CN116287275B - Ptgr1作为cdk4/6抑制剂与二甲双胍联合用药指导标志物的应用 - Google Patents
Ptgr1作为cdk4/6抑制剂与二甲双胍联合用药指导标志物的应用 Download PDFInfo
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Abstract
本发明属于生物医药以及肿瘤相关领域,具体涉及PTGR1作为CDK4/6抑制剂与二甲双胍联合用药指导标志物的应用,本发明以PTGR1作为CDK4/6抑制剂与二甲双胍联合用药指导标志物,相对于盲目给予二甲双胍作辅助治疗的方式,本发明为前列腺癌患者是否需要使用二甲双胍联合CDK4/6抑制剂作辅助内分泌治疗提供依据,细胞周期依赖性激酶4/6抑制剂能增加PTGR1高表达前列腺癌细胞对二甲双胍的敏感性,有利于提高药物的使用效率,降低其毒副作用。
Description
技术领域
本发明属于生物医药以及肿瘤相关领域,具体涉及PTGR1作为CDK4/6抑制剂与二甲双胍联合用药指导标志物的应用。
背景技术
前列腺癌是男性中最常见和第二致死性的恶性肿瘤,目前临床上前列腺癌的治疗手段以手术治疗配合雄激素剥夺治疗(也称为去势治疗)、放射治疗为主。虽然局限性前列腺癌患者经治疗后的五年总体生存率接近100%,但是一旦疾病发生进展则骤降至30%,相比局限性前列腺癌,临床上对于进展性前列腺癌(局部进展期前列腺癌、转移性前列腺癌)的治疗效果不佳,因此对于前列腺癌的治疗方案需要不断优化,也需要我们继续探索新的治疗手段提高临床疗效。
为了适应其快速增殖和转移的能量需求肿瘤细胞具有独特的代谢重编程特征。前列腺癌在从上皮内瘤变发展到转移过程中,细胞的能量来源不断变化。例如:在健康前列腺细胞中三羧酸循环由于其原料柠檬酸盐被用于前列腺液合成而被抑制。侵袭性前列腺癌细胞中的能量产生主要通过三羧酸循环和氧化磷酸化实现。在转移性前列腺癌细胞中氧化磷酸化水平降低,而糖酵解水平增加。总结先前研究结果发现,不同阶段的前列腺癌可能依赖于不同的代谢途径来产生能量,这为精准治疗提供了潜在的靶点。
代谢治疗是现今的研究热点,许多临床试验也表明干扰肿瘤细胞代谢疗法对患者有积极的成效。其中,二甲双胍作为现今一线口服降糖药物,因其有效的抑制糖酵解能力、副作用少和成本低的特点,被广泛应用于肿瘤治疗的研究。二甲双胍已被证明可直接作用于肿瘤细胞内线粒体,影响呼吸电子链从而改变ATP/ADP和NADH/NAD+比值,降低肿瘤细胞的能量代谢和抑制糖酵解。除此之外还能调控AMPK、mTOR和IGF等重要细胞信号通路,从整体上达到抑制肿瘤细胞周期的效果。虽然,二甲双胍被大量研究证明能抑制肿瘤生长,但它目前还不是临床上用于抗肿瘤治疗的指南用药。另一方面,由于肿瘤细胞异质性的存在,肿瘤细胞对药物作用的应答反应是不一致的,长期服用甚至导致细胞对药物产生抗性。因此,如何开展应用和提高二甲双胍在肿瘤治疗上的效果仍是目前的难题。
发明内容
针对现有技术存在的问题,本发明目的在于提供PTGR1作为CDK4/6抑制剂与二甲双胍联合用药指导标志物中的应用。
基于上述目的,本发明采用的技术方案如下:
第一方面,本发明提供PTGR1的检测试剂在制备指导治疗前列腺癌用药的试剂盒中的应用。
前列腺素还原酶1(PTGR1)属于中链脱氢酶/还原酶超家族,研究发现PTGR1在包括前列腺癌在内的多种恶性肿瘤中表达上调,而且PTGR1表达升高的肿瘤患者预后不良。我们随后通过对比多个前列腺癌公共数据库进行分析,发现PTGR1的mRNA表达量升高与前列腺癌患者的无生化复发生存率呈负相关,即PTGR1高表达的前列癌患者预后不良,提示PTGR1具有促进前列腺癌进展的潜在作用。此外,PTGR1还有调节细胞周期和抑制氧化应激的作用。
本发明首次发现单纯的二甲双胍用药使得细胞产生抗药性,对于PTGR1高表达的前列腺癌肿瘤细胞尤其如此,而CDK4/6抑制剂能增加PTGR1高表达前列腺癌肿瘤细胞对二甲双胍的敏感性,故而本发明以PTGR1作为CDK4/6抑制剂与二甲双胍联合用药指导标志物,可为前列腺癌患者是否需要使用二甲双胍联合CDK4/6抑制剂作辅助内分泌治疗提供依据。
优选地,PTGR1的检测试剂为检测PTGR1的mRNA的检测试剂或为检测PTGR1蛋白的检测试剂。
优选地,检测PTGR1的mRNA的检测试剂包括PTGR1的检测引物,PTGR1的检测引物包括上游引物和下游引物:上游引物序列如SEQ ID NO.3所示,下游引物序列如SEQ ID NO.4所示。
优选地,检测PTGR1蛋白的检测试剂包括一抗和二抗,其中,一抗为兔来源PTGR1多克隆抗体,二抗为羊抗兔二抗。
本发明通过对PTGR1的mRNA及蛋白的检测,分析其是否高表达,进而为前列腺癌的用药指导提供依据。
优选地,前列腺癌为局部进展期前列腺癌(肿瘤突破前列腺包膜但未发生转移)或转移性前列腺癌。
优选地,前列腺癌组织中PTGR1的高表达指示治疗前列腺癌用药为CDK4/6抑制剂与二甲双胍联合用药。
第二方面,本发明提供一种PTGR1的检测试剂盒,所述试剂盒用于检测PTGR1的mRNA的表达量或检测PTGR1蛋白的表达量;
所述检测PTGR1的mRNA的表达量的试剂盒包括PTGR1的检测引物,所述PTGR1的检测引物包括如下:上游引物序列如SEQ ID NO.3所示,下游引物序列如SEQ ID NO.4所示;
所述检测PTGR1蛋白的表达量的试剂盒包括一抗和二抗,所述一抗为兔来源PTGR1多克隆抗体,所述二抗为羊抗兔二抗。
第三方面,本发明提供CDK4/6抑制剂与二甲双胍在联合用于制备治疗前列腺癌药物中的应用。
第四方面,本发明提供一种用于治疗前列腺癌的药物,所述药物包括CDK4/6抑制剂与二甲双胍。
与现有技术相比,本发明的有益效果如下:
(1)相对于盲目给予二甲双胍作辅助治疗的方式,本发明以PTGR1作为CDK4/6抑制剂与二甲双胍联合用药指导标志物,可为前列腺癌患者是否需要使用二甲双胍联合CDK4/6抑制剂作辅助内分泌治疗提供依据;有利于提高药物的使用效率,而且细胞周期依赖性激酶4/6抑制剂能增加PTGR1高表达前列腺癌细胞对二甲双胍的敏感性,提高药物的使用效率,可适当下调二甲双胍的用量,进一步降低其对胃肠道的毒副作用。
(2)本发明提供的用于检测PTGR1表达量的试剂盒,可实现体外精确检测患者前列腺癌组织中PTGR1的mRNA和蛋白表达情况及阳性表达程度,实验结果证明PTGR1表达程度与二甲双胍敏感性成反相关;使用本试剂盒对PTGR1蛋白进行检测,染色强度越强表明对二甲双胍治疗敏感性越低;使用本试剂盒对PTGR1蛋白进行检测,染色强度越强,可为前列腺癌患者是否需要使用二甲双胍联合CDK4/6抑制剂作辅助内分泌治疗提供依据。
(3)本发明首次将细胞周期依赖性激酶4/6抑制剂结合二甲双胍联合用于治疗前列腺癌,尤其是对进展性前列腺癌如局部进展期前列腺癌(肿瘤突破前列腺包膜但未发生转移)或转移性前列腺癌具有更优用药指导作用。细胞周期依赖性激酶4/6抑制剂结合二甲双胍治疗还能在一定程度上补充PTGR1高表达的负面效果;在经济效益方面,CDK4/6抑制剂已被证明在多种恶性肿瘤的临床前模型具有抑制肿瘤生长的作用,且目前在乳腺癌临床治疗中具有良好的疗效,本试剂盒能拓展其在癌症治疗中的应用范围。
附图说明
图1为长期二甲双胍刺激前列腺癌DU145(上)以及22RV1(下)细胞后进行裸鼠皮下移植瘤实验结果;
图2为前列腺癌DU145与22RV1细胞株对二甲双胍产生耐药性后PTGR1的mRNA(上)以及蛋白(下)表达鉴定结果图;
图3为高表达PTGR1的DU145与22RV1细胞株的mRNA(左)以及蛋白(右)鉴定结果图;
图4为高表达PTGR1的DU145与22RV1细胞株在不同二甲双胍浓度刺激条件下的细胞存活率实验的结果图;
图5为高表达PTGR1的DU145与22RV1细胞株在二甲双胍刺激条件下细胞周期实验结果图;
图6为前列腺癌旁组织(左)和前列腺癌(右)的肿瘤组织蛋白免疫印迹实验结果图;
图7为四个公共数据库中PTGR1高表达患者以及与低表达患者无生化复发生存期结果图;
图8为PTGR1表达阴性(左)和阳性(右)的肿瘤组织免疫组化结果图;
图9为高表达PTGR1的前列腺癌细胞在三种CDK4/6抑制剂刺激条件下的生长曲线结果图;
图10为PTGR1高表达细胞对二甲双胍联合CDK4/6抑制剂治疗的生长曲线结果图;
图11为细胞周期依赖性激酶4/6抑制剂联合二甲双胍作前列腺癌辅助内分泌治疗的具体分子机制。
具体实施方式
本发明提供了一种应用于二甲双胍辅助癌症治疗的检测试剂盒,通过对前列腺癌组织进行PTGR1的mRNA与蛋白阳性程度的检测,可初步评估前列腺癌患者是否需要使用二甲双胍联合CDK4/6抑制剂作辅助内分泌治疗提供依据,为除抗雄激素治疗外的内分泌治疗提供新的治疗靶点。本发明检测试剂盒对临床上获得的前列腺癌组织均可进行检测,可以通过诊断性前列腺穿刺检查或手术获得前列腺病灶组织。并且本试剂盒可通过体外实验进行检测,对患者危险性少,无毒副作。在一些实施例中,用于二甲双胍辅助癌症治疗的检测试剂盒包含PTGR1多克隆一抗,特异性高,而且实验步骤简单、快捷,十分高效,可对PTGR1的阳性颜色强度进行评分,初步预测二甲双胍对前列腺癌患者疗效。
在一些实施例中,本发明提供的PTGR1 mRNA以及蛋白表达检测试剂盒,可实现体外精确检测患者前列腺癌组织中的PTGR1 mRNA和蛋白表达情况及阳性表达程度。相对于传统盲目给予二甲双胍作辅助治疗的方式,检测PTGR1的表达量能为是否使用二甲双胍联合CDK4/6抑制剂作辅助内分泌治疗提供有力的依据,有利于提高药物的使用效率。在经济效益方面,CDK4/6抑制剂已被证明在多种恶性肿瘤的临床前模型具有抑制肿瘤生长的作用,且目前在乳腺癌临床治疗中具有良好的疗效,本试剂盒能拓展其在癌症治疗中的应用范围。因此,本专利对临床上内分泌治疗前列腺癌提供新的治疗靶点产生重大意义。
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的实验方法如无特殊说明,均为常规方法;本发明中的试剂浓度均为质量浓度;所用的材料、试剂等,如无特别说明,本发明中的试剂或材料均可从市场上或其它公开渠道获得。
实施例1
首先在体外利用两种前列腺癌细胞系DU145-WT、22RV1-WT分别构建持续二甲双胍治疗的细胞系模型,持续刺激培养6个月后,得到耐药组细胞DU145-MetR、22RV1-MetR。
我们使用DU145和22RV1的野生组细胞(DU145-WT、22RV1-WT)与耐药组细胞(DU145-MetR、22RV1-MetR)构建裸鼠皮下移植瘤模型,并在接种后同时采用250mg/kg(1.25mg/mL)二甲双胍(简称:Met)喂养,每隔三天记录小鼠肿瘤大小,结果显示DU145野生组细胞的皮下肿瘤体积在第30天后明显小于耐药组细胞,两者差异具有统计学意义(图1)。22RV1野生组细胞的皮下肿瘤体积在第18天后明显小于耐药组细胞,两者差异具有统计学意义(图1),证明持续二甲双胍治疗后前列腺癌细胞对二甲双胍产生抵抗性。
随后设计PTGR1和内参ACTB的引物序列备用:
ACTB:
Forward primer(5'-3'):AGCGAGCATCCCCCAAAGTT(SEQ ID NO.1);
Reverse primer(5'-3'):GGGCACGAAGGCTCATCATT(SEQ ID NO.2);
PTGR1:
Forward primer(5'-3'):GACAACGCACTCCATTTCTGA(SEQ ID NO.3);
Reverse primer(5'-3'):TGCTGCATTAACCATCACTGTT(SEQ ID NO.4)。
材料准备充分后利用逆转录试剂盒(Thermo Fisher Scientific)分别对4种细胞株的总RNA进行逆转录(总反应体系20μl,分别是11μl的DEPC水含1μg RNA、Oligo(dt)1μl、5×buffer 4μl、核糖核苷酸酶抑制剂1μl、dNTP MIX 2μl、M-Mulu逆转录酶1μl)及实时荧光定量聚合酶链式反应(25μl的反应体系,分别是Primer 1 1μl、Primer 2 1μl、2×Master12.5μl、ddH2O补至总体系25μl)检测PTGR1的mRNA表达量(图2,左)。
利用蛋白免疫印迹实验检测4种细胞株PTGR1蛋白表达量。操作方法如下:首先提取蛋白,按照RIPA:100x PMSF:Loading buffer=100:1.25:25的比例配制裂解液,充分裂解细胞,100℃水浴锅中加热20分钟,利用蛋白电泳分离目的蛋白,准备转印夹,转膜液,海绵垫及滤纸,待电泳结束后,设置转膜电流为280-320mA转膜时间65-85分钟。蛋白转印结束后洗涤3遍,利用脱脂牛奶封闭120分钟,利用兔来源PTGR1多克隆抗体(ab222818,Abcam)4℃孵育过夜。一抗孵育结束后,洗涤3遍,在摇床上室温利用羊抗兔二抗500μg/ml(ab205718,abcam)孵育,洗涤。最后配置化学底物发光液进行化学发光(图2,右)。结果证明前列腺癌细胞DU145和22RV1对二甲双胍产生抵抗性后PTGR1的mRNA以及蛋白表达量升高。
实施例2
通过包装过表达质粒载体(嘌呤霉素,绿色荧光蛋白元件和PTGR1 ORF序列),利用慢病毒转染技术构建了PTGR1高表达的DU145、22RV1的细胞株(DU145-PTGR1,22RV1-PTGR1)和二者的对照组细胞株(图2,DU145-CG,22RV1-CG),并通过qRT-PCR实验(实时荧光定量聚合酶链式反应)检测四种细胞株的PTGR1的mRNA表达量,包括步骤如下:
材料准备充分后利用逆转录试剂盒(Thermo Fisher Scientific)分别对4种细胞株的总RNA进行逆转录(总反应体系20μl,分别是11μl的DEPC水含1μg RNA、Oligo(dt)1μl、5×buffer 4μl、核糖核苷酸酶抑制剂1μl、dNTP MIX 2μl、M-Mulu逆转录酶1μl)及聚合酶链反应PCR(25μl的反应体系,分别是Primer 1 1μl、Primer2 1μl、2×Master 12.5μl、ddH2O补至总体系25μl)检测PTGR1的mRNA表达量(图3,左)。
利用蛋白免疫印迹实验检测4种细胞株PTGR1蛋白表达量。操作方法如下:首先提取蛋白,按照RIPA:100x PMSF:Loading buffer=100:1.25:25的比例配制裂解液,充分裂解细胞,100℃水浴锅中加热20分钟,利用蛋白电泳分离目的蛋白,准备转印夹,转膜液,海绵垫及滤纸,待电泳结束后,设置转膜电流为280-320mA转膜时间65-85分钟。蛋白转印结束后洗涤3遍,利用脱脂牛奶封闭120分钟,利用兔来源PTGR1多克隆抗体(ab222818,Abcam)4℃孵育过夜。一抗孵育结束后,洗涤3遍,在摇床上室温利用羊抗兔二抗500μg/ml(ab205718,abcam)孵育,洗涤。最后配置化学底物发光液进行化学发光(图3,右)。结果证明前列腺癌DU145及22RV1高表达PTGR1细胞构建成功。
实施例3
利用实施例2成功构建的PTGR1高表达前列腺癌细胞株(DU145-PTGR1、22RV1-PTGR1)和对照组前列腺癌细胞株(DU145-CG、22RV1-CG)进行细胞CCK-8增殖实验,取对数生长期细胞消化,取96孔板,根据细胞种类设置每孔加入细胞的数量:3000个/孔;每孔加入100μL全血清培养基,将细胞稀释成所需的浓度,周围一圈细胞孔因挥发量比较大故一般不接种细胞,用200μL dPBS代替。待细胞贴壁后加入二甲双胍刺激,同时将培养基体积补充至200μL,待到刺激时间结束后配置CCK-8工作液:CCK-8:全血清RPMI 1640=1:9。加入工作液,待反应时间结束后用酶标仪测定在450nm处OD值。结果显示PTGR1高表达细胞对二甲双胍抵抗性更强(图4)。
实施例4
利用实施例2成功构建的PTGR1高表达前列腺癌细胞株(DU145-PTGR1、22RV1-PTGR1)和对照组前列腺癌细胞株(DU145-CG、22RV1-CG)进行细胞周期实验,收集对数生长期细胞后,利用细胞周期试剂盒对细胞进行处理(联科生物,CCS012),利用BDFACSAriaTMFusion流式细胞仪样本上机分析,拟合周期曲线,最后绘制统计图,结果证明二甲双胍对PTGR1高表达前列腺癌细胞细胞周期的抑制作用降低(图5)。
实施例5
本实施例提供一种检测PTGR1蛋白表达量的试剂盒,该试剂盒内容包括:1.兔来源PTGR1多克隆抗体(ab222818,Abcam);2.羊血清1ml(Gibco);3.羊抗兔二抗500μg/ml(ab205718,abcam);用蛋白免疫印迹实验检测前列腺癌组织蛋白表达量。
操作方法如下:首先提取前列腺癌组织(PCa)或癌旁组织(Adj.T),按照RIPA:100xPMSF:Loading buffer=100:1.25:25的比例配制裂解液,充分研磨裂解组织,100℃水浴锅中加热20分钟,利用蛋白电泳分离目的蛋白,准备转印夹,转膜液,海绵垫及滤纸,待电泳结束后,设置转膜电流为280-320mA转膜时间65-85分钟。蛋白转印结束后洗涤3遍,利用脱脂牛奶封闭120分钟,利用兔来源PTGR1多克隆抗体(ab222818,Abcam)4℃孵育过夜。一抗孵育结束后,洗涤3遍,在摇床上室温利用羊抗兔二抗500μg/ml(ab205718,abcam)孵育,洗涤。最后配置化学底物发光液进行化学发光(图6),结果显示前列腺癌旁组织(左)中PTGR1表达量较前列腺癌组织(右)低。图7展示了高表达PTGR1患者在公共数据库中无生化复发生存时间较短。
实施例6
本实施例提供一种检测PTGR1蛋白表达量的试剂盒,该试剂盒内容包括:1.兔来源PTGR1多克隆抗体(ab222818,Abcam);2.羊血清1ml(Gibco);3.羊抗兔二抗500μg/ml(ab205718,abcam);4.EDTA抗原修复液100ml(Beyotime);5.Biotin-Avidin封闭试剂盒(Vector La bora tories);6.ABC系统复合物(Vector Laboratories);7.DAB染色剂(DakoProducts);8.TBS漂洗缓冲液(TBST)(BOSTER Biological Technology)。
以手术获取前列腺癌组织作石蜡切片为例,采用上述试剂盒检测PTGR1蛋白表达情况,其具体步骤如下:
1.组织包埋,石蜡切片切好后,置于60摄氏度烘箱中烘烤30-45分钟;
2.人工操作对烘烤后的切片进行脱蜡水化(自上而下):
(1)二甲苯Ⅰ---5mins;
(2)二甲苯Ⅱ---5mins;
(3)酒精100%Ⅰ---5mins;
(4)酒精100%Ⅱ---5mins;
(5)酒精95%Ⅰ---5mins;
(6)酒精95%Ⅱ---5mins;
(7)酒精70%---2mins;
(8)H2OⅠ---2mins;
(9)H2OⅡ---2mins;
3.将切片慢慢地置于EDTA抗原修复液中进行抗原修复,再在高压蒸汽锅中加热15min,让其在室温下自然冷却1.5-2小时。
4.把切片放到蒸馏水里并在摇床上洗3次,每次2分钟。
5.把切片放到TBST里并在摇床上洗3次,每次5分钟。
6.把切片放到3%的H2O2中在摇床上室温孵育20分钟。
7.把切片放到蒸馏水里并在摇床上洗3次,每次2分钟。
8.把切片放到TBST里并在摇床上洗3次,每次5分钟。
9.用阻水笔小心地把组织圈住,每张切片分别加用含Avidin溶液的含5%的血清的TBST溶液中,室温下摇床孵育封闭30分钟。
10.甩去Avidin溶液,每张切片分别加兔来源PTGR1多克隆抗体(ab222818,Abcam)(1:200,抗体稀释于混有Biotin的含5%的动物血清的TBST溶液中)。
11.室温下孵育1小时。
12.把切片放到TBST里并在摇床上洗3次,每次5分钟。
13.甩掉TBST溶液,每张切片分别加入羊抗兔二抗500μg/ml(ab205718,abcam),于室温下摇床上孵育30分钟。
14.把切片放到TBST里并在摇床上洗3次,每次5分钟。
15.甩掉TBST溶液,加入ABC溶液,于室温下摇床上孵育30分钟。
16.把切片放到TBST里并在摇床上洗3次,每次5分钟。
17.甩掉TBST溶液,每张切片分别滴加适量新鲜配制的DAB,于镜下观察到目标染色后将切片浸入蒸馏水以中止染色反应。
18.将切片放入苏木精复染5-10秒,在温水中冲洗干净,在碳酸锂里浸润一下再次用温水冲洗干净。
19.人工操作复染后的切片进行脱水(自下而上)
取出二甲苯中的切片,用中性树脂封片,于显微镜下观察,若PTGR1表达呈阳性反应,则提示患者可以服用细胞周期依赖性激酶4/6抑制剂联合二甲双胍作前列腺癌辅助治疗,图中展示PTGR1阴性(图8,左)与阳性(图8,右)的前列腺癌组织免疫组化图。
实施例7
本实施例探究PTGR1与细胞周期依赖性激酶4/6抑制剂的相互作用。利用实施例2成功构建的PTGR1高表达前列腺癌细胞株(DU145-PTGR1、22RV1-PTGR1)和对照组前列腺癌细胞株(DU145-CG、22RV1-CG)进行细胞CCK-8增殖实验,取对数生长期细胞消化,取96孔板,根据细胞种类设置每孔加入细胞的数量:DU145:3000个/孔,22RV1:5000/孔;每孔加入100μL全血清培养基,将细胞稀释成所需的浓度,周围一圈细胞孔因挥发量比较大故一般不接种细胞,用200μLdPBS代替。待细胞贴壁后加入细胞周期依赖性激酶4/6抑制剂刺激,同时将培养基体积补充至200μL,待到刺激时间结束后配置CCK-8工作液:CCK-8:10%血清RPMI 1640=1:9。加入工作液,待反应时间结束后用酶标仪测定在450nm处OD值。结果显示PTGR1高表达细胞在细胞周期依赖性激酶4/6抑制剂刺激下生长更快(图9)。
实施例8
本实施例探究细胞周期依赖性激酶4/6抑制剂对PTGR1高表达前列腺癌细胞的二甲双胍敏感性。利用实施例2成功构建的PTGR1高表达前列腺癌细胞株(DU145-PTGR1)进行细胞CCK-8增殖实验,取对数生长期细胞消化,取96孔板,根据细胞种类设置每孔加入细胞的数量:DU145:3000个/孔;每孔加入100μL全血清培养基,将细胞稀释成所需的浓度,周围一圈细胞孔因挥发量比较大故一般不接种细胞,用200μL dPBS代替。待细胞贴壁后加入细胞周期依赖性激酶4/6抑制剂刺激,同时将培养基体积补充至200μL,待到刺激时间结束后配置CCK-8工作液:CCK-8:10%血清RPMI 1640=1:9。加入工作液,待反应时间结束后用酶标仪测定在450nm处OD值。结果显示细胞周期依赖性激酶4/6抑制剂能提高PTGR1高表达细胞对二甲双胍刺激的敏感性(图10)。细胞周期依赖性激酶4/6抑制剂联合二甲双胍作前列腺癌辅助内分泌治疗的具体分子机制如图11所示,PTGR1通过促进细胞周期从G0/G1期进入S和G2/M期,从而抵抗二甲双胍的抗肿瘤作用,CDK4/6能有效抵抗PTGR1的促周期进展作用。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (6)
1.PTGR1的检测试剂在制备指导治疗前列腺癌用药的试剂盒中的应用,其特征在于,所述治疗前列腺癌的药物为二甲双胍。
2.如权利要求1所述的应用,其特征在于,所述PTGR1的检测试剂为检测PTGR1的mRNA的检测试剂或为检测PTGR1蛋白的检测试剂。
3.如权利要求2所述的应用,其特征在于,所述检测PTGR1的mRNA的检测试剂包括PTGR1的检测引物,所述PTGR1的检测引物包括如下:上游引物序列如SEQ ID NO.3所示,下游引物序列如SEQ ID NO.4所示。
4.如权利要求2所述的应用,其特征在于,所述检测PTGR1蛋白的检测试剂包括一抗和二抗,所述一抗为兔来源PTGR1多克隆抗体,所述二抗为羊抗兔二抗。
5.如权利要求1所述的应用,其特征在于,所述前列腺癌为局部进展期前列腺癌或转移性前列腺癌。
6.CDK4/6抑制剂与二甲双胍在联合用于制备治疗前列腺癌药物中的应用,其特征在于,所述CDK4/6抑制剂为瑞博西尼、帕博西尼或阿贝西利。
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