水產科學重慶市重點實驗室,重慶 400715)

使用InP/ZnS QDs以雌性稀有鮈鯽()為實驗動物,通過腹腔注射染毒,設計了(200, 400, 800nmol/L)3個實驗組,在量子點暴露4和7d時取卵受精.以胚胎受精率、存活率、仔魚體長、全長為指標,對仔魚進行阿利新藍染色和HE(Hematoxylin Eosin)染色,通過檢測發育相關基因(,)的相對表達量,研究InP/ZnS QDs對稀有鮈鯽子代軟骨發育的影響.結果表明:高濃度組仔魚的體長減少6.2%、全長減少5.9%;顱面PQ-Meckel角增加24.8%,下頜骨長度減小14.6%和15.2%、寬度減小10.0%和10.7%;顱面軟骨細胞腫大、數量減少.并發現QDs在不同發育時間對相關基因的相對表達量影響不同.總之InP/ZnS QDs會對稀有鮈鯽子代軟骨發育產生不良影響.

InP/ZnS Quantum Dots；稀有鮈鯽；毒性；軟骨發育

量子點(QDs)作為新型熒光納米物質,具有獨特光學性質,如光量子效率高、熒光發射強度高、穩定性好等[1-3],使其得到廣泛的應用.然而,通過環境釋放、職業暴露等途徑,人們接觸QDs的機會越來越多,其毒性效應也受到關注.研究表明,QDs可以穿透生物屏障對生物體的肝臟、脾臟、神經系統等造成影響,引起炎癥反應,刺激ROS(Reactive Oxygen Species)產生[4].據有關報道,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,對后代的生長產生不利影響[5].無鎘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、廣闊的顏色調節性和低毒性等特點[6].25μg/kg體重的InP/ZnS QDs注射進小鼠體內84d后,仍可在其脾臟、腎臟和肝臟中檢測到,但對小鼠的生理指標無顯著影響[7].在72h 內2.5μg/mLInP/ZnS QDs可使神經母細胞(SH-SY5Y)的活性顯著降低[8],200～800nmol/L InP/ZnS QDs在稀有鮈鯽胚胎發育過程中均引起致畸效應和死亡[9],20mg/kg體重的 InP/ZnS QDs可以在小鼠體內激活巨噬細胞并引起炎癥反應[10].不同表面基團修飾的2μg/mL InP/ZnS QDs均對人類肺癌細胞(HCC-15)和肺泡II型上皮細胞(RLE-6TN)產生細胞毒性,羧基和氨基修飾的QDs比羥基修飾的毒性更大[11].說明InP/ZnS QDs具有一定毒性,有必要進行深入研究.

骨形成蛋白2b()在控制骨外器官細胞形成及骨骼修復過程中起主導作用[12-13],(-)是最早被用來作為軟骨形成的細胞指標.(Runt-related transcription factor 2b)是成骨細胞分化調節網絡的重要位點之一[14],可作為胚胎發育后期的軟骨細胞終末分化、成骨分化和骨形成的標志[15].本文重點研究InP/ZnS QDs處理稀有鮈鯽親本對子一代胚胎與仔魚骨骼生長發育的影響.利用軟骨染色、HE染色探究仔魚在不同時期軟骨發育狀況,利用實時熒光定量PCR技術探究InP/ZnS QDs對子代骨形成蛋白基因()軟骨細胞轉錄因子() mRNA表達量變化,探討InP/ZnS QDs對稀有鮈鯽子代骨骼發育的影響,為InP/ZnS QDs的廣泛使用與研究提供基礎科學依據.

1 材料與方法

1.1 實驗準備

本實驗采用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,熒光最大發射波長為628nm,購于蘇州星爍納米科技有限公司.

實驗用魚來自西南大學淡水魚類生殖與發育教育部重點實驗室,在養殖體系中選取健康1齡親魚飼養于循環水養殖系統中,每缸水體1.5L.每個實驗組選取18尾魚,設置3個平行,每個平行組6尾魚,雄:雌=1:1,雄性平均體重(1.05±0.21)g,平均體長(3.80±0.14)cm;雌性平均體重(1.38±0.29)g,平均體長(3.73±0.24)cm:每日早晚投喂1次飼料(四川正大集團S3號飼料),中午投喂1次豐年蟲,水溫控制(27±0.5)℃,光照時間為12h:12h.對其進行腹腔注射投毒,由于InP/ZnS QDs在現實環境中的濃度未見報道,所以本實驗根據毒理濃度設置的一般原則:將胚胎半致死濃度的1/2即800nmol/L設置為最高濃度,并根據相等對數間距設置1個空白組和3個實驗組:0,200, 400和800nmol/L.

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

1.2 顯微觀察和數據收集

InP/ZnS QDs暴露后,在投毒后的4,7d進行取卵,與正常雄性稀有鮈鯽精子進行受精.在受精后4h統計受精率;在胚胎發育過程中每12h挑出死卵并統計個數,發育至72h統計存活率.96h在體式顯微鏡(SMZ25Nikon,日本)下對仔魚進行拍照,并測量仔魚體長、全長.

1.3 軟骨染色

當胚胎發育至96h.各濃度選取6尾仔魚進行軟骨染色,觀察其頭部軟骨發育情況.軟骨染色方法為無酸雙染色法[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.換無酸雙染色溶液,由A、B兩種溶液混合而成,現用現配,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液的無酸雙染色溶液混合.加入1mL染液于無酶管中,室溫下在搖床放置過夜.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.

在體式顯微鏡下進行拍照,用Image J測量仔魚下頜骨長度、寬度和PQ-Meckel角.每個濃度組測量6尾,用SPSS進行統計分析.

1.4 組織學觀察

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進行透明.隨后用石蠟進行包埋,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仔魚的軟骨細胞發育情況.

1.5 相關基因表達量測定

將36,48,72hpf的胚胎根據總RNA抽提試劑盒(上海生工生物工程有限公司)說明提取總RNA,并用PrimeScript?RT reagent Kit With gDNA Eraser(TaKaRa,大連,中國)試劑盒將各樣品RNA進行逆轉錄.用稀有鮈鯽管家基因作為內參,參考FAN等[17]所設計的引物(表1).RT-PCR 反應體系如下(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,無菌水6μL.擴增條件參照說明書,各基因擴增效率范圍在95%～ 97%,并分析結果.

表1 real-time PCR所用引物

2 結果與分析

2.1 胚胎早期發育影響

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

表2 InP/ZnS QDs對稀有鮈鯽子代發育的影響

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

2.2 軟骨染色結果

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)與對照組(圖1A,B)無顯著性差異.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果

A:對照組,PQ-Meckel角;B:對照組,ab:下頜骨寬度,cd:下頜骨長度;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.標尺=200μm.

圖2 InP/ZnS QDs暴露后稀有鮈鯽子代顱面軟骨染色測量結果

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

2.3 組織學觀察結果

為探究InP/ZnS QDs對軟骨細胞結構形態的影響,利用HE染色對仔魚切片進行觀察.結果顯示,對照組軟骨細胞排列密集、細胞核完整清晰(圖3A).隨著QDs濃度升高,實驗組仔魚的軟骨細胞發生腫大、數量減少的現象(圖3C～G).這種組織學結構改變的發生率見表3.

圖3 InP/ZnS QDs對稀有鮈鯽子代咽軟骨組織學影響

A:對照組;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.標尺=200μm

表3 咽軟骨組織學結構改變的發生率

2.4 骨發育相關基因的相對表達量

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

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

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

圖4 InP/ZnS QDs對稀有鮈鯽子代bmp2b、sox9a、runx2b基因的影響

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

3 討論

本研究將雌性稀有鮈鯽暴露于InP/ZnS QDs 4和7d,發現QDs會影響子一代胚胎骨骼發育,使其體長、全長短于對照組,顱骨形態結構發生變化;仔魚軟骨細胞形態結構發生變化、數量減少;骨發育相關基因的mRNA表達量也受到影響.

QDs的毒性與包被其的物質和核心金屬離子等都有關系,其毒性會影響胚胎發育的多個方面.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育,包括畸形率增加、顱面骨發育異常、骨骼發育相關基因表達水平顯著下降[18].將稀有鮈鯽胚胎直接暴露于5～40nmol/LInP/ ZnS QDs中,會導致胚胎畸形率增加,并使其肝臟發育異常[19].50～800nmol/LCuInS2/ZnS QDs可使稀有鮈鯽胚胎畸形率顯著升高[20],但InP/ZnS QDs對稀有鮈鯽子代骨骼發育的影響未有研究.

魚類骨骼與人類骨骼的基本信號通路都高度保守[21],如斑馬魚()常被作為同時出現顱面和肢體缺陷的人類綜合征的研究模式動物[22].母體稀有鮈鯽暴露于15和255μg/L的雙酚A會使子代畸形率增加、抑制顱面軟骨發育[17].本研究中,僅800nmol/L InP/ZnS QDs暴露7d時對稀有鮈鯽胚胎孵化率產生顯著影響,400nmol/L濃度組的體長和全長顯著變短.頭部軟骨作為魚類軟骨重要組成部分,其形態結構也作為軟骨發育重要指標之一.顱面異常與許多疾病都有關聯,顱面異常會影響攝食、呼吸等.本研究結果表明,高濃度組仔魚的PQ-Meckel角顯著增大,下頜骨寬度和長度顯著減小.說明InP/ZnS QDs影響了稀有鮈鯽子代的下頜骨發育.顱骨畸形與軟骨細胞形態、數量有直接關系[23].高濃度組仔魚軟骨細胞形態發生變化、細胞數量減少.

是家族的一員,可誘導間充質干細胞(MSC)定向分化為成軟骨細胞或成骨細胞,對背側正常發育十分重要[24],在骨愈合過程中發揮關鍵作用.可誘導ALP活性,促進礦化[25].可通過與基因家族相關產物結合來調節下游基因的表達,如[26-27].基因是軟骨分化中必需的轉錄因子,對軟骨缺損修復也有積極作用,在軟骨細胞中持續表達[28].可使骨祖細胞分化為軟骨細胞[29],在發育前期參與MSC的細胞凝結,細胞凝結對軟骨細胞后續發育分化是必需的條件[30].是成骨細胞分化和軟骨細胞成熟中必須的轉錄因子之一,對軟骨細胞分化和骨吸收十分重要[31].過表達導致軟骨細胞早熟,并使小鼠出現顱骨發育不良等骨骼畸形的現象[32]本研究中,在各時間點的表達趨勢與較為相同,但因的主要功能為促使MSC向成軟骨細胞分化,的主要功能為促使MSC向成骨細胞分化,并且信號通路在成骨分化過程中也存在著負反饋調節[33],所以推測基因間相互作用,導致基因表達趨勢受到影響.各基因在仔魚軟骨發育階段的表達量不同,如褐牙鲆()在胚胎和仔魚時期幾乎檢測不到的表達,而在仔魚時期表達量較高,在出膜后仔魚中表達量先升高后遞減[34].本研究中在仔魚孵化前QDs多為促進這三個基因的表達,而在72hpf多為抑制其表達.說明QDs可能在子一代胚胎發育早期通過上調骨發育相關基因,將軟骨細胞成熟和成骨細胞分化過程提前.72hpf為胚胎出膜時期,此時基因 mRNA表達量對仔魚出膜可能產生影響,也會直接影響出膜后仔魚骨骼發育.在仔魚出膜后的36～48h,仔魚開始開口攝食獲取營養,這對于仔魚后續生長發育十分重要.本文中,在72hpf QDs顯著抑制骨骼發育相關的三個基因表達,這很有可能抑制下頜骨發育,與軟骨染色、組織學觀察結果一致.

QDs具有生殖毒性.之前研究表明,400 和800nmol/L ZnSe/ZnS QDs對雄性稀有鮈鯽造成生殖毒性,使其精子質量下降,并損傷子一代的胚胎DNA[35].800nmol/LInP/ZnS QDs可對稀有鮈鯽睪丸造成損傷,表現為線粒體腫脹、生殖細胞減少等[36]. CdSe/ZnS QDs使小鼠卵巢中的促卵泡激素受體()和黃體()mRNA的表達量顯著下調,并減少成熟卵母細胞數量[37].本文通過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通過腹腔注射進入尼羅羅非魚()體內7d內,各時間點均可觀測QDs分布在卵母細胞周圍的結締組織內[38];大于2.0μg/mL InP/ZnS QDs可抑制體外小鼠卵母細胞的成熟[39].所以推測本實驗中QDs可能進入卵巢對卵子發育過程產生影響.QDs對生殖和發育的影響機制包括:改變配子發生和胚胎發育基因的表達[40].本文InP/ZnS QDs通過改變胚胎時期骨骼發育相關基因表達,影響子一代顱骨發育.

Gottschalk 等[41]用模型預測出在自然界地表水(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]通過模型預測2014年歐洲7個地區的地表水中QDs濃度范圍為9.6～ 530fg/L(1fg/L=10-15g/L),在飛塵中濃度為環境中最高,范圍在6.6～350μg/kg.研究表明,預估每年會有0.057～1.14t的QDs釋放到環境中[43].QDs可以通過飲食暴露從浮游動物轉移到斑馬魚,說明QDs可以通過潛在食物鏈轉移[44].InP/ZnS QDs常被用于生物化學分析,如檢測多巴胺[45]、腺苷[46]和辣根過氧化物酶[47],具有操作簡便、靈明度高、選擇性優異的優勢.所以以稀有鮈鯽為實驗對象探究InP/ZnS QDs的生物毒性是十分必要的.

4 結論

InP/ZnS QDs進入稀有鮈鯽母體后,400nmol/L實驗組仔魚的體長減少6.2%、全長減少5.9%;高濃度則顱面PQ-Meckel角增加24.8%,下頜骨長度減小14.6%和15.2%、寬度減小10.0%和10.7%. InP/ZnS QDs使子代顱面軟骨細胞形態發生變化;并影響子代、和的mRNA表達量,推測在發育早期InP/ZnS QDs促進軟骨細胞成熟、成骨細胞分化,而在出膜時抑制骨發育相關基因表達,影響仔魚顱骨發育,從而影響其生長發育.

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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對稀有鮈鯽子代軟骨發育的影響 [J]. 中國環境科學, 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,西南大學碩士研究生,主要從事從事動物形態發育與魚類毒理學方面的研究.發表論文3篇. 970952060@qq.com.