CN113916857A - 利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法 - Google Patents
利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法 Download PDFInfo
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Abstract
本发明提供了利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,包括如下步骤:将发育至3天的斑马鱼胚胎暴露于含有菊酯类杀虫剂的胚胎培养液中,采用半静态培养法,在特定温度、周期性明暗环境下培养,待斑马鱼胚胎发育至5天时,对斑马鱼胚胎的肝脏发育情况进行评价。本发明将野生AB型斑马鱼品系与转基因斑马鱼品系相结合准确评估菊酯类杀虫剂的肝脏毒性,通过转基因斑马鱼品系对暴露后斑马鱼的肝脏形式进行观察;同时采用油红染色方法评价野生AB型斑马鱼品系脂质代谢情况,以及检测菊酯类杀虫剂暴露后斑马鱼谷丙转氨酶、谷草转氨酶活性,为菊酯类杀虫剂的肝脏毒性研究提供理论依据,完善菊酯类杀虫剂的安全风险评价体系。
Description
技术领域
本发明属于毒性评价技术领域,具体涉及一种利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法。
背景技术
菊酯类杀虫剂是上世纪70年代人工合成的一类重要的杀虫剂,迄今已商品化的拟除虫菊酯有50多种,因其具有高效、低毒、广谱、击倒快、残留少等特点,被广泛应用于农业生产和家庭生活中。在高毒有机磷农药被禁止使用后,菊酯杀虫剂有了更广泛的使用空间,但在使用的同时,也带来了环境污染和食品安全等问题。由于某些菊酯类杀虫剂残留期较长,不仅对蜜蜂、家蚕和蝶类等农业有益生物的毒性较大,对鱼、虾、蟹、贝等水生生物毒性也较高,而且大部分拟除虫菊酯类农药属于环境激素类物质,对哺乳类动物的正常生理活动有很大影响,所以长期接触即使是低剂量也会引起慢性疾病,有些类型还有致畸、致癌、致突变的作用。目前蚊香类卫生杀虫剂主要成分为拟除虫菊酯,由于与人类的长期接触,菊酯类杀虫剂的毒性需要密切关注。
肝脏是生物体内的重要器官,具有合成蛋白质、储存脂质等功能,同时也是物质代谢的重要枢纽。斑马鱼作为一种模式生物,与人类具有很高的同源性,斑马鱼的脏器和细胞功能和人类有很高的相似性,并且斑马鱼的肝脏代谢与人类的代谢也极为相似,因此可以作为研究环境污染物或者农药肝脏毒性的理想模型。目前对于菊酯类杀虫剂的毒性研究主要在发育毒性、神经毒性、生殖毒性和免疫毒性等,对于肝脏发育毒性并没有研究报道。
传统的斑马鱼肝脏毒性研究方法主要使用野生AB型斑马鱼品系,使用显微镜观察不能够准确定位;并且斑马鱼幼鱼体型小,不能够进行解剖,无法准确观察肝脏的形态、面积等。因此急需一种能够能够评价菊酯类杀虫剂肝脏毒性的方法。
发明内容
本发明为解决上述技术问题进行,提供了一种新的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,实现菊酯类杀虫剂对肝脏损伤的直观评价。
本发明将野生AB型斑马鱼品系与转基因斑马鱼品系相结合准确评估菊酯类杀虫剂的肝脏毒性,通过转基因斑马鱼品系对暴露后斑马鱼的肝脏形式进行观察;同时采用油红染色方法评价野生AB型斑马鱼品系脂质代谢情况,以及检测菊酯类杀虫剂暴露后斑马鱼谷丙转氨酶、谷草转氨酶活性,为菊酯类杀虫剂的肝脏毒性研究提供理论依据,完善菊酯类杀虫剂的安全风险评价体系。
为了实现上述目的,本发明提供的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法包括如下步骤:将发育至3天的斑马鱼胚胎暴露于含有菊酯类杀虫剂的胚胎培养液中,采用半静态培养法,在特定温度、周期性明暗环境下培养,待斑马鱼胚胎发育至5天时,对斑马鱼胚胎的肝脏发育情况进行评价。
优选的,含有菊酯类杀虫剂的胚胎培养液的配置方法如下:
1)采用二甲基亚砜作为溶剂将菊酯类杀虫剂配制成浓度为40μg/mL的高浓度母液,于-20℃保存;
2)将配置好的母液用胚胎培养液稀释为不同浓度(10μg/L、20μg/L、30μg/L和40μg/L)的工作液,溶液现配现用,保持二甲基亚砜的浓度小于千分之一;
3)将含有菊酯类杀虫剂的培养液与不含菊酯类杀虫剂的培养液注入6cm的培养皿中,培养液的体积控制在8~9mL。
优选的,稀释后菊酯类杀虫剂的浓度不低于10μg/L;所述菊酯类杀虫剂包括天然除虫菊素、溴氰菊酯、氯氰菊酯或氯菊酯。
优选的,斑马鱼胚胎为野生斑马鱼AB品系胚胎和转基因斑马鱼胚胎Tg(fabp10a:ds;Red;ela3l:EGFP)进行胚胎选择时,选取发育良好的,由斑马鱼繁殖发育至三天已孵化的胚胎进行暴露实验,每个培养皿中放置20个胚胎。
优选的,进行培养时,将培养容器置于恒温光控培养箱中,温度控制在28±1℃,光照与黑暗周期为14/10h,采取半静态培养法,每隔24h更换培养液,暴露时长为48h。
优选的,对斑马鱼胚胎的肝脏发育情况进行评价的方法包括:检测转基因斑马鱼胚胎肝脏面积和荧光强度;检测野生斑马鱼AB品系胚胎肝脏部位脂质油红染色情况、肝功能相关酶谷丙转氨酶及谷草转氨酶的数据结果并分析。
在该评价方法中,A、斑马鱼肝脏面积和荧光强度采集是通过荧光显微镜采集转基因斑马鱼仔鱼的2D图像,然后通过ImageJ软件勾勒出红色荧光的肝脏部分,计算红色荧光的面积及荧光强度。
B、检测野生斑马鱼AB品系胚胎肝脏部位脂质油红染色情况的方法包括如下步骤:
1)按质量体积比3:1000,取Oil Red O粉末充分溶解于体积分数为60%的丙二醇或PBS溶液中,配制成染色液,用滤纸过滤待用,该溶液现配现用;
2)收集暴露后的斑马鱼仔鱼,加入麻醉剂进行麻醉;
3)加入4%PFA,于4℃固定过夜后,PBS清洗3次,每次5分钟;
4)加入体积分数为60%的丙二醇或PBS溶液,室温孵育30分钟;
5)加入过滤后的新鲜Oil Red O染色液,室温孵育4小时;
6)PBS漂洗多次,每次20分钟,直至去除背景噪音信号
7)将胚胎置于载玻片上调整位置,在体式显微镜下观察并采集图像。
C、肝功能相关酶谷丙转氨酶及谷草转氨酶的样品采集与检测方法为:收集暴露后的斑马鱼仔鱼置于冰水中进行安乐死,每个样品加10倍体积PBS冰浴匀浆,3000g离心10分钟后,收集上清液,根据试剂盒说明对样品中谷丙转氨酶、谷草转氨酶活性进行检测。
本发明的有益效果如下:
本发明中所用的模式生物斑马鱼,具有产卵量大,胚胎透明,发育时间短,有助于提高实验效率和降低实验成本。同时相比于传统方法仅采用野生斑马鱼AB品系,本发明所提供的的方法选用野生斑马鱼AB品系和转基因Tg(fabp10a:dsRed;ela3l:EGFP)品系,将两个品系相结合,可将肝脏可视化,准确得出肝脏的形状和面积,从而评价肝脏毒性。同时利用油红染色可以准确评价拟除虫菊素作用后斑马鱼的脂质代谢情况,并且检测谷丙转氨酶和谷草转氨酶的活性可以评价拟除虫菊素作用后斑马鱼的肝功能。
本发明能够实现菊酯类杀虫剂肝脏毒性的评价,具有代表性、普遍性、准确性高等特点,具有推广应用前景。
附图说明
图1为天然除虫菊素暴露后转基因斑马鱼品系肝脏形态示意图。
图2为天然除虫菊素暴露后转基因斑马鱼品系肝脏面积。
图3为天然除虫菊素暴露后转基因斑马鱼品系肝脏部位荧光强度。
图4为天然除虫菊素暴露后野生斑马鱼AB品系肝脏部位油红染色示意图。
图5为天然除虫菊素暴露后野生斑马鱼AB品系谷丙转氨酶活性。
图6为天然除虫菊素暴露后野生斑马鱼AB品系谷草转氨酶活性。
具体实施方式
下面结合本发明的附图和实施例对本发明的实施作详细说明,以下实施例是在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体操作过程,但本发明的保护范围不限于下述的实施例。
为了更好地理解本发明而不是限制本发明的范围,在本申请中所用的表示用量、百分比的所有数字、以及其他数值,在所有情况下都应理解为以词语“大约”所修饰。因此,除非特别说明,否则在说明书和所附权利要求书中所列出的数字参数都是近似值,其可能会根据试图获得的理想性质的不同而加以改变。各个数字参数至少应被看作是根据所报告的有效数字和通过常规的四舍五入方法而获得的。
下以天然除虫菊素为例,利用斑马鱼评价其对肝脏发育毒性的方法,其他菊酯类杀虫剂对肝脏发育毒性的评价方法类似。
实施例1菊酯类杀虫剂的暴露
1)采用二甲基亚砜(DMSO)作为溶剂将天然除虫菊素杀虫剂配制成浓度为40μg/mL的高浓度母液,于-20℃保存;
2)将配置好的母液用胚胎培养液稀释为10μg/L、20μg/L、30μg/L和40μg/L的工作液,溶液现配现用,保持DMSO的浓度小于千分之一;
3)将含有菊酯类杀虫剂的培养液与不含菊酯类杀虫剂的培养液(DMSO)注入6cm的培养皿中,培养液的体积控制在8~9mL;
4)选择试验模型为野生斑马鱼AB型和转基因斑马鱼Tg(fabp10a:dsRed;ela3l:EGFP);
5)选取发育良好的,由斑马鱼繁殖发育至三天已孵化的胚胎,进行暴露实验,每个培养皿中放置20个胚胎;
6)将培养皿置于恒温光控培养箱中,温度控制在28±1℃,光照与黑暗周期为14/10h,采取半静态培养法,每隔24h更换培养液,暴露时长为48h。
实施例2转基因斑马鱼肝脏形态评估
在天然除虫菊素暴露转基因斑马鱼48h后,使用荧光显微镜采集转基因斑马鱼仔鱼的2D图像,红色荧光表示斑马鱼的肝脏部位。
结果如图1所示,与对照组相比,在10μg/L、20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,斑马鱼的肝脏形态均发生了显著变化,其中10μg/L天然除虫菊素暴露后,斑马鱼肝脏发生皱缩现象,20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,斑马鱼肝脏发生胀大现象,斑马鱼幼鱼肝脏变形,表明天然除虫菊素暴露诱导肝脏变性或坏死,证明天然除虫菊素具有肝脏毒性。
实施例3转基因斑马鱼肝脏面积和荧光强度评估
在天然除虫菊素暴露转基因斑马鱼48h后,使用荧光显微镜采集转基因斑马鱼仔鱼的2D图像,通过ImageJ软件勾勒出红色荧光的肝脏部分,计算红色荧光的面积及荧光强度。
天然除虫菊素暴露后斑马鱼肝脏面积如图2所示,10μg/L天然除虫菊素暴露后,斑马鱼肝脏面积减小,20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,斑马鱼肝脏面积增大,表明天然除虫菊素暴露诱导斑马鱼肝脏变形,产生肝脏毒性。
天然除虫菊素暴露后斑马鱼肝脏荧光强度如图3所示,10μg/L、20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,肝脏部位的荧光强度明显增加,表明天然除虫菊素暴露诱导斑马鱼肝脏变形,产生肝脏毒性。
实施例4野生AB型斑马鱼油红染色评价斑马鱼肝脏脂质代谢情况
采用油红染色步骤如下:
1)取0.3g Oil Red O粉末充分溶解于100mL 60%丙二醇/PBS,配制成染色液,用滤纸过滤待用(现配现用);
2)收集暴露后的斑马鱼仔鱼,加入麻醉剂;
3)加入4%PFA,于4℃固定过夜;
4)PBS清洗3次,每次5分钟;
5)加入60%丙二醇/PBS,室温孵育30分钟;
6)加入过滤后的新鲜Oil Red O染色液,室温孵育4小时;
7)PBS漂洗多次,每次20分钟,直至去除背景噪音信号;
8)将胚胎置于载玻片上调整位置,在体式显微镜下观察并采集图像。
天然除虫菊素暴露后斑马鱼肝脏脂质代谢情况如图4所示,10μg/L、20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,肝脏部位油红染色加深,Oil Red O为脂溶性染料,可使组织内甘油三酯等中性脂肪特异性着色而被广泛应用于观察器官或组织内的中性脂肪的分布情况,表明天然除虫菊素暴露诱导斑马鱼产生肝脏脂质代谢紊乱现象,证明天然除虫菊素具有肝脏毒性。
实施例5谷丙转氨酶的样品采集与检测
收集暴露后的斑马鱼仔鱼置于冰水中进行安乐死,每个样品加10倍体积PBS冰浴匀浆,离心(3000g,10分钟)。收集上清液,样品中谷丙转氨酶活性采用相应的试剂盒(本实验使用南京建成公司的谷丙转氨酶测试盒)进行检测。
天然除虫菊素暴露后斑马鱼谷丙转氨酶活性如图5所示,10μg/L、20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,斑马鱼谷丙转氨酶活性升高,且呈浓度依赖关系。ALT为一种参与蛋白质新陈代谢的酶,起加快体内蛋白质氨基酸在体内转化的作用,肝脏发炎时,转氨酶就会从肝细胞释放到血液中,血清转氨酶增高表明肝脏发生病变。天然除虫菊素暴露后,斑马鱼谷丙转氨酶活性升高,表明天然除虫菊素诱导斑马鱼产生肝脏毒性现象。
实施例6谷草转氨酶的样品采集与检测。
收集暴露后的野生AB品系斑马鱼仔鱼置于冰水中进行安乐死,每个样品加10倍体积PBS冰浴匀浆,离心(3000g,10分钟)。收集上清液,样品中谷草转氨酶活性采用相应的试剂盒(本实验使用南京建成公司的谷草转氨酶测试盒)进行检测。
天然除虫菊素暴露后斑马鱼谷草转氨酶活性如图5所示,10μg/L、20μg/L、30μg/L和40μg/L天然除虫菊素暴露后,斑马鱼谷草转氨酶活性升高,且呈浓度依赖关系。谷草转氨酶存在肝脏肝细胞中,肝脏损害时谷草转氨酶血清浓度升高。天然除虫菊素暴露后,斑马鱼谷草转氨酶活性升高,表明天然除虫菊素诱导斑马鱼产生肝脏毒性现象。
本发明提供的评价菊酯类杀虫剂肝脏毒性的方法,将AB品系和转基因Tg(fabp10a:dsRed;ela3l:EGFP)品系相结合,将肝脏可视化。以斑马鱼肝脏面积大小、荧光强度、肝脏部位油红染色和谷丙转氨酶和谷草转氨酶的活性为评价指标,能够快速、直观地检测菊酯类杀虫剂暴露后肝脏形态变化,肝脏变性情况,脂质代谢情况,肝脏功能损伤情况,提高了检测的灵敏度和结果的准确性。
建立的菊酯类杀虫剂对斑马鱼肝脏毒性的评价模型具有简单、快速、稳定可靠和重复性好的优点。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (9)
1.一种利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于,包括如下步骤:将发育至3天的斑马鱼胚胎暴露于含有菊酯类杀虫剂的胚胎培养液中,采用半静态培养法,在特定温度、周期性明暗环境下培养,待斑马鱼胚胎发育至5天时,对斑马鱼胚胎的肝脏发育情况进行评价,
其中,所述斑马鱼胚胎为野生斑马鱼AB品系胚胎和转基因斑马鱼胚胎的组合。
2.根据权利要求1所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,含有菊酯类杀虫剂的胚胎培养液的配置方法如下:
1)采用二甲基亚砜作为溶剂将菊酯类杀虫剂配制成高浓度母液,于-20℃保存;
2)将配置好的母液用胚胎培养液稀释为不同浓度的工作液,溶液现配现用,保持二甲基亚砜的浓度小于千分之一;
3)将含有菊酯类杀虫剂的培养液与不含菊酯类杀虫剂的培养液注入6cm的培养皿中,培养液的体积控制在8~9mL。
3.根据权利要求2所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,稀释后菊酯类杀虫剂的浓度不低于10μg/L,
所述菊酯类杀虫剂包括天然除虫菊素、溴氰菊酯、氯氰菊酯或氯菊酯。
4.根据权利要求2所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,进行胚胎选择时,选取发育良好的,由斑马鱼繁殖发育至三天已孵化的胚胎进行暴露实验,每个培养皿中放置20个胚胎。
5.根据权利要求1所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,进行培养时,将培养容器置于恒温光控培养箱中,温度控制在28±1℃,光照与黑暗周期为14/10h,采取半静态培养法,每隔24h更换培养液,暴露时长为48h。
6.根据权利要求2所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,对斑马鱼胚胎的肝脏发育情况进行评价的方法包括:检测转基因斑马鱼胚胎肝脏面积和荧光强度;检测野生斑马鱼AB品系胚胎肝脏部位脂质油红染色情况、肝功能相关酶谷丙转氨酶及谷草转氨酶的数据结果并分析。
7.根据权利要求5所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,斑马鱼肝脏面积和荧光强度采集是通过荧光显微镜采集转基因斑马鱼仔鱼的2D图像,然后通过ImageJ软件勾勒出红色荧光的肝脏部分,计算红色荧光的面积及荧光强度。
8.根据权利要求5所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,检测野生斑马鱼AB品系胚胎肝脏部位脂质油红染色情况的方法包括如下步骤:
1)按质量体积比3:1000,取Oil Red O粉末充分溶解于体积分数为60%的丙二醇或PBS溶液中,配制成染色液,用滤纸过滤待用,该溶液现配现用;
2)收集暴露后的斑马鱼仔鱼,加入麻醉剂进行麻醉;
3)加入4%PFA,于4℃固定过夜后,PBS清洗3次,每次5分钟;
4)加入体积分数为60%的丙二醇或PBS溶液,室温孵育30分钟;
5)加入过滤后的新鲜Oil Red O染色液,室温孵育4小时;
6)PBS漂洗多次,每次20分钟,直至去除背景噪音信号
7)将胚胎置于载玻片上调整位置,在体式显微镜下观察并采集图像。
9.根据权利要求5所述的利用斑马鱼评价菊酯类杀虫剂肝脏发育毒性的方法,其特征在于:
其中,肝功能相关酶谷丙转氨酶及谷草转氨酶的样品采集与检测方法为:收集暴露后的斑马鱼仔鱼置于冰水中进行安乐死,每个样品加10倍体积PBS冰浴匀浆,3000g离心10分钟后,收集上清液,根据试剂盒说明对样品中谷丙转氨酶、谷草转氨酶活性进行检测。
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