CN112156105A - 一种抑制剂联合小干扰rna抑制结肠癌细胞活性的新方法 - Google Patents
一种抑制剂联合小干扰rna抑制结肠癌细胞活性的新方法 Download PDFInfo
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Abstract
本发明涉及一种通过NAMPT抑制剂与PARP抑制剂联合小干扰RNA抑制结肠癌细胞活性的新方法。本方法的优势在于创造性的把抗肿瘤分子靶向药物和RNA干扰技术结合起来,利用肿瘤细胞与正常细胞之间分子生物学上的差异,使肿瘤细胞特异性死亡。本发明依据结肠癌细胞中NAMPT高表达和对氧化损伤高敏感性的特点进行药物组合设计,所得的联合用药组合方法与传统化疗药物相比,抗结肠癌细胞活性更强,同时对正常结肠上皮细胞活性的抑制作用较弱,在细胞水平上,具有更高的生物安全性。
Description
技术领域
本发明涉及生物医药领域,是一种通过NAMPT抑制剂与PARP抑制剂联合小干扰RNA抑制结肠癌细胞活性的新方法。
背景技术
结肠癌是人类第三大癌症,也是死亡率较高的癌症之一,全球每年大约有120万结肠癌新发病例。随着我国经济的发展,人们的饮食结构和生活习惯发生了巨大变化,结肠癌在我国的发病率逐年提高,位居全部恶性肿瘤第三位,死亡率为0.01%。
奥沙利铂(Oxaliplatin)是治疗结肠癌的首选药物之一,它通过产生烷化结合物作用于DNA,形成链内和链间交联,从而抑制DNA的合成及复制,发挥抗肿瘤作用。奥沙利铂存在毒副作用大,耐药性等缺点。急需寻求更新、更安全、效果更佳的治疗措施。
NAMPT是合成NAD+前体NMN的限速酶,在多种恶性肿瘤中高表达。因为NAD+是细胞物质和能量代谢的重要辅酶,和正常细胞相比,肿瘤细胞对NAD+的需求量更大,所以肿瘤细胞会表达更多的NAMPT以满足自身对NAD+的高需求,这也使肿瘤细胞更容易受到NAD+生成抑制的影响。通过抑制NAMPT的表达减少肿瘤细胞内NAD+的含量已成为治疗癌症的新思路。
目前,已经有NAMPT抑制剂FK866用于结肠癌的实验性治疗,FK866通过抗肿瘤细胞的转移,肿瘤细胞血管生成以及增加化疗药物对肿瘤细胞的敏感性而发挥抗肿瘤作用,但其治疗效果不佳,目前未在临床上推广应用。
本实验室在研究中发现FK866通过提高细胞中活性氧(ROS)的水平达到抑制结肠癌细胞活性的作用。癌细胞对氧化打击具有高度敏感性,癌细胞中的活性氧水平远远高于正常细胞活动所需的含量,过量的活性氧如果没有得到有效清除就会造成癌细胞中DNA的氧化损伤,导致DNA产生突变,从而影响或抑制癌细胞的生长和存活。
研究也发现,当细胞中活性氧水平升高后,会激活特异性转运NMN的蛋白SLC12A8表达,增加其运输NMN的能力,进而增加NAD+的含量,降低细胞内活性氧水平。利用小干扰RNA干扰SLC12A8基因的表达,会进一步加剧癌细胞中DNA的氧化损伤。当细胞中DNA产生氧化损伤后,细胞会进行自我修复,PARP是DNA损伤修复过程中的一种关键蛋白,PARP抑制剂能有效阻断癌细胞中DNA的损伤修复。
本研究使用PARP抑制剂与促DNA损伤药物的联合应用。应用FK866抑制结肠癌细胞活性的基础上干扰SLC12A8的表达,降低细胞中NAD+含量,进一步升高癌细胞中活性氧水平,产生DNA的氧化损伤;在此基础上,联合PARP抑制剂奥拉帕尼(Olaparib),进一步加重DNA的氧化损伤,进而启动癌细胞的凋亡通路,造成癌细胞的死亡。
发明内容
本发明的目的在于克服现有技术的不足之处,提供一种抑制结肠癌细胞活性的新方法。
本发明解决其技术问题所采用的技术方案是:一种通过NAMPT抑制剂与PARP抑制剂联合小干扰RNA抑制结肠癌细胞活性的新方法,步骤如下:
本发明所涉及的细胞株为结肠癌细胞HT29、HCT116、CACO2和正常结肠上皮细胞NCM460。
本发明所涉及的联合用药组合方法为,抗肿瘤分子靶向药物NAMPT抑制剂FK866和PARP抑制剂奥拉帕尼,干扰SLC12A8基因表达的siSLC12A8。
研究结果显示,该联合用药组合方法与化疗药物奥沙利铂相比,抗结肠癌细胞活性,抑制细胞增殖,促进细胞凋亡的作用更强,同时对正常肠上皮细胞的损伤更少,为后续进行结肠癌体内实验提供新的策略和思路。
本发明取得的优点和积极效果为:
1.本发明首次提出NAMPT抑制剂FK866和PARP抑制剂奥拉帕尼联合干扰SLC12A8基因表达的siSLC12A8药物联用的新方法。FK866通过抑制NAMPT活性减少细胞中NAD+含量,增加细胞中活性氧水平,提高癌细胞对氧化损伤的敏感性,siSLC12A8干扰SLC12A8基因的表达,进一步提高细胞中活性氧水平,产生DNA的氧化损伤,同时应用奥拉帕尼抑制PARP活性阻止癌细胞在DNA氧化损伤后进行自我修复。三种药物在抑制结肠癌细胞活性上具有协同增效的作用,解决了FK866在抑制结肠癌细胞活性上效果不佳的缺点,同时提供了奥拉帕尼和siSLC12A8在抑制结肠癌细胞活性上的新应用。
2.本发明所涉及的联合用药组合方法,创造性的把抗肿瘤分子靶向药物和RNA干扰技术结合起来。利用肿瘤细胞与正常细胞之间分子生物学上的差异,使肿瘤细胞特异性死亡。本发明依据结肠癌细胞中NAMPT高表达和对氧化损伤的高敏感性的特点进行药物组合设计,所得的联合用药组合方法与传统化疗药物奥沙利铂相比,抗结肠癌细胞活性更强,同时对正常结肠上皮细胞活性的抑制作用较弱,在细胞水平上,具有更高的生物安全性。
附图说明
图1为实施例1中,用药处理细胞后细胞的存活率。
图2为实施例2中,用药处理细胞后细胞的增殖迁移率。
图3为实施例3中,用药处理细胞后细胞的凋亡率。
图4为实施例4中,用药处理细胞后细胞的周期分布变化
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应该视为对本发明的具体限制。
实施例1、实施例2、实施例3、实施例4所需的实验材料和试剂如下:
实验细胞株:人结肠癌细胞HT29、HCT116,人结直肠腺癌细胞CACO2,人正常结肠上皮细胞NCM460。
化学药品和试剂:奥沙利铂,Annexin V-FITC/PI细胞凋亡检测试剂盒,细胞周期与细胞凋亡检测试剂盒,genOFF st-h-SLC12A8_001。
生物制剂:奥拉帕尼,FK866
实施例1
MTT法检测不同药物组合对细胞存活率的影响。
用完全培养基调整细胞浓度为2x104/ml,接种于96孔板,每孔200ul,培养过夜,次日分别用不同的药物组合处理细胞。药物组合分别是:FK866、奥拉帕尼、FK866+奥拉帕尼、奥沙利铂、siSLC12A8、siSLC12A8+FK866、siSLC12A8+奥拉帕尼、siSLC12A8+FK866+奥拉帕尼、siSLC12A8+奥沙利铂。同时用等体积的完全培养基作为对照组Control。每组设5个复孔,5%CO2,37℃孵育24和48h后。每孔加入20μL MTT溶液(5mg/mL),继续培养4h。若药物与MTT能够反应,可先离心后弃去培养液,小心用PBS冲2-3遍后,再加入含MTT的培养液。终止培养,小心吸去孔内培养液。每孔加入150μLDMSO(二甲基亚砜),置摇床上避光低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪OD 490nm处测量各孔的吸光值(A),取平均值。
细胞存活率计算方法:对照组的细胞存活率记作1,实验组细胞生存率=(药物处理组A490/对照组A490)。
不同药物组合对细胞存活率的影响如图1所示,siSLC12A8+FK866+奥拉帕尼抑制结肠癌细胞活性的作用效果比奥沙利铂强,作用效果与时间成正比。同时发现siSLC12A8+FK866+奥拉帕尼对正常结肠上皮细胞活性的抑制作用较弱。siSLC12A8干扰SLC12A8的表达后,显著增加癌细胞对化疗药物的敏感性,但也显著抑制了正常结肠上皮细胞的活性。为此接下来的实验会进一步对比siSLC12A8+FK866+奥拉帕尼与奥沙利铂对细胞的作用效果。
实施例2
细胞划痕实验(WoundHealing),检测不同药物组合对细胞迁移的影响。
先用marker笔在6孔板背后,用直尺比着,均匀地划横线,大约每隔1cm一道,横穿过孔。每孔至少穿过5条线。用完全培养基调整细胞浓度为5x105/ml,接种于6孔板,每孔2ml,培养过夜。次日用枪头比着直尺,尽量垂至于背后的横线划痕,枪头要垂直,不能倾斜。用PBS洗细胞3次,去除划下的细胞。加入不同的药物组合,药物组合为奥沙利铂和siSLC12A8+FK866+奥拉帕尼。同时用等体积的完全培养基作为对照组Control。放入37度5%CO2培养箱,培养。按0,12,24小时取样,拍照。
不同药物组合对细胞迁移的影响如图2所示,2种药物均能抑制细胞的迁移能力,siSLC12A8+FK866+奥拉帕尼对细胞迁移能力的抑制作用更强。
实施例3
流式细胞术检测不同药物组合对细胞凋亡的影响。
Annexin V-FITC/PI细胞凋亡检测试剂盒分别检测,奥沙利铂和siSLC12A8+FK866+奥拉帕尼对细胞的凋亡情况的影响。
不同药物组合对细胞凋亡的影响如图3所示,2种药物均能促进细胞的凋亡,siSLC12A8+FK866+奥拉帕尼对细胞促凋亡的能力更强。
实施例4
流式细胞术检测不同药物组合对细胞周期分布变化的影响。
细胞周期与细胞凋亡检测试剂盒分别检测,奥沙利铂和siSLC12A8+FK866+奥拉帕尼对细胞周期分布变化的影响。
不同药物组合对细胞周期分布变化的影响如图4所示,2种药物均能诱导细胞周期的阻滞,从而抑制细胞的生长。奥沙利铂通过铂原子与DNA形成交叉联结,拮抗DNA的复制和转录,诱导细胞G1期和S期的阻滞。siSLC12A8+FK866+奥拉帕尼通过增加细胞中的氧化损伤,同样具有诱导细胞G1期和S期的阻滞的作用,但作用效果略低于奥沙利铂。
综合实施例1、实施例2、实施例3、实施例4的实验结果表明,siSLC12A8+FK866+奥拉帕尼在抑制细胞迁移,促进细胞凋亡的作用上强于奥沙利铂,但在阻滞细胞周期的作用上弱于奥沙利铂。siSLC12A8+FK866+奥拉帕尼在有效抑制结肠癌细胞HT29,HCT116,CACO2活性的同时,对正常结肠上皮细胞活性的抑制较弱。这是因为在结肠癌细胞中NAMPT高表达并且癌细胞对氧化损伤高敏感,所以siSLC12A8+FK866+奥拉帕尼对结肠癌细胞活性的抑制具有靶向性,也说明siSLC12A8+FK866+奥拉帕尼联合使用相比奥沙利铂具有更好的抗结肠癌细胞活性的作用。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (8)
1.一种通过NAMPT抑制剂与PARP抑制剂联合小干扰RNA抑制结肠癌细胞活性的新方法,其特征在于,所述联合用药组合方法包括抗肿瘤分子靶向药物和RNA干扰技术。
2.如权利要求1所述的联合用药组合方法,其特征在于,所述抗肿瘤分子靶向药物包括NAMPT抑制剂和PARP抑制剂,所述RNA干扰技术包括小干扰RNA。
3.如权利要求2所述的联合用药组合方法,其特征在于,所述NAMPT抑制剂包括FK866,所述PARP抑制剂包括奥拉帕尼(Olaparib),所述小干扰RNA包括干扰SLC12A8基因表达的siSLC12A8。
4.如权利要求3所述的联合用药组合方法,其特征在于,这种联合药物组合方法包括siSLC12A8+FK866+奥拉帕尼。
5.如权利要求1至4任一项所述的一种通过NAMPT抑制剂与PARP抑制剂联合小干扰RNA抑制结肠癌细胞活性的新方法,其特征在于,所述细胞株包括人结肠癌细胞HT29、HCT116,人结直肠腺癌细胞CACO2,人正常结肠上皮细胞NCM460。
6.如权利要求4所述的联合用药组合方法,在抑制癌细胞活性,促进细胞凋亡和阻滞细胞周期上的应用。
7.如权利要求6所述的联合用药组合方法,其特征在于,奥拉帕尼,siSLC12A8在抗结肠癌细胞活性上的新用途。
8.如权利要求2.3.4.7所述的联合用药组合方法,siSLC12A8+FK866+奥拉帕尼抗结肠癌细胞活性的作用优于传统化疗药物奥沙利铂(Oxaliplatin),更具有生物安全性。
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