CN106434832A - 一种用于检测节球藻毒素生物毒性的制备方法 - Google Patents
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Abstract
一种用于检测节球藻毒素生物毒性的制备方法，提供一种用于测试水中节球藻毒素生物毒性的检测方法，按如下步骤进行：(1)人脐静脉内皮细胞置于温度为37℃，空气湿度为95％(含5％CO2)的细胞培养箱中，培养于含10‑20％胎牛血清、60μg/mL内皮细胞生长添加剂和1％青霉素‑链霉素双抗的RPMI1640培养液。(2)取传代2‑3代的细胞，分别加入磷酸缓冲液(PBS)为空白样品A,加入节球藻毒素水样为检测样品B，放置于混匀器上震荡2小时；等。该方法运用人脐静脉内皮细胞针对节球藻毒素进行生物毒性检测，最终结果准确、稳定、重现性好，影响因素少，是对现有方法的有效改进，具有较高的实用价值和应用潜力。
Description
技术领域
本发明属于环境污染物的检测方法领域，具体涉及一种节球藻毒素生物毒性的检测方法。该方法运用人脐静脉内皮细胞(HUVECs)进行检测，建立了准确检测节球藻毒素水样生物毒性的方法。本方法最终结果准确、稳定、重现性好，是对现有方法的有效改进。。
背景技术
淡水蓝藻水华过程中产生的蓝藻毒素威胁着水域生态系统的健康，其中尤以蓝藻毒素危害最为严重。其中节球藻毒素是最近发现的新型藻毒素。节球藻毒素是由泡沫节球藻产生的一种环五肽肝毒素，具有严重的肝毒性、基因毒性、胚胎毒性和遗传毒性，同时还是肿瘤的引发剂和促进剂，并可以在生物和环境中进行富集和转化。通常情况下水体中节球藻毒素含量极低，而在水华水体中却可以达到几百甚至几千μg/L。此类毒素具有水溶性，长期摄入会对人体造成危害。我国目前常用的水处理工艺难以将其彻底去除，使人们的身体健康受到威胁。
目前采用藻类毒素的毒性检测通常采用生物检测法，如小鼠口服或注射来间接衡量毒性，毒性强弱用半致死剂量表示。此外还有用无脊椎动物如虾、贝、水蚤及其卵进行毒性评价的研究，以发光细菌进行毒性试验也有报道。此类方法只可较粗略地判断藻提取物是否有急性毒性，灵敏度较低，毒素消耗量大，需要大量的动物试验品，存在伦理问题，并且只能检测总的毒性，不能检测出细胞凋亡和坏死，对慢性毒性缺乏定量评价。而细胞毒性检测技术不仅可以对毒素进行定性鉴定，并且能对其进行精确的定量测定。目前报道有使用鲫鱼巨噬细胞、草鱼淋巴细胞等水生动物细胞检测毒性机理[1]，本发明将人脐静脉内皮细胞作为生物标志物进行节球藻毒素的生物毒性评价，实现定量化检测节球藻毒素对人体心血管的毒性效应，更具有针对性，水质生物毒性评价的灵敏性和准确性更高，能为水体污染提供预警技术支持，保障供水水质安全。
[1].张杭君等,节球藻毒素对草鱼淋巴细胞凋亡毒性效应及机理.应用生态学报,2013(10):第2977-2982页.
发明内容
本发明所要解决的技术问题是针对现有节球藻毒素生物毒性检测方法中存在结果粗略，灵敏度较低，毒素消耗量大，需要大量的动物试验品等问题，提供一种用于测试水中节球藻毒素生物毒性的检测方法。本发明将人脐静脉内皮细胞作为生物标志物进行节球藻毒素的生物毒性评价，实现利用细胞凋亡率和坏死率定量化检测节球藻毒素对人体心血管的毒性效应。
本发明技术方案为：
一种用于检测节球藻毒素生物毒性的制备方法，其特征在于，按如下步骤进行：
(1)人脐静脉内皮细胞置于温度为37℃，空气湿度为95％(含5％CO2)的细胞培养箱中，培养于含10-20％胎牛血清、60μg/mL内皮细胞生长添加剂和1％青霉素-链霉素双抗的RPMI1640培养液。
(2)取传代2-3代的细胞，分别加入磷酸缓冲液(PBS)为空白样品A,加入节球藻毒素水样为检测样品B，放置于混匀器上震荡2小时。
(3)将样品A和B用PBS洗涤细胞二次，并分别配成浓度为1×105-1×106cells/mL细胞悬液；
(4)A、B样品均加入5μL Annexin V-FITC混匀后，再加入5μL碘化丙啶(PI)，混匀；室温、避光、反应5-15min；在1小时内，进行流式细胞仪检测。
(5)用流式细胞术(激发波长Ex＝488nm；发射波长Em＝530nm)检测死细胞、活细胞和凋亡细胞的比例。
(6)结果计算：通过流式细胞仪自带软件(BD FACSCanto II Flow Cytometer,为现有通用方法)测出的样品B的凋亡率和死亡率对照空白样品A，即可简便、可靠的判定水质毒性。
本发明方法具有如下优点：
(1)人脐静脉内皮细胞是介于血管壁组织和血液之间的一层单核细胞，它是血液中的化合物或毒素从血管管腔进入血管壁深层的第一道屏障，因而也将成为进入血液的藻毒素进攻的首道屏障。因此可用其细胞的凋亡率和死亡率简便、可靠的判定水质判断对人体心血管的毒性作用。
(2)能定量测定细胞凋亡率和坏死率，结果准确、稳定、重现性好，能更细致的判断节球藻毒素生物毒性。
该方法运用人脐静脉内皮细胞针对节球藻毒素进行生物毒性检测，最终结果准确、稳定、重现性好，影响因素少，是对现有方法的有效改进，具有较高的实用价值和应用潜力。
附图说明
图1为实施例1检测的结果示意图。
图2为实施例2检测的结果示意图。
具体实施方式
实施例1
本发明应用于5μg/mL节球藻毒素(NOD)生物毒性检测：
(1)人脐静脉内皮细胞置于温度为37℃，空气湿度为95％(含5％CO2)的细胞培养箱中，培养于含20％胎牛血清、60μg/mL内皮细胞生长添加剂和1％青霉素-链霉素双抗的RPMI1640培养液。
(2)取传代3代的细胞，分别加入磷酸缓冲液(PBS)为空白样品A,加入节球藻毒素水样为检测样品B，放置于混匀器上震荡2小时。
(3)将样品A和B用PBS洗涤细胞二次，并分别配成浓度约为1×105cells/mL细胞悬液；
(4)A、B样品均加入5μL Annexin V-FITC混匀后，再加入5μL碘化丙啶(PI)，混匀；室温、避光、反应5-15min；在1小时内，进行流式细胞仪检测。
(5)用流式细胞术(激发波长Ex＝488nm；发射波长Em＝530nm)检测死细胞、活细胞和凋亡细胞的比例。
(6)结果计算：通过流式细胞仪自带软件(BD FACSCanto II Flow Cytometer,为现有通用方法)测出的样品B的凋亡率和死亡率对照空白样品A，即可简便、可靠的判定水质毒性。
图1是按上述方法检测的结果示意图。
将上述方法应用于模拟节球藻毒素水质生物毒性检测，得出细胞总死亡率为55.58％。
实施例2
本发明应用于10μg/mL节球藻毒素(NOD)生物毒性检测：
(1)人脐静脉内皮细胞置于温度为37℃，空气湿度为95％(含5％CO2)的细胞培养箱中，培养于含10％胎牛血清、60μg/mL内皮细胞生长添加剂和1％青霉素-链霉素双抗的RPMI1640培养液。
(2)取传代3代的细胞，分别加入磷酸缓冲液(PBS)为空白样品A,加入节球藻毒素水样为检测样品B，放置于混匀器上震荡2小时。
(3)将样品A和B用PBS洗涤细胞二次，并分别配成浓度约为5×105cells/mL细胞悬液，；
(4)A、B样品均加入5μL Annexin V-FITC混匀后，再加入5μL碘化丙啶(PI)，混匀；室温、避光、反应5-15min；在1小时内，进行流式细胞仪检测。
(5)用流式细胞术(激发波长Ex＝488nm；发射波长Em＝530nm)检测死细胞、活细胞和凋亡细胞的比例。
(6)结果计算：通过流式细胞仪自带软件(BD FACSCanto II Flow Cytometer,为现有通用方法)测出的样品B的凋亡率和死亡率对照空白样品A，即可简便、可靠的判定水质毒性。
图2是按上述方法检测的结果示意图。
将上述方法应用于模拟节球藻毒素水质生物毒性检测，得出细胞总死亡率为77.74％。
Claims (1)
1.一种用于检测节球藻毒素生物毒性的制备方法，其特征在于，按如下步骤进行：
(1)人脐静脉内皮细胞置于温度为37℃，空气湿度为95％的细胞培养箱中，培养于含10-20％胎牛血清、60μg/mL内皮细胞生长添加剂和1％青霉素-链霉素双抗的RPMI 1640培养液；
(2)取传代2-3代的细胞，分别加入磷酸缓冲液(PBS)为空白样品A,加入节球藻毒素水样为检测样品B，放置于混匀器上震荡2小时；
(3)将样品A和B用PBS洗涤细胞二次，并分别配成浓度为1×105-1×106cells/mL细胞悬液；
(4)A、B样品均加入5μL Annexin V-FITC混匀后，再加入5μL碘化丙啶(PI)，混匀；室温、避光、反应5-15min；在1小时内，进行流式细胞仪检测；
(5)用流式细胞术检测死细胞、活细胞和凋亡细胞的比例；
(6)结果计算：通过流式细胞仪自带软件测出的样品B的凋亡率和死亡率对照空白样品A，判定水质毒性。
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| CN112322694A (zh) * | 2020-10-21 | 2021-02-05 | 桂林理工大学 | 一种检测绿藻细胞凋亡的方法 |
| CN115524500A (zh) * | 2022-08-11 | 2022-12-27 | 合肥学院 | 一种快速评估水体中蓝藻毒素致突风险的方法 |
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| CN105483202A (zh) * | 2016-01-25 | 2016-04-13 | 上海艾耐基新能源科技有限公司 | 用于测试高藻水生物毒性的方法 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112322694A (zh) * | 2020-10-21 | 2021-02-05 | 桂林理工大学 | 一种检测绿藻细胞凋亡的方法 |
| CN115524500A (zh) * | 2022-08-11 | 2022-12-27 | 合肥学院 | 一种快速评估水体中蓝藻毒素致突风险的方法 |
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