CN111487096A - 利用斑马鱼分析微塑料分布及损伤程度的方法 - Google Patents
利用斑马鱼分析微塑料分布及损伤程度的方法 Download PDFInfo
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Abstract
本发明公开了一种利用斑马鱼分析微塑料分布及损伤程度的方法。所述方法通过使用微塑料颗粒对斑马鱼进行暴露实验,对斑马鱼组织样品的采集,控制脱水与透明处理时间,经染色后通过显微镜观察微塑料分布及对斑马鱼的损伤程度。本发明能最大程度的保留斑马鱼摄取微塑料后在体内最原始的分布状态和损伤程度,为后续准确评价微塑料毒性提供基础数据。
Description
技术领域
本发明属于生物体内污染物代谢分析技术领域,涉及一种利用斑马鱼分析微塑料分布及损伤程度的方法。
背景技术
微塑料,是对尺寸小于5mm的不同形态塑料的统称,由于其尺寸微小、分布广泛,易与环境中其他污染物发生相互作用,破坏生态系统的稳定性。近年来,微塑料已成为全球海洋生态环境中备受关注的新型污染物。海洋生物对微塑料无法辨别,会通过多种途径摄入体内,包括呼吸摄入、饮食摄入和胞吞作用等方式。
目前,追踪微塑料在生物体内的方法主要有:1)利用身体透明的实验动物作为模型,如浮游动物桡足类Centropages typicus、桡足类Calanus helgolandicus、轮虫等(Environmental Science&Technology,2013,47(12):6646-6655),此方法仅能观察微塑料在体内的分布,无法有效判断微塑料所造成的机体损伤;2)使用荧光微塑料对生物体进行染毒后解剖,通过组织消解,测定荧光强度,分析各组织中微塑料的成分与含量,此方法只保留了对微塑料的定量,缺乏对机体损伤的分析(张宴.定量分析微塑料在哺乳动物体内富集和分布的方法.CN201610045344)。
石蜡组织切片是组织学中应用最广泛的一种切片制作方法,其价格低廉,操作简便。石蜡切片不仅用于观察正常细胞组织的形态结构,也是病理学和法医学等学科用以研究、观察及判断细胞组织的形态变化的主要方法,广泛地用于其他领域的研究中。斑马鱼作为生物学中的模式生物,其基因与人的相似度高达87%。在实际应用中,斑马鱼已经作为人类疾病动物模型,涉及到神经系统疾病(如阿尔茨海默症、帕金森氏病等)、肿瘤、心脏病等领域。现有文献报道的组织切片方法适用于其他生物体,如小鼠的组织切片方法,并不适用于斑马鱼的组织切片。另外,传统方法制作的斑马鱼组织石蜡切片经常出现收缩、脆裂等情况,导致所得切片的具体分析产生误差(生物学杂志,2006,23(1):45-46;江苏农业科学,2013,41(11):260-263)。综上所述,现有追踪微塑料在生物体内的方法无法准确判断微塑料对生物体造成损伤的具体情况。
发明内容
本发明的目的在于提供一种利用斑马鱼分析微塑料分布及损伤程度的方法,该方法能直观地将微塑料在斑马鱼体内的分布及损伤状态展示出来,简便、快速且成本低,为后续的微塑料毒性评价提供基础数据。
实现本发明目的的技术方案如下:
利用斑马鱼分析微塑料分布及损伤程度的方法,包括斑马鱼暴露实验,样品采集,组织固定,脱水与透明,浸蜡,包埋,切片,展片和烤片,脱蜡及复水,染色,脱水与透明,镜检等过程,具体步骤如下:
步骤1,斑马鱼暴露实验和样品采集:将斑马鱼暴露在微塑料水溶液环境中,暴露实验结束后,对斑马鱼解剖并分段;
步骤2,组织固定:采用4%多聚甲醛固定液对斑马鱼分段进行固定;
步骤3,脱水与透明:将固定后的斑马鱼分段依次置于梯度浓度的乙醇溶液中,脱水处理30~60分钟,脱水结束后,先置于等体积的乙醇与二甲苯的混合溶液中,再置于二甲苯中,进行透明处理,处理时间均为30~60分钟;
步骤4,将脱水与透明处理后的斑马鱼分段浸蜡,包埋,切片,展片和烤片,脱蜡及复水,染色,脱水与透明,荧光显微镜下观察斑马鱼体内微塑料分布及组织的损伤情况。
步骤1中,所述的微塑料为聚苯乙烯、聚乙烯、聚氯乙烯、聚酯、聚酰胺、聚对苯二甲酸乙二醇酯中的一种或者多种。
步骤1中,所述的暴露时间为7~14天,所述的分段为将斑马鱼横切分成头段、胸腹段和尾段。
步骤3中,乙醇溶液的梯度浓度为75%,85%,95%和100%。
步骤4中,脱水与透明处理为依次置于梯度浓度的乙醇溶液中,脱水处理1~5分钟,再在二甲苯中透明处理5~30分钟。
步骤4中,所述的切片、展片和烤片过程,选择5~20μm进行切片,并在温水中展片,在30~60℃烤片12~24小时。
步骤4中,所述的脱蜡及复水过程,使用二甲苯处理10~30分钟,再置于梯度浓度的乙醇溶液中,处理1~10分钟、水处理1~10分钟。
步骤4中,所述的染色试剂为苏木素染液和伊红染液,染色时间为1~10分钟。
与现有技术相比,本发明具有以下优点:
本发明通过控制组织脱水与透明时间,改进了传统斑马鱼组织切片不佳,易出现脆裂空洞等情况,直观地展示斑马鱼体内的微塑料分布情况及微塑料对斑马鱼机体造成的损伤情况,填补了当下尚无分析斑马鱼体内微塑料分布富集与微塑料对斑马鱼造成损伤的有机结合的方法空缺,为后续微塑料的环境健康风险以及人体健康风险评价提供准确的基础数据支持。
附图说明
图1为本发明所使用的聚苯乙烯微塑料在扫描电镜下的照片;
图2为本发明的斑马鱼样品采集方式;
图3为空白对照组斑马鱼头段切片后染色结果图(40×);
图4为空白对照组斑马鱼头段(鳃组织)切片后染色结果图(400×);
图5为空白对照组斑马鱼胸腹段切片后染色结果图(40×);
图6为空白对照组斑马鱼胸腹段(肠组织)切片后染色结果图(400×);
图7为暴露实验组斑马鱼头段切片后染色结果图(40×);
图8为暴露实验组斑马鱼头段(鳃组织)切片后染色结果图(400×);
图9为暴露实验组斑马鱼胸腹段切片后染色结果图(40×);
图10为暴露实验组斑马鱼胸腹段(肠组织)切片后染色结果图(400×);
图11为脱水步骤处理时间为1分钟的斑马鱼(肠组织)切片后染色效果图(40×);
图12为脱水步骤处理时间为2小时的斑马鱼(肠组织)切片后染色效果图(40×)。
具体实施方式
以下结合具体实施例和附图对本发明作进一步详述。
本发明所使用的主要实验试剂和设备有:
表1主要实验试剂
表2实验仪器
实施例1
1、制备相关试剂:
20mg/L聚苯乙烯微塑料水溶液:用量筒量取998mL自来水,移取1%(w/v)聚苯乙烯微塑料母液2mL加入其中,现配现用。
磷酸缓冲液:称取NaCl 8g,KCl 0.2g,Na2HPO4 1.44g,KH2PO4 0.24g,溶于800mL去离子水中,调pH至7.0,定容至1L去离子水中,高温高压灭菌后,4℃保存备用。
4%多聚甲醛溶液:100mL磷酸缓冲液中加入4g多聚甲醛,水浴60℃加热助溶,约2小时完全溶解后,4℃保存备用。
2、微塑料暴露实验:在花鸟鱼虫市场,购买成年斑马鱼作为实验动物,挑选匀称健康鱼体。以尺寸为1μm、浓度为20mg/L聚苯乙烯微塑料水溶液为暴露环境,随机将斑马鱼分为暴露组和空白组,每组6只,暴露周期为7天。
3、组织样品采集:按照图1所述方式,将斑马鱼横切分成头段、胸腹段和尾段,取头段和胸腹段用于下一步实验。
4、石蜡切片制作:
(1)组织固定:斑马鱼头段和胸腹段组织经4%多聚甲醛固定液固定12小时后,自来水冲洗30分钟,洗去其固定液;
(2)脱水与透明:将经(1)处理过的组织依次使用下列试剂处理:75%乙醇30分钟、85%乙醇30分钟、95%乙醇30分钟、无水乙醇30分钟、无水乙醇30分钟、无水乙醇一份+二甲苯一份10分钟、二甲苯30分钟;
(3)浸蜡:将经(2)处理过的组织放入熔化好石蜡的浸蜡缸中进行浸蜡,依次使用下列试剂处理:55℃石蜡60分钟,55℃石蜡60分钟;
(4)包埋:将经(3)处理过的组织放入熔化好石蜡的包埋盒中进行包埋,转移到-20℃冷冻台上使石蜡凝固,把包埋好组织的蜡块从盒中取出进行修整;
(5)切片、展片和烤片:将经(4)处理过的蜡块固定于切片机上,与切片刀平行,选取5μm的厚度进行切片,选择完整的蜡片,放入45℃温水中展片,将载玻片放入水中捞出展开的蜡片,使组织样品整齐排列在载玻片上,放置于切片架中,烘箱40℃烤片12小时,自然冷却;
(6)脱蜡及复水:将经(5)处理过的载玻片放入染缸中,依次使用下列试剂处理:二甲苯10分钟、二甲苯10分钟、无水乙醇5分钟、95%乙醇2分钟、85%乙醇2分钟、75%乙醇2分钟、自来水2分钟;
(7)染色:将经(6)处理过的载玻片依次使用下列试剂处理:苏木素染液30秒、自来水5分钟、95%乙醇1分钟、伊红染液1分钟;
(8)脱水与透明:将经(7)处理过的载玻片依次使用下列试剂处理:75%乙醇1分钟、85%乙醇1分钟、95%乙醇1分钟、无水乙醇5分钟、二甲苯5分钟;
(9)镜检:将经(8)处理过的载玻片晾干后,在显微镜下观察并拍照。
对比例1
本对比例与实施例1基本相同,唯一不同的是4.(2)脱水与透明过程中,脱水处理时间为1min。
对比例2
本对比例与实施例1基本相同,唯一不同的是4.(2)脱水与透明过程中,脱水处理时间为2h。
图3和图7分别为40倍显微镜下空白对照组头段和实验组头段的切片效果图,可以看出,斑马鱼头段切片组织保存完整,结构清晰,附近的肌肉组织平滑舒张,切片整体效果良好。
图4和图8分别为400倍显微镜下空白对照组鳃组织和实验组鳃组织的切片效果图,由图8对比图4可以看出,图8鳃组织的鳃丝外围附着可见的聚苯乙烯微塑料颗粒,鳃细胞由对照组图4的正常形态转变为核质分布不均、肿大等形态。
图5和图9分别为40倍显微镜下空白对照组胸腹段和实验组胸腹段的切片效果图,可以看出,肠组织内黏膜具有发达的褶皱结构,排列有序,肠道整体结构完整,外围附近肌肉组织平滑舒展,切片整体效果良好。
图6和图10分别为400倍显微镜下空白对照组肠组织和实验组肠组织的切片效果图,由图10对比图6可以看出,图10肠组织的微绒毛结构附着可见的聚苯乙烯微塑料颗粒,微绒毛结构由图6对照组的正常形态转变为结构破损,细胞坏死等状态。
图11对比例1,所采用的脱水步骤,各试剂处理时间为1分钟的条件下,40倍显微镜下空白对照组肠组织切片效果图,可以看出,脱水时间过短,会导致组织脱水不全,组织粘附在一起,不易分析损伤情况。
图12对比例2,所采用的脱水步骤,各试剂处理时间为2小时的条件下,40倍显微镜下空白对照组肠组织切片效果图,可以看出,脱水时间过长,会造成组织出现脆裂、空洞等情况,导致切片质量较低,并对组织的结构产生误判。
综上所述,采用本发明方法在优化斑马鱼石蜡组织切片质量的基础上,既能观察到微塑料在斑马鱼体内的分布,也能观察到微塑料的摄入对斑马鱼机体造成的损伤情况。
以上结合具体实施例和附图对本发明的实施方式作了详细的说明,但是本发明并不限于上述实施方式,在所述技术领域普通技术人员所具备的知识范围内所作的任何等同替换、改进等均应包含在本发明的保护范围之内。
Claims (9)
1.利用斑马鱼分析微塑料分布及损伤程度的方法,其特征在于,具体步骤如下:
步骤1,斑马鱼暴露实验和样品采集:将斑马鱼暴露在微塑料水溶液环境中,暴露实验结束后,对斑马鱼解剖并分段;
步骤2,组织固定:采用4%多聚甲醛固定液对斑马鱼分段进行固定;
步骤3,脱水与透明:将固定后的斑马鱼分段依次置于梯度浓度的乙醇溶液中,脱水处理30~60分钟,脱水结束后,先置于等体积的乙醇与二甲苯的混合溶液中,再置于二甲苯中,进行透明处理,处理时间均为30~60分钟;
步骤4,将脱水与透明处理后的斑马鱼分段浸蜡,包埋,切片,展片和烤片,脱蜡及复水,染色,脱水与透明,荧光显微镜下观察斑马鱼体内微塑料分布及组织的损伤情况。
2.根据权利要求1所述的方法,其特征在于,步骤1中,所述的微塑料为聚苯乙烯、聚乙烯、聚氯乙烯、聚酯、聚酰胺、聚对苯二甲酸乙二醇酯中的一种或者多种。
3.根据权利要求1所述的方法,其特征在于,步骤1中,所述的暴露时间为7~14天。
4.根据权利要求1所述的方法,其特征在于,步骤1中,所述的分段为将斑马鱼横切分成头段、胸腹段和尾段。
5.根据权利要求1所述的方法,其特征在于,步骤4中,脱水与透明处理为依次置于梯度浓度的乙醇溶液中,脱水处理1~5分钟,再在二甲苯中透明处理5~30分钟。
6.根据权利要求1或5所述的方法,其特征在于,步骤3中和步骤4中,乙醇溶液的梯度浓度为75%,85%,95%和100%。
7.根据权利要求1所述的方法,其特征在于,步骤4中,所述的切片、展片和烤片过程,选择5~20μm进行切片,并在温水中展片,在30~60℃烤片12~24小时。
8.根据权利要求1所述的方法,其特征在于,步骤4中,所述的脱蜡及复水过程,使用二甲苯处理10~30分钟,再置于梯度浓度的乙醇溶液中,处理1~10分钟、水处理1~10分钟。
9.根据权利要求1所述的方法,其特征在于,步骤4中,所述的染色试剂为苏木素染液和伊红染液,染色时间为1~10分钟。
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