CN111235269A - Plin2及定量检测plin2的试剂的应用和试剂盒 - Google Patents

Plin2及定量检测plin2的试剂的应用和试剂盒 Download PDF

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CN111235269A
CN111235269A CN201811430343.6A CN201811430343A CN111235269A CN 111235269 A CN111235269 A CN 111235269A CN 201811430343 A CN201811430343 A CN 201811430343A CN 111235269 A CN111235269 A CN 111235269A
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plin2
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张丽华
赵宝锋
孙瑞
褚宏伟
杨开广
张玉奎
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Dalian Institute of Chemical Physics of CAS
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Abstract

本发明涉及PLIN2作为肿瘤耐药检测分子靶点的应用，本发明发现肿瘤耐药细胞株中PLIN2表达量上调，定量检测PLIN2可作为检测肿瘤耐药的用途。化疗仍是肿瘤治疗的主要手段之一，化疗药物耐药性是影响肿瘤患者治疗效果的重大障碍。本发明提供了一种可诊断肿瘤耐药的标志物。

Description

PLIN2及定量检测PLIN2的试剂的应用和试剂盒
技术领域
本发明属于分子生物学和肿瘤防治领域，更具体而言，本发明涉及预测肿瘤耐药性领域。本发明提供了PLIN2可作为一种检测肿瘤耐药性的应用，利用定量检测PLIN2分子的表达可设计出预测肿瘤耐药性的试剂。
背景技术
恶性肿瘤是危害人类健康的一类疾病，化疗是目前治疗恶性肿瘤的主要手段之一。然而在治疗过程中出现的肿瘤对化疗药物的耐药性是影响肿瘤治疗效果的主要障碍。因此，为了提高肿瘤化疗疗效，对肿瘤耐药机制进行研究找到肿瘤耐药关键靶点，并根据这些靶点进行设计提高肿瘤化疗敏感性的新药是本领域重点研究方向。
PLIN2又称脂肪分化相关蛋白，是脂滴包被蛋白PAT家族蛋白成员之一。人的PLIN2基因位于9p22.1-p22.3，基因总长5003bp。PLIN2蛋白的PAT结构域高度保守，位于N端区域的第1～115位氨基酸。PAT结构域与脂质蓄积以及脂滴、蛋白的稳定有关。在细胞脂质蓄积的过程中PLIN2的表达增加，一方面是因为脂质蓄积诱导其表达，另一方面是由于其定位于脂滴时蛋白质会更加稳定。PLIN2表达增加也会保护脂滴水解。PLIN2可以通过调节脂质储存以维持内质网稳态，进而促进肾癌细胞增殖，特别是在营养和氧气限制的条件下，促进肿瘤细胞的存活(Cancer Discov.2015；5(6):652-67.)。进一步的研究表明，PLIN2高表达是肾癌潜在的诊断标志物(Proteomics Clin Appl.2017；11(11-12).)，PLIN2敲除可以促进肾癌细胞增殖、侵袭和迁移(Int J Oncol.2018；53(1):137-147.)。然而PLIN2是否可以作为检测肿瘤耐药性方面的研究未见报道，本发明首次公开了PLIN2在肿瘤耐药细胞中高表达，定量检测PLIN2表达可以作为检测肿瘤耐药诊断的应用。
发明内容
基于上述肿瘤在化学药物治疗过程中出现的肿瘤耐药问题，本发明的一个目的是提供一种肿瘤耐药的关键基因靶点和蛋白质靶点，为检测肿瘤耐药性提供有效工具。
本发明首先通过定量蛋白质组学技术发现耐药相关靶点分子PLIN2，通过检测不同来源的肿瘤细胞及其耐药株，验证PLIN2在耐药细胞株中高表达，可以作为肿瘤耐药的检测靶点。
本发明中的实验操作通常是按照常规实验条件进行，也可按照实施例中实验条件操作，试剂使用可按照制造厂商所附说明使用。
基于此，本发明提供了一种肿瘤耐药的关键靶点，PLIN2。针对该靶点，本发明一个目的是设计PLIN2定量检测试剂盒。
在本发明的PLIN2定量检测试剂盒，所采用定量检测方法为RNA转录水平上定量检测PLIN2表达水平和蛋白质表达水平上检测PLIN2蛋白质表达水平。
本发明的有益效果是：
由于目前化疗仍是大部分肿瘤的主要治疗手段，肿瘤化疗耐药性现已成为影响肿瘤患者预后的重要因素。本发明公开了一个特异性的分子标志物PLIN2，定量检测PLIN2表达可作为预测肿瘤耐药的重要分子标志物。
具体实施形式
下面结合具体实施例，具体阐述本发明。下列实施例中，未注明的具体条件的实验方法，通常是按照常规条件，如Sambrook等人著，分子克隆：实验室指南(New York：ColdSpring Harbor Laboratory Press,1989)中所述条件，或按照制造厂商所建议的条件。所用溶液中百分比如无特殊说明均为体积比。
实施例1.PLIN2在卵巢癌顺铂耐药细胞中蛋白质水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，首先通过蛋白质印迹法(western blot)检测了卵巢癌细胞系SKOV3及其化疗药物顺铂耐药细胞系SKOV3/DDP中的PLIN2蛋白质表达水平。
RIPA裂解液：
50mM Tris(pH 7.4)，150mM NaCl，1％NP-40，0.5％sodium deoxycholate，0.1％SDS，以及1％蛋白酶抑制剂。
试验方法：
将一个长满15cm培养皿的细胞用PBS(pH 7.4)(购买于美国Gibco公司)洗3次，加入300μl的RIPA裂解液(购买于南京凯基生物科技发展有限公司)，冰上裂解细胞30分钟，12000g离心15min去除沉淀获得蛋白质上清液。用BCA试剂(购买于南京凯基生物科技发展有限公司)测定蛋白质浓度。每个蛋白样品上样量60μg，PLNE2抗体购自美国GeneTex公司，采用蛋白质印迹法western blot(参考《分子克隆》)检测蛋白质表达水平。PLIN2的抗体用溶解了5％的脱脂奶粉的TBST缓冲液1:500稀释。20×TBST缓冲液用180gNaCl和122gTris(购买于美国Sigma公司)溶解于800ml的水，调节pH到7.6，最后定容至1000ml。Biorad电泳仪，Biorad转膜仪(购买于美国Biorad公司)PLIN2的抗体购买于美国Gene Tex公司，loading buffer,PVDF膜(购买于南京凯基生物科技发展有限公司)
试验结果：
结果表明PLIN2在卵巢癌顺铂耐药细胞系中蛋白质表达水平均高于其对照非耐药细胞系。而且表达差异在2倍以上,且差异具有统计学意义(P<0.05)。
实施例2.PLIN2在宫颈癌顺铂耐药细胞中RNA转录水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，首先通过PCR方法检测了宫颈癌细胞系SiHa及其化疗药物顺铂耐药细胞系SiHa/DDP中的RNA转录水平的表达。
检测方法：
1.细胞RNA提取
首先收取细胞2.5×106个，移入1.5mlRNase-free的EP管中，加入1mlTrizol,(购买于美国Invitrogen公司)均匀混合以后再室温下静止5min。加入0.2ml的氯仿震荡15s后在室温下静置2min，在低温离心机中4℃离心，12000g×15min后取出上清。加入0.5ml的异丙醇，均匀混合后在室温下静置10min。在低温离心机中4℃离心，12000g×15min后去除上清。加入1ml75％的乙醇轻轻洗涤并在低温离心机下7500g×5min后弃去上清。晾干后加入适当的DEPC水溶解，测定RNA浓度，稀释RNA样品至同一浓度并使其浓度不能高于500ng/μl。获取之后保存于-80℃备用。
2.RNA反转录
RNA反转录试剂盒购自TAKARA公司，按照TAKARA公司提供的反转录试剂盒说明书进行。
3.实时定量PCR检测PLIN2表达水平
PLIN2引物序列：正向:CACCCTCCTGTCCAACATCC
反向：TGGCTGCTCTTGTCCATCTC
实时定量PCR试剂盒购自TAKARA公司，按照TAKARA公司提供的试剂盒说明书进行操作。反应条件包括预变性过程：在95℃下10s进行1个cycle，然后紧接着PCR反应过程：从95℃,10s,降至60℃，30s,一共40个cycle。
试验结果：
结果表明PLIN2在宫颈癌顺铂耐药细胞中RNA表达水平明显高于其对照非耐药细胞系，且差异具有统计学意义(P<0.05)。
实施例3.PLIN2在肺癌吉非替尼耐药细胞中蛋白质水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，发明人首先通过蛋白质印迹法(western blot)检测了肺癌细胞系PC-9及其化疗药物吉非替尼耐药细胞系PC-9/GR中的PLIN2蛋白质表达水平。
RIPA裂解液：
50mM Tris(pH 7.4)，150mM NaCl，1％NP-40，0.5％sodium deoxycholate，0.1％SDS，以及1％蛋白酶抑制剂。
试验方法：
将一个长满15cm培养皿的细胞用PBS(pH 7.4)(购买于美国Gibco公司)洗3次，加入300μl的RIPA裂解液(购买于南京凯基生物科技发展有限公司)，冰上裂解细胞30分钟，12000g离心15min去除沉淀获得蛋白质上清液。用BCA试剂(购买于南京凯基生物科技发展有限公司)测定蛋白质浓度。每个蛋白样品上样量60μg，PLIN2抗体购自美国GeneTex公司，用蛋白质印迹法western blot(参考《分子克隆》)检测蛋白质表达水平。PLIN2的抗体用溶解了5％的脱脂奶粉的TBST缓冲液1:500稀释。20×TBST缓冲液用180gNaCl和122gTris(购买于美国Sigma公司)溶解于800ml的水，调节pH到7.6，最后定容至1000ml。Biorad电泳仪，Biorad转膜仪(购买于美国Biorad公司)PLIN2的抗体购买于美国Gene Tex公司，loadingbuffer,PVDF膜(购买于南京凯基生物科技发展有限公司)
试验结果：
结果表明PLIN2在肺癌顺铂耐药细胞系中蛋白质表达水平均于其对照非耐药细胞系。而且表达差异在2倍以上，且差异具有统计学意义(P<0.05)。
实施例4.PLIN2在肝癌阿霉素耐药细胞中蛋白质水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，发明人首先通过蛋白质印迹法(western blot)检测了肝癌细胞系HEPG2及其化疗药物阿霉素耐药细胞系HEPG2/ADR中的PLIN2蛋白质表达水平。
RIPA裂解液：
50mM Tris(pH 7.4)，150mM NaCl，1％NP-40，0.5％sodium deoxycholate，0.1％SDS，以及1％蛋白酶抑制剂。
试验方法：
将一个长满15cm培养皿的细胞用PBS(pH 7.4)(购买于美国Gibco公司)洗3次，加入300μl的RIPA裂解液(购买于南京凯基生物科技发展有限公司)，冰上裂解细胞30分钟，12000g离心15min去除沉淀获得蛋白质上清液。用BCA试剂(购买于南京凯基生物科技发展有限公司)测定蛋白质浓度。每个蛋白样品上样量60μg,用蛋白质印迹法western blot(参考《分子克隆》)检测蛋白质表达水平。PLIN2的抗体用溶解了5％的脱脂奶粉的TBST缓冲液1:500稀释。20×TBST缓冲液用180gNaCl和122gTris(购买于美国Sigma公司)溶解于800ml的水，调节pH到7.6，最后定容至1000ml。Biorad电泳仪，Biorad转膜仪(购买于美国Biorad公司)PLIN2的抗体购买于美国Gene Tex公司，loading buffer,PVDF膜(购买于南京凯基生物科技发展有限公司)。
试验结果：
结果表明PLIN2在肝癌阿霉素耐药细胞系中蛋白质表达水平高于其对照非耐药细胞系。且差异具有统计学意义(P<0.05)。
实施例5.PLIN2在宫颈癌阿霉素耐药细胞中RNA转录水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，发明人首先通过PCR方法检测了宫颈癌细胞系HeLa及其化疗药物阿霉素耐药细胞系HeLa/ADR中的RNA转录水平的表达。
检测方法：
1.细胞RNA提取
首先收取细胞2.5×106个，移入1.5mlRNase-free的EP管中，加入1mlTrizol,(购买于美国Invitrogen公司)均匀混合以后再室温下静止5min。加入0.2ml的氯仿震荡15s后在室温下静置2min，在低温离心机中4℃离心，12000g×15min后取出上清。加入0.5ml的异丙醇，均匀混合后在室温下静置10min。在低温离心机中4℃离心，12000g×15min后去除上清。加入1ml75％的乙醇轻轻洗涤并在低温离心机下7500g×5min后弃去上清。晾干后加入适当的DEPC水溶解，测定RNA浓度，稀释RNA样品至同一浓度并使其浓度不能高于500ng/μl。获取之后保存于-80℃备用。
2.RNA反转录
RNA反转录试剂盒购自TAKARA公司，按照TAKARA公司提供的反转录试剂盒说明书进行。
3.实时定量PCR检测PLIN2表达水平
PLIN2引物序列：正向:CACCCTCCTGTCCAACATCC
反向：TGGCTGCTCTTGTCCATCTC
实时定量PCR试剂盒购自TAKARA公司，按照TAKARA公司提供的试剂盒说明书进行操作。反应条件包括预变性过程：在95℃下10s进行1个cycle，然后紧接着PCR反应过程：从95℃,10s,降至60℃，30s,一共40个cycle。
试验结果：
结果表明PLIN2在宫颈癌阿霉素耐药细胞中RNA表达水平高于其对照非耐药细胞系，且差异具有统计学意义(P<0.05)。
实施例6.PLIN2在肝癌5氟尿嘧啶耐药细胞中蛋白质水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，发明人首先通过蛋白质印迹法(western blot)检测了肝癌细胞系Bel7402及其化疗药物5-Fu耐药细胞系Bel/5-Fu中的PLIN2蛋白质表达水平。
RIPA裂解液：
50mM Tris(pH 7.4)，150mM NaCl，1％NP-40，0.5％sodium deoxycholate，0.1％SDS，以及1％蛋白酶抑制剂。
试验方法：
将一个长满15cm培养皿的细胞用PBS(pH 7.4)(购买于美国Gibco公司)洗3次，加入300μl的RIPA裂解液(购买于南京凯基生物科技发展有限公司)，冰上裂解细胞30分钟，12000g离心15min去除沉淀获得蛋白质上清液。用BCA试剂(购买于南京凯基生物科技发展有限公司)测定蛋白质浓度。每个蛋白样品上样量60μg，用蛋白质印迹法western blot(参考《分子克隆》)检测蛋白质表达水平。PLIN2的抗体用溶解了5％的脱脂奶粉的TBST缓冲液1:500稀释。20×TBST缓冲液用180gNaCl和122gTris(购买于美国Sigma公司)溶解于800ml的水，调节pH到7.6，最后定容至1000ml。Biorad电泳仪，Biorad转膜仪(购买于美国Biorad公司)PLIN2的抗体购买于美国Gene Tex公司，loading buffer,PVDF膜(购买于南京凯基生物科技发展有限公司)。
试验结果：
结果表明PLIN2在肝癌5氟尿嘧啶耐药细胞系中蛋白质表达水平高于其对照非耐药细胞系，且差异具有统计学意义(P<0.05)。
实施例7.PLIN2在肺癌紫杉醇耐药细胞中RNA转录水平高表达
为研究PLIN2在正常肿瘤细胞和耐药细胞中的表达水平，发明人首先通过PCR方法检测了肺癌细胞系A549及其化疗药物紫杉醇耐药细胞系A549/taxol中的RNA转录水平的表达。
检测方法：
1.细胞RNA提取
首先收取细胞2.5×106个，移入1.5mlRNase-free的EP管中，加入1mlTrizol,(购买于美国Invitrogen公司)均匀混合以后再室温下静止5min。加入0.2ml的氯仿震荡15s后在室温下静置2min，在低温离心机中4℃离心，12000g×15min后取出上清。加入0.5ml的异丙醇，均匀混合后在室温下静置10min。在低温离心机中4℃离心，12000g×15min后去除上清。加入1ml75％的乙醇轻轻洗涤并在低温离心机下7500g×5min后弃去上清。晾干后加入适当的DEPC水溶解，测定RNA浓度，稀释RNA样品至同一浓度并使其浓度不能高于500ng/μl。获取之后保存于-80℃备用。
2.RNA反转录
RNA反转录试剂盒购自TAKARA公司，按照TAKARA公司提供的反转录试剂盒说明书进行。
3.实时定量PCR检测PLIN2表达水平
PLIN2引物序列：正向:CACCCTCCTGTCCAACATCC
反向：TGGCTGCTCTTGTCCATCTC
实时定量PCR试剂盒购自TAKARA公司，按照TAKARA公司提供的试剂盒说明书进行操作。反应条件包括预变性过程：在95℃下10s进行1个cycle，然后紧接着PCR反应过程：从95℃,10s,降至60℃，30s,一共40个cycle。
试验结果：
结果表明PLIN2在肺癌紫杉醇耐药细胞中RNA表达水平高于其对照非耐药细胞系。
综上所述，PLIN2在各组织来源的肿瘤耐药细胞株中的表达均高于其对照非耐药细胞株，定量检测PLIN2能够作为检测肿瘤耐药的用途。据此可制备相应肿瘤耐药检测试剂盒，该些试剂盒中含有定量检测PLIN2的RNA转录水平的试剂、定量检测PLIN2的蛋白表达水平的试剂等。通过检测患者肿瘤组织中PLIN2的RNA和蛋白质表达水平，从而判断该患者是否对肿瘤化疗药物治疗具有化疗耐药性。进而通过抑制患者肿瘤组织中的PLIN2，以增强肿瘤的化疗治疗效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (8)

1.PLIN2的应用，其特征在于，PLIN2作为检测肿瘤耐药分子靶点的应用。
2.定量检测PLIN2的试剂在制备肿瘤耐药检测试剂盒中的应用。
3.如权利要求1或2所述的应用，其特征在于，所述肿瘤耐药药物为顺铂、吉非替尼、阿霉素、紫杉醇、5-氟尿嘧啶(5-Fu)中的一种或二种以上。
4.如权利要求1或3所述的应用，其特征在于，所述肿瘤包括肝癌、卵巢癌、肺癌、宫颈癌中的一种或二种以上。
5.如权利要求2所述的应用，其特征在于，所述定量检测PLIN2的试剂选自定量检测PLIN2的RNA转录水平试剂或定量检测PLIN2的蛋白质表达水平的试剂。
6.一种肿瘤细胞PLIN2定量检测试剂盒。
7.如权利要求6所述的PLIN2定量检测试剂盒，其特征在于，
所述定量检测PLIN2的试剂选自定量检测PLIN2的RNA转录水平试剂或定量检测PLIN2的蛋白质表达水平的试剂。
8.如权利要求6或7所述的PLIN2定量检测试剂盒，其特征在于，
所采用定量检测方法为RNA转录水平上定量检测PLIN2表达水平和蛋白质表达水平上检测PLIN2蛋白质表达水平。
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