CN104142405A - 硼替佐米对k562细胞株中dna甲基转移酶表达的检测方法 - Google Patents

硼替佐米对k562细胞株中dna甲基转移酶表达的检测方法 Download PDF

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CN104142405A
CN104142405A CN201410371771.1A CN201410371771A CN104142405A CN 104142405 A CN104142405 A CN 104142405A CN 201410371771 A CN201410371771 A CN 201410371771A CN 104142405 A CN104142405 A CN 104142405A
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吴圣豪
郑翠苹
陈松燕
蔡小平
石岳坚
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Wenzhou Central Hospital
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Abstract

本发明公开了一种硼替佐米对K562细胞株中DNA甲基转移酶表达的检测方法，设置对照组与实验组：将K562细胞置于含10％灭活新鲜小牛血清RPMI-1640培养液，在37℃，体积分数为5％CO2、饱和湿度条件下培养，保持细胞活力＞97％，取指数生长期的K562细胞（1×105/ml）分为对照组与实验组（3）Annexin-V法流式细胞仪（FCM）检测K562细胞凋亡率。本申请的方法能确定硼替佐米对K562细胞株中DNA甲基转移酶表达的抑制效果，从而为硼替佐米在白血病中的研究提供了可靠依据。

Description

硼替佐米对K562细胞株中DNA甲基转移酶表达的检测方法
技术领域
本发明属于生物学检测技术，具体是指一种硼替佐米对K562 细胞株DNA甲基转移酶表达的检测方法。
背景技术
DNA甲基化异常是人类恶性肿瘤及白血病的一种最常见的可以查明的分子异常，DNA 甲基转移酶（DNA methyltransferase，DNMTs）活性增高是其重要原因，硼替佐米（bortezomib，商品名：万珂）是蛋白酶体抑制剂类抗肿瘤药物，临床主要用于治疗复发及难治性多发性骨髓瘤，在白血病治疗方面的应用仍处于理论研究和探索阶段。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足，而提供一种硼替佐米对K562细胞株DNA甲基转移酶表达的检测方法。通过该方法能确定硼替佐米对K562细胞株中DNA甲基转移酶表达的抑制效果，从而为硼替佐米在白血病中的研究提供了可靠依据。
为实现上述目的，本发明的技术方案是
（1）设置对照组与实验组：将K562细胞置于含10％灭活新鲜小牛血清RPMI-1640 培养液，在37 ℃，体积分数为5％CO2、饱和湿度条件下培养，保持细胞活力＞ 97％，取指数生长期的K562细胞（1×105/ml）分为对照组与实验组，实验组分别加入6nmol/L、20nmol/L和60 nmol/L的硼替佐米，分别作用时间分别为12 h、24 h和36 h；对照组加入无硼替佐米的细胞培养液，相同条件下培养；
本步骤获得以下实验组和对照组：
A 组，不加任何处理的对照组；B 组，6 nmol/L 硼替佐米处理组；C 组，20 nmol/L 硼替佐米处理组；D 组，60nmol/L 硼替佐米处理组；
（2）蛋白印迹法检测胞内DNMT1 表达：收集硼替佐米作用12 h 后的各处理组细胞，Western 及IP细胞裂解液提取胞内蛋白，参照试剂盒说明用BCA方法测定蛋白浓度，并调节浓度，每泳道取100 μg上样，于10％十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后，电转至硝酸纤维膜，以β-actin 为内参蛋白，加入兔抗人DNMT1 多克隆抗体、β-actin 一抗，碱性磷酸酶标记山羊抗兔二抗，BCIP/NBT 碱性磷酸酯酶显色试剂盒显色，采用BandScan 软件分析各组蛋白条带与相应β-actin 的A 值之比。实验重复3 次；
（3）Annexin-V 法流式细胞仪（FCM）检测K562细胞凋亡率：收集硼替佐米各浓度处理12-36 h 的K562 细胞1×106 个，用磷酸盐缓冲液洗涤2次， 离心弃上清，加入100 μl Binding Buffer 和FITC 标记的Annexin-V （20 mg/L）10 μl，室温避光30 min，再加入PI（50 mg/L）5 μl，避光反应5 min后，加入400 μl Binding Buffer，立即用FCM 进行定量检测，同时以不加AnnexinV-FITC 及PI 的一管作为阴性对照。单纯FITC 阳性（FITC＋/PI-）细胞群为早期凋亡细胞。
 
总之，本检测方法研究证实了在硼替佐米的干预下，白血病K562 细胞株的DNMT1 的表达水平下降，肿瘤细胞的生长受到抑制，凋亡率增加，本研究为硼替佐米在白血病治疗中的应用以及其他以DNMT1 为靶点药物在白血病中的研究提供了可靠依据，同时为白血病的基因治疗提供新思路。
下面结合具体实施方式对本发明做进一步介绍。
具体实施方式
    下面通过实施例对本发明进行具体的描述，只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限定，该领域的技术工程师可根据上述发明的内容对本发明作出一些非本质的改进和调整。
实施例
材料的选择：
K562 细胞株由浙江大学医学院血液病实验室赠送；RPMI1640 培养基、蛋白提取试剂盒及实验相关引物合成由南京凯基生物公司提供。RPMI-1640培养基为美国Gibco 公司产品，AnnexinⅤ-异硫氰酸
荧光素（FITC）凋亡试剂盒为美国Bipec 公司产品，硼替佐米为美国Millennium 公司产品。兔抗人DNMT1、β-actin 多克隆抗体为美国Cell-signaling 公司产品，碱性磷酸酶标志山羊抗兔二抗，BCIP/NBT
碱性磷酸酯酶显色试剂盒购自上海优宁维生物科技有限公司。
检测方法：
（1）设置对照组与实验组：将K562细胞置于含10％灭活新鲜小牛血清RPMI-1640 培养液，在37 ℃，体积分数为5％CO2、饱和湿度条件下培养，保持细胞活力＞ 97％，取指数生长期的K562细胞（1×105/ml）分为对照组与实验组，实验组分别加入6nmol/L、20nmol/L和60 nmol/L的硼替佐米，分别作用时间分别为12 h、24 h和36 h；对照组加入无硼替佐米的细胞培养液，相同条件下培养；
本步骤获得以下实验组和对照组：
A 组，不加任何处理的对照组；B 组，6 nmol/L 硼替佐米处理组；C 组，20 nmol/L 硼替佐米处理组；D 组，60nmol/L 硼替佐米处理组；
（2）蛋白印迹法检测胞内DNMT1 表达：收集硼替佐米作用12 h 后的各处理组细胞，Western 及IP细胞裂解液提取胞内蛋白，参照试剂盒说明用BCA方法测定蛋白浓度，并调节浓度，每泳道取100 μg上样，于10％十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后，电转至硝酸纤维膜，以β-actin 为内参蛋白，加入兔抗人DNMT1 多克隆抗体、β-actin 一抗，碱性磷酸酶标记山羊抗兔二抗，BCIP/NBT 碱性磷酸酯酶显色试剂盒显色，采用BandScan 软件分析各组蛋白条带与相应β-actin 的A 值之比。实验重复3 次；
（3）Annexin-V 法流式细胞仪（FCM）检测K562细胞凋亡率：收集硼替佐米各浓度处理12-36 h 的K562 细胞1×106 个，用磷酸盐缓冲液洗涤2次， 离心弃上清，加入100 μl Binding Buffer 和FITC 标记的Annexin-V （20 mg/L）10 μl，室温避光30 min，再加入PI（50 mg/L）5 μl，避光反应5 min后，加入400 μl Binding Buffer，立即用FCM 进行定量检测，同时以不加AnnexinV-FITC 及PI 的一管作为阴性对照。单纯FITC 阳性（FITC＋/PI-）细胞群为早期凋亡细胞。
本申请检测了硼替佐米干预K562 细胞株后，DNMT1 酶催化活性和细胞生活状态变化。用蛋白印迹法分析硼替佐米作用12 h 后，发现胞内DNMT1的活性均下降，与对照组相比，差异有统计学意义（P＜0．05）。提示DNMT1 转录水平及催化甲基转移能力下降。同时，研究还发现该作用在一定的浓度范围内呈现明显的浓度依赖性。
本申请的检测方法亦证实经硼替佐米处理K562 细胞后，肿瘤细胞增殖减缓，凋亡率增加。硼替佐米能有效地诱导K562 细胞凋亡，细胞凋亡率随硼替佐米浓度的升高而增大，硼替佐米作用36 h 后凋亡细胞比例显著高于其他时间，细胞凋亡率与作用时间呈正相关趋势。

Claims (1)

1.一种硼替佐米对K562细胞株中DNA甲基转移酶表达的检测方法，其特征在于：
（1）设置对照组与实验组：将K562细胞置于含10％灭活新鲜小牛血清RPMI-1640 培养液，在37 ℃，体积分数为5％CO2、饱和湿度条件下培养，保持细胞活力＞ 97％，取指数生长期的K562细胞（1×105/ml）分为对照组与实验组，实验组分别加入6nmol/L、20nmol/L和60 nmol/L的硼替佐米，分别作用时间分别为12 h、24 h和36 h；对照组加入无硼替佐米的细胞培养液，相同条件下培养；
本步骤获得以下实验组和对照组：
A 组，不加任何处理的对照组；B 组，6 nmol/L 硼替佐米处理组；C 组，20 nmol/L 硼替佐米处理组；D 组，60nmol/L 硼替佐米处理组；
（2）蛋白印迹法检测胞内DNMT1 表达：收集硼替佐米作用12 h 后的各处理组细胞，Western 及IP细胞裂解液提取胞内蛋白，参照试剂盒说明用BCA方法测定蛋白浓度，并调节浓度，每泳道取100 μg上样，于10％十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后，电转至硝酸纤维膜，以β-actin 为内参蛋白，加入兔抗人DNMT1 多克隆抗体、β-actin 一抗，碱性磷酸酶标记山羊抗兔二抗，BCIP/NBT 碱性磷酸酯酶显色试剂盒显色，采用BandScan 软件分析各组蛋白条带与相应β-actin 的A 值之比；实验重复3 次；
（3）Annexin-V 法流式细胞仪（FCM）检测K562细胞凋亡率：收集硼替佐米各浓度处理12-36 h 的K562 细胞1×106 个，用磷酸盐缓冲液洗涤2次， 离心弃上清，加入100 μl Binding Buffer 和FITC 标记的Annexin-V （20 mg/L）10 μl，室温避光30 min，再加入PI（50 mg/L）5 μl，避光反应5 min后，加入400 μl Binding Buffer，立即用FCM 进行定量检测，同时以不加AnnexinV-FITC 及PI 的一管作为阴性对照，单纯FITC 阳性（FITC＋/PI-）细胞群为早期凋亡细胞。
CN201410371771.1A 2014-07-31 2014-07-31 硼替佐米对k562细胞株中dna甲基转移酶表达的检测方法 Pending CN104142405A (zh)

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CN114350633B (zh) * 2021-12-31 2024-04-26 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一种dna甲基转移酶1的抗原肽及其多克隆抗体

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Application publication date: 20141112