CN116609525B - 基于细胞内aldh3b1蛋白水平以筛选染色质重塑复合体抑制剂的方法 - Google Patents
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Abstract
本发明公开了基于细胞内ALDH3B1蛋白水平以验证小分子化合物为染色质重塑复合物抑制剂的方法，包括a)细胞铺板、b)药物处理、c)细胞预处理、d)细胞封闭、e)一抗孵育、f)二抗孵育和g)成像和数据分析。本发明利用细胞内western检测原理，可直接先在培养板上对细胞内蛋白进行原位免疫荧光染色，再通过荧光扫描成像仪对荧光信号进行定量分析，从而根据ALDH3B1蛋白水平开发出一种在细胞水平上验证筛选得到的小分子化合物是否为染色质重塑复合体抑制剂的方法，与传统的筛选方法相比，无需提取蛋白或RNA，具有操作简单、灵敏度高、特异性强和通量高等优点，能够为进一步开发靶向染色质重塑复合体小分子药物提供有效的验证手段，具有临床药物开发价值。
Description
【技术领域】
本发明涉及分子细胞生物学和生物化学技术的技术领域，特别是筛选染色质重塑复合体抑制剂的技术领域。
【背景技术】
哺乳动物的染色质重塑复合体通过改变DNA与核小体的结合构象，来调控DNA可及性，从而影响转录起始复合物结合DNA，控制许多关键的生物过程，包括基因转录、细胞增殖、DNA损伤修复和肿瘤生成等。阻断染色质重塑复合物功能，能调控肿瘤细胞基因转录，发挥抑制肿瘤细胞生长的作用。
由于小分子化合物在抑制细胞染色质重塑复合物功能后，可特异性抑制ALDH3B1基因的表达，因此可通过ALDH3B1基因的表达水平变化来判断小分子化合物对染色质重塑复合物功能是否有抑制作用，从而评估该化合物是否为染色质重塑复合物的抑制剂。传统验证小分子化合物是否为染色质重塑复合物抑制剂的方法包括实时荧光定量PCR和Western blot两种方法。然而，上述两种方法均存在实验操作复杂、不能进行定量分析，且无法进行高通量检测等缺点。
【发明内容】
本发明的目的就是解决现有技术中的问题，提出基于原位细胞内ALDH3B1蛋白水平变化，以验证小分子化合物为染色质重塑复合物抑制剂的方法。本发明通过对ALDH3B1蛋白进行原位细胞内标记，通过免疫荧光染色和荧光成像分析，能有效验证小分子化合物对ALDH3B1蛋白水平的影响，从而判定该化合物是否为染色质重塑复合物抑制剂。本发明具有高通量、强特异性、易操作和经济适用的优点，可补足现有验证方法不足。
为实现上述目的，本发明通过以下技术方案来实现：
基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，包括如下步骤：
a)细胞铺板：在细胞培养腔室中加入癌细胞悬液并培养至肿瘤细胞完全贴壁；
b)药物处理：将待验证的小分子化合物进行梯度稀释并分别滴加至不同的肿瘤细胞培养腔室，将滴加有不同浓度的待验证的小分子化合物的各个细胞培养腔室共同作为药物检测组，将未滴加有待验证的小分子化合物的细胞培养腔室作为阴性对照组，分别对药物检测组和阴性对照组进行培养；
c)细胞预处理：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加固定剂以进行细胞固定，在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加溶脂剂以进行细胞增透；所述固定剂为3.5～4.5％的多聚甲醛且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，固定时间为10～20min，所述溶脂剂为0.4～0.6％的Triton X100且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，溶脂时间为3～10min；
d)细胞封闭：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加封闭剂以进行细胞封闭；所述封闭剂为2.5～3.5％的牛血清白蛋白且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，封闭时间为15～45min；
e)一抗孵育：将ALDH3B1抗体经稀释剂稀释后再分别滴加至药物检测组的各个培养腔室内，在阴性对照组的各个培养腔室内仅滴加与药物检测组中经稀释剂稀释后的ALDH3B1抗体等体积的稀释剂，分别对药物检测组和阴性对照组进行孵育；
f)二抗孵育：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加经稀释剂稀释后的山羊抗兔IgG H&L并进行孵育；
g)成像和数据分析：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后分别对药物检测组和阴性对照组进行信号扫描和荧光成像并以阴性对照组的信号结果作为背景信号；若待验证的小分子化合物与ALDH3B1蛋白表达水平呈现剂量依赖性，则待验证的小分子化合物对染色质重塑复合物具有抑制作用且为该复合物抑制剂。
作为优选，在所述步骤a)至g)中，采用带有若干个培养孔的黑色透明培养板作为培养容器并以各个培养孔分别作为各个培养腔室。
作为优选，在所述步骤c)、d)、f)和g)中，润洗剂为PBS缓冲液。
作为优选，在所述步骤a)中，采用混入10％胎牛血清的RPMI 1640作为细胞培养基，肿瘤细胞悬液的密度为2×104～10×104个/ml且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，培养温度为36.5～37.5℃，培养时间为12～24h，CO2培养浓度为4～6％。
作为优选，在所述步骤b)中，培养温度为36.5～37.5℃，培养时间为36～60h，CO2培养浓度为4～6％。
作为优选，在所述步骤e)中，稀释剂为2.5～3.5％的牛血清白蛋白，ALDH3B1抗体与稀释剂按体积比1：500～700进行稀释，经稀释剂稀释后的ALDH3B1抗体的在药物检测组的各个培养腔室内的滴入均为40～60μl，稀释剂在阴性对照组的各个培养腔室内的滴入均为40～60μl，孵育时间为0.5～1.5h。
作为优选，在所述步骤f)中，稀释剂为2.5～3.5％的牛血清白蛋白，山羊抗兔IgGH&L与稀释剂按体积比1：800～1200进行稀释且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为40～60μl，孵育时间为0.5～1.5h。
作为优选，在所述步骤g)中，信号扫描和荧光成像的激光光源的波长为630～640nm。
本发明的有益效果：
本发明利用细胞内western检测原理，可直接先在培养板上对细胞内蛋白进行原位免疫荧光染色，再通过荧光扫描成像仪对荧光信号进行定量分析，从而根据ALDH3B1蛋白水平开发出一种在细胞水平上验证筛选得到的小分子化合物是否为染色质重塑复合物抑制剂的方法，与传统的筛选方法相比，无需提取蛋白或RNA，具有操作简单、灵敏度高、特异性强和通量高等优点，能够为进一步开发靶向染色质重塑复合物小分子药物提供有效的验证手段，具有临床药物开发价值。
本发明的特征及优点将通过实施例结合附图进行详细说明。
【附图说明】
图1是本发明实施例中测试的染色质重塑复合物抑制剂结构式
图2是本发明实施例中测试的染色质重塑复合物抑制剂对NCI-H1299细胞中ALDH3B1蛋白水平抑制的荧光成像结果；
图3是发明实施例中测试的染色质重塑复合物抑制剂对NCI-H1299细胞中ALDH3B1蛋白水平抑制的荧光信号曲线图。
【具体实施方式】
采用本发明基于细胞内ALDH3B1蛋白水平以验证小分子化合物为染色质重塑复合物抑制剂的方法，测试已知的染色质重塑复合物抑制剂处理后对H1299细胞内ALDH3B1蛋白表达的抑制作用。具体的仪器列表以及主要的细胞、试剂和耗材列表分别参阅下表1和2：
| 名称 | 型号 | 厂家 |
| 二氧化碳培养箱 | CLM-170B-CN | ESCO |
| 生物安全柜 | AC2-4S8-CN | ESCO |
| 分液仪 | Multidrop Pico 8 | Thermo Scientific |
| 多功能激光扫描仪 | Typhoon 5 | GE |
表1仪器列表
表2主要的细胞、试剂、耗材列表
基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，包括如下步骤：
a)细胞铺板：采用RPMI 1640细胞培养基+10％胎牛血清(FBS)作为液体培养基制备人非小细胞肺癌细胞NCI-H1299的癌细胞悬液(密度为6×104个/ml)，在培养板(黑色透明底96孔)中于各个培养孔内分别加入100μl的癌细胞悬液，将培养板置于37℃的5％的CO2的二氧化碳培养箱中培养过夜以待癌细胞完全贴壁；
b)药物处理：采用分液仪分别在培养板的各个培养孔内滴加经PBS缓冲液梯度稀释后的染色质重塑复合物抑制剂且工作浓度分别为3333.33、1111.11、370.37、123.46、41.15、13.72、4.57和1.52nM，将滴加有不同浓度的染色质重塑复合物抑制剂的各个培养孔共同作为药物检测组，将未滴加有染色质重塑复合物抑制剂的各个培养孔作为阴性对照组，每个条件做两个复孔，再将培养板置于37℃的5％的CO2的二氧化碳培养箱中培养48h；
c)细胞预处理：弃除培养板内的残液并于药物检测组和阴性对照组的各个培养孔内分别加入100μl的PBS缓冲液润洗1次再于药物检测组和阴性对照组的各个培养孔内分别加入100μl的4％的多聚甲醛并室温静置15min，弃除培养板内的残液并于药物检测组和阴性对照组的各个培养孔内分别加入100μl的PBS缓冲液润洗1次再于药物检测组和阴性对照组的各个培养孔内分别加入100μl的0.5％的Triton X100并室温静置5min；
d)细胞封闭：弃除培养板内的残液并于药物检测组和阴性对照组的各个培养孔内分别加入100μl的PBS缓冲液润洗1次再于药物检测组和阴性对照组的各个培养孔内分别加入100μl的3％的牛血清白蛋白(BSA)并室温静置30min；
e)一抗孵育：将ALDH3B1抗体由3％的BSA进行1：600稀释再于药物检测组的各个培养孔内分别滴加50μl并室温静置1h，在阴性对照组的各个培养孔内不加入ALDH3B1抗体而仅滴加50μl的3％的BSA并室温静置1h；
f)二抗孵育：弃除培养板内的残液并于药物检测组和阴性对照组的各个培养孔内分别加入100μl的PBS缓冲液润洗2次，将山羊抗兔IgG H&L由3％的BSA进行1：1000稀释再于药物检测组和阴性对照组的各个培养孔内分别滴加50μl并室温静置1h；
g)成像和数据分析：弃除培养板内的残液并于药物检测组和阴性对照组的各个培养孔内分别加入100μl的PBS缓冲液润洗2次，在Typhoon 5多功能激光扫描仪内选择635nm激光光源再分别对药物检测组和阴性对照组进行信号扫描和荧光成像并以阴性对照组的信号结果作为背景信号。
参见图2和图3，结果说明染色质重塑复合物抑制剂处理能显著抑制NCI-H1299细胞中的ALDH3B1蛋白表达水平且呈现剂量依赖性，提示染色质重塑复合物抑制剂为染色质重塑复合物小分子抑制剂。
上述实施例是对本发明的说明，不是对本发明的限定，任何对本发明简单变换后的方案均属于本发明的保护范围。
Claims (8)
1.基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于，包括如下步骤：
a)细胞铺板：在培养腔室中加入人非小细胞肺癌细胞 NCI-H1299的癌细胞悬液并培养至人非小细胞肺癌细胞 NCI-H1299完全贴壁；
b)药物处理：将待验证的小分子化合物进行梯度稀释并分别滴加至不同的培养腔室，将滴加有不同浓度的待验证的小分子化合物的各个培养腔室共同作为药物检测组，将未滴加有待验证的小分子化合物的培养腔室作为阴性对照组，分别对药物检测组和阴性对照组进行培养；
c)细胞预处理：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加固定剂以进行细胞固定，在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加溶脂剂以进行细胞增透；所述固定剂为3.5～4.5%的多聚甲醛且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，固定时间为10～20min，所述溶脂剂为0.4～0.6%的Triton X100且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，溶脂时间为3～10min；
d)细胞封闭：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加封闭剂以进行细胞封闭；所述封闭剂为2.5～3.5%的牛血清白蛋白且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，封闭时间为15～45min；
e)一抗孵育：将ALDH3B1抗体经稀释剂稀释后再分别滴加至药物检测组的各个培养腔室内，在阴性对照组的各个培养腔室内仅滴加与药物检测组中经稀释剂稀释后的ALDH3B1抗体等体积的稀释剂，分别对药物检测组和阴性对照组进行孵育；
f)二抗孵育：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后再分别滴加经稀释剂稀释后的山羊抗兔IgG H&L并进行孵育；
g)成像和数据分析：在利用润洗剂分别对药物检测组和阴性对照组的各个培养腔室进行润洗后分别对药物检测组和阴性对照组进行信号扫描和荧光成像并以阴性对照组的信号结果作为背景信号；若待验证的小分子化合物能显著抑制NCI-H1299细胞中的ALDH3B1蛋白表达水平且呈现剂量依赖性，则待验证的小分子化合物对染色质重塑复合体具有抑制作用且为该复合体抑制剂。
2.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤a)至g)中，采用带有若干个培养孔的黑色透明培养板作为培养容器并以各个培养孔分别作为各个培养腔室。
3.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤c)、d)、f)和g)中，润洗剂为PBS缓冲液。
4.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤a)中，采用混入10%胎牛血清的RPMI 1640作为细胞培养基，人非小细胞肺癌细胞 NCI-H1299的癌细胞悬液的密度为2×104～10×104个/ml且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为80～120μl，培养温度为36.5～37.5℃，培养时间为12～24h，CO2培养浓度为4～6%。
5.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤b)中，培养温度为36.5～37.5℃，培养时间为36～60h，CO2培养浓度为4～6%。
6.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤e)中，稀释剂为2.5～3.5%的牛血清白蛋白，ALDH3B1抗体与稀释剂按体积比1：500～700进行稀释，经稀释剂稀释后的ALDH3B1抗体的在药物检测组的各个培养腔室内的滴入体积均为40～60μl，稀释剂在阴性对照组的各个培养腔室内的滴入体积均为40～60μl，孵育时间为0.5～1.5h。
7.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤f)中，稀释剂为2.5～3.5%的牛血清白蛋白，山羊抗兔IgGH&L与稀释剂按体积比1：800～1200进行稀释且在药物检测组和阴性对照组的各个培养腔室内的滴入体积均为40～60μl，孵育时间为0.5～1.5h。
8.如权利要求1所述的基于细胞内ALDH3B1蛋白水平以筛选染色质重塑复合体抑制剂的方法，其特征在于：在所述步骤g)中，信号扫描和荧光成像的激光光源的波长为630～640nm。
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