CN106939301A - Parp1抑制剂耐药细胞株的建立方法及其应用 - Google Patents

Parp1抑制剂耐药细胞株的建立方法及其应用 Download PDF

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CN106939301A
CN106939301A CN201710305074.XA CN201710305074A CN106939301A CN 106939301 A CN106939301 A CN 106939301A CN 201710305074 A CN201710305074 A CN 201710305074A CN 106939301 A CN106939301 A CN 106939301A
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王宁
王然
王一然
王雅杰
刘传
吴家雪
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Abstract

本发明提供一种PARP1抑制剂耐药细胞株的建立方法，其特征在于，包括如下步骤：步骤一：对B细胞感染puro‑chek2载体，向B细胞中转入puro基因，同时将细胞自身表达的背景chek2去掉；步骤二：感染后进行puromycin筛选，筛选后得到chek2沉默的B细胞；步骤三：对Chek2‑null的B细胞感染带有GFP的Chek2Y394C；补充具体步骤：步骤四：筛选带有GFP标记的细胞，得到PARP1抑制剂耐药细胞株。按照本发明的PARP1抑制剂耐药细胞株的建立方法得到的PARP1抑制剂耐药细胞株对研究PARP1抑制剂耐药提供了体外细胞模型。

Description

PARP1抑制剂耐药细胞株的建立方法及其应用
技术领域
本发明涉及一种PARP1抑制剂耐药细胞株的建立方法，本发明还涉及PARP1抑制剂耐药细胞株的应用，属于生物技术领域。
背景技术
乳腺癌的药物治疗已经进入了分子分型指导下的精准化治疗时代。通过组化四要素(ER、PR、HER2和Ki67)而代替基因芯片检测的简易分子分型，已广泛应用于指导临床药物治疗方案的制定。但三阴型乳腺癌(TNBC)因其ER、PR以及HER2均阴性，对靶向激素受体的内分泌治疗和靶向HER2的系列药物治疗无效。曾被列为靶向治疗的弃儿。而三阴型乳腺癌又较其他分子类型发病更年轻化，遗传倾向更明显，有更强的侵袭性，2年内局部复发和内脏转移的风险更高。传统的化疗虽然显示出较高的近期有效率，但缓解时间较短，总体生存差，亟待开发针对性强的靶向治疗药物。
BRCA1/2的突变率在三阴型乳腺癌中波动在9％到28％。因为BRCA1/2是DNA同源重组修复(Homologous recombination,HR)的主要基因，其突变导致三阴型乳腺癌对DNA损伤的药物敏感性显著增加。抑制PARP1(poly ADP-ribose polymerase family member 1,聚腺苷二磷酸-核糖转移酶1)抑制剂可使BRCA1/2基因突变的细胞选择性凋亡，以PARP1为靶点的分子治疗可能成为三阴型乳腺癌新的治疗手段，引领三阴型乳腺癌步入了靶向治疗的里程。基于此理论，应用PARP1抑制剂联合DNA损伤化疗药物卡铂治疗BRCA1/2突变率相对较高的三阴性乳腺癌应该具有更好的疗效。然而，O’Shaughnessy教授牵头开展的国际多中心III期临床研究，并未显示出PARP1抑制剂iniparib联合致DNA损伤化疗药物卡铂及吉西他滨治疗三阴性乳腺癌的优势.随后Astra Zeneca公布的另一个PARP1抑制剂olaparib治疗三阴性乳腺癌II期临床结果，也未达到预期的疗效。因此，对于一种理论上对三阴性乳腺癌有效的药物是否在实际中也同样有效，目前缺乏一种有效的判断机制。
发明内容
本发明的目的在于提供一种PARP1抑制剂耐药细胞株的建立方法及其应用，用以研究PARP1抑制剂耐药的问题，同时判断药物是否对三阴性乳腺癌有效。
本发明采用了如下技术方案：
一种PARP1抑制剂耐药细胞株的建立方法，其特征在于，包括如下步骤：
步骤一：对B细胞感染puro-chek2载体，向B细胞中转入puro基因，同时将细胞自身表达的背景chek2去掉；
步骤二：感染后进行puromycin筛选，筛选后得到chek2沉默的B细胞；
步骤三：对Chek2-null的B细胞感染带有GFP的Chek2Y394C；
步骤四：筛选带有GFP标记的细胞，得到PARP1抑制剂耐药细胞株。
作为优选，本发明的PARP1抑制剂耐药细胞株的建立方法，还包括：验证的步骤：对步骤四中得到的有GFP标记的细胞分别使用顺铂、表柔比星和AZD2281进行验证，如果在上述三种药物的刺激下，步骤四中得到的细胞的存活率比Chek2WT细胞的存活率更高，说明成功得到了PARP1抑制剂的耐药细胞株。
作为优选，本发明的PARP1抑制剂耐药细胞株的建立方法，还可以具有这样的特征：其中，所述B细胞为Eμ-Myc p19Arf-/-B细胞。
本发明还提供一种上述的耐药细胞株的建立方法建立的PARP1抑制剂耐药细胞株在药物筛选中的应用。
作为优选，上述的应用，还包括建立野生型和空载体对照组的步骤，具体步骤是：除了将Chek2-null的B细胞带有GFP的Chek2Y394C之外，另取两组Chek2-null的B细胞，分别感染Chek2WT以及Chek2VEC。
作为优选，上述的应用，还包括：使用不同浓度梯度的抗癌药物同时对Chek2Y394C细胞、Chek2WT细胞以及Chek2VEC细胞进行刺激，检测三种细胞的存活率，然后判断抗癌药物是否对Chek2Y394C细胞有杀伤作用。
发明的有益效果
本发明的PARP1抑制剂耐药细胞株的建立方法，CHEK2的Y390C突变通过影响细胞周期阻滞和凋亡，导致PARP1抑制剂的耐药，而重新过表达野生型CHEK2后，细胞对PARP1抑制剂的敏感性得到恢复。该细胞模型的建立对研究PARP1抑制剂耐药提供了体外细胞模型。另外，还可以利用此位点的基因突变来筛选抗肿瘤药物。
附图说明
图1是Chek2表达抑制后，细胞对表柔比星、顺铂以及PARP1抑制剂均表现出耐药的结果图；
图2是Chek2在使用shRNA处理后表达下调的结果图；
图3是Chek2的Y394C位点突变后的细胞对不同药物的敏感性结果；
图4是使用AZD2281处理后的细胞存活率结果；
图5是使用AZD2281处理一段时间后再用其它药物处理的细胞存活比例；
图6是Y394C细胞对底物P53 Ser20的磷酸化结果；
图7是Y394C细胞中Cdc25A的磷酸化结果。
具体实施方式
以下结合附图来说明本发明的具体实施方式。需要说明的是，本发明中未详细描述的实验，均按照生物化学的标准操作规程或者相应试剂盒的说明书进行操作。
实施例一：PARP1抑制剂耐药细胞株的建立方法
步骤一：首先采用逆转录RNAi方法。逆转录病毒载体为pMSCV-IRES-GFP。重组的病毒由感染质粒的Phonix细胞产生。对Eμ-Myc p19Arf-/-B细胞感染包含puro-chek2的shRNA的质粒，只含puromycin抗性，不含GFP，向B细胞中转入puro基因，同时将细胞自身表达的背景Chek2去掉。shRNA感染后进行嘌呤霉素(puromycin)筛选，筛选后得到Chek2-null的Bcell。Chek2-null表示敲低或者沉默Chek2。
3’端CHEK2 cDNA的发卡目的片段序列为：
CCAGAAACACATAATCATTAA。
步骤二：
由于人的Chek2 Y390位点在鼠中对应的为Y394位点，因此在本实施方式中构建小鼠的Chek2的cDNA时，应当将394位点的酪氨酸碱基突变为半胱氨酸即TAT→TGT；得到chek2Y394C的cDNA。
对Chek2-null的B细胞分别感染带有GFP基因的Chek2WT，Chek2Y394C和Chek2VEC，WT表示野生型，Y394C表示394C位点突变，VEC表示空载体，作为对照。对感染的Chek2WT，Chek2Y394C和Chek2VEC进行流式细胞分选，含GFP的细胞被筛选出来。含有GFP的细胞表示感染成功。
步骤三：药物敏感性检测：使用顺铂浓度为3μM、表柔比星浓度为15nM和AZD2281浓度为1nM分别对上述三种细胞进行刺激。结果如图3所示，图3中的MLP即无CHEK2表达的空载体细胞。结果表明：Chek2Y394C和Chek2WT细胞对PARP1抑制剂在内的药物敏感性明显不同，Chek2Y394C对AZD2281耐药，Chek2WT对药物敏感。
步骤四：细胞存活率检测：采用不同浓度的AZD2281处理CHEK2Y390C和Chek2WT细胞48小时，结果如图4所示，发现和Chek2WT相比，CHEK2Y394C细胞存活率更高。图4中AZD2281的浓度梯度是：0.7nM，2.1nM，3.5nM，4.9nM和7nM。
表1：细胞存活率检测结果
mock AZD1 AZD2 AZD3 AZD4 AZD5
chek-Y394C 88.87％ 61.02％ 58.83％ 40.02％ 18.91％ 0.87％
chek2-WT 88.87％ 52.70％ 22.62％ 17.31％ 0.88％ 0.77％
control 88.87％ 59.12％ 57.12％ 39.29％ 18.92％ 0.86％
步骤五：细胞存活率检测：使用PARP1抑制剂AZD2281对Chek2Y394C、Chek2WT、Chek2VEC三种细胞处理6小时后。结果如图5所示，不同Chek2表达状态的细胞存活比例不同，CHEK2Y394C细胞存活比例大，而Chek2WT细胞存活比例小。
构建CHEK2的shRNA，进行药物实验，选择DNA损伤的化疗药物顺铂Cisplatin，表柔比星Dox和PARP1抑制剂。具体流程如下：采用顺铂Cisp和表柔比星DOX，用48孔板进行试验，每孔加100μL B细胞培养基，0.2M B细胞和100μL的顺铂：20ug/ml，或者表柔比星：10ug/ml。结果提示CHEK2表达抑制后，细胞对表柔比星，顺铂以及PARP1抑制剂AZD2281均表现出耐药，如图1所示：验证了CHEK2 Y394位点突变确实对PARP1抑制剂形成了耐药性。
对感染的shRNA进行puromycin筛选后，抽提RNA反转录成cDNA，进行real-timePCR结果如图2所示：结果显示CHEK2确实有下调。
实施例二：PARP1抑制剂耐药细胞株在研究PARP1抑制剂抗药性中的应用举例。
如图6所示，Western的结果显示Y394C细胞对底物P53Ser20的磷酸化大大降低。如图7所示，表达Y394C的细胞对底物CDC25A的磷酸化大大降低。DNA双链受到损伤时，激活后的CHEK2可以磷酸化Cdc25A，从而增强Cdc25A的泛素化并导致其降解，这样Cdc25A不能去除CDK2上抑制其活性位点的磷酸基团，使得CDK2没有活性，发挥G1/S期检测点效应，阻断DNA合成。
因此，实验结果显示Y394C突变降低了CHEK2对DNA损伤药物的修复功能。在PARP1抑制剂AZD2281作用下，Y394C突变后细胞对p53的Ser20的磷酸化水平降低，同时我们在研究中发现，AZD2281处理6小时后，表达Y394C的细胞对底物CDC25A的磷酸化大大降低。因此，实验结果显示PARP1抑制剂治疗后，表达Y394C突变的细胞的G1→S细胞阻滞受到明显的影响。
总之，通过以上实验，证实了CHEK2 Y394C过表达的细胞对PARP1抑制剂AZD2281耐药，而CHEK2野生型的细胞对PARP1抑制剂敏感。通过该细胞模型，可以深入研究PARP1抑制剂耐药的机制，为PARP1抑制剂更好应用于临床提供研究基础。
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<110> 上海长海医院
<120> PARP1抑制剂耐药细胞株的建立方法及其应用
<130> JSP1712093
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> 人工序列
<400> 1
ccagaaacac ataatcatta a 21

Claims (6)

1.一种PARP1抑制剂耐药细胞株的建立方法，其特征在于，包括如下步骤：
步骤一：对B细胞感染puro-chek2载体，向B细胞中转入puro基因，同时将细胞自身表达的背景chek2去掉；
步骤二：感染后进行puromycin筛选，筛选后得到chek2沉默的B细胞；
步骤三：对Chek2-null的B细胞感染带有GFP的Chek2Y394C；
步骤四：筛选带有GFP标记的细胞，得到PARP1抑制剂耐药细胞株。
2.如权利要求1所述的PARP1抑制剂耐药细胞株的建立方法，其特征在于，还包括：
验证的步骤：对步骤四中得到的有GFP标记的细胞分别使用顺铂、表柔比星和AZD2281进行验证，如果在上述三种药物的刺激下，步骤四中得到的细胞的存活率比Chek2WT细胞的存活率更高，说明成功得到了PARP1抑制剂的耐药细胞株。
3.如权利要求1所述的PARP1抑制剂耐药细胞株的建立方法，其特征在于：
其中，所述B细胞为Eμ-Myc p19Arf-/-B细胞。
4.如权利要求1所述的PARP1抑制剂耐药细胞株在药物筛选中的应用。
5.如权利要求4所述的应用，其特征在于，还包括建立野生型和空载体对照组的步骤，具体步骤是：除了将Chek2-null的B细胞带有GFP的Chek2Y394C之外，另取两组Chek2-null的B细胞，分别感染Chek2WT以及Chek2VEC。
6.如权利要求5所述的应用，其特征在于，还包括：使用不同浓度梯度的抗癌药物同时对Chek2Y394C细胞、Chek2WT细胞以及Chek2VEC细胞进行刺激，检测三种细胞的存活率，然后判断抗癌药物是否对Chek2Y394C细胞有杀伤作用。
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