CN115252636A - 一种寡聚脱氧核苷酸及其在制备抗肿瘤药物中的应用 - Google Patents
一种寡聚脱氧核苷酸及其在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,具体涉及一种寡聚脱氧核苷酸及其在制备抗肿瘤药物中的应用。该寡聚脱氧核苷酸由复数N个5’‑CATT‑3’碱基序列组成,N大于等于2。本发明提供了新靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸能够抑制肿瘤细胞MIF的表达,从而抑制肿瘤恶性进展。这开拓了核酸类药物在抗肿瘤防治中的新领域,从而为肿瘤靶向MIF药物的开发提供新的思路及方向,并为临床抗肿瘤治疗提供新思路。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及寡聚脱氧核苷酸及在制备抗肿瘤的药物中应用。
背景技术
巨噬细胞移动抑制因子(Macrophage migration inhibitory factor,MIF)是一种多效细胞因子,因能够限制体内巨噬细胞的活动而得名,但MIF还具有调控炎症反应,抑制糖皮质激素作用,调控细胞凋亡等多种不同的功能。研究证实,肿瘤细胞(如肺肿瘤、鼻咽癌、膀胱癌、神经母细胞瘤、前列腺癌、结肠癌、食管鳞状细胞癌、胃癌、肝细胞癌、颅咽管瘤等)MIF呈高表达,随着肿瘤分化程度越低、其MIF的表达越高,而且MIF的高表达预示着肿瘤患者预后不良。MIF可通过多种机制促进肿瘤恶性进展,主要体现(1)直接促癌:促进肿瘤增值、转移、凋亡、血管新生等:如MIF可作为p53基因(抑癌基因)的一个负调节因子,MIF的表达具有抑制p53介导的肿瘤细胞凋亡,从而促进肿瘤进展;同时,MIF具有氧化还原酶的活性,能抑制氧化剂导致的肿瘤细胞凋亡,从而增强肿瘤细胞的抵抗能力。(2)抑制机体免疫系统,促进肿瘤逃逸免疫抑制:研究已经证实MIF调控肿瘤内T淋巴细胞、B淋巴细胞、NK细胞等免疫细胞正常抗肿瘤功能的发挥。
目前,仍然没有一种有效抑制MIF过量表达的药物,亟需一种物质能够抑制癌细胞的MIF表达,从而为肿瘤的药物开发提供新的思路及方向,为抗肿瘤药物的开发提供科学依据,并为临床抗肿瘤治疗提供新思路。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此,本发明提出一种寡聚脱氧核苷酸及其在制备抗肿瘤药物中的应用,该寡聚脱氧核苷酸及在制备抗肿瘤的药物中能够抑制肿瘤细胞的巨噬细胞移动抑制因子(MIF)的表达,从而抑制肿瘤的恶性进展。
本发明的发明构思为:发现出一种寡聚脱氧核苷酸能够抑制肿瘤细胞/癌细胞的MIF表达,从而抑制肿瘤的恶性进展。
本发明的第一方面提供一种寡聚脱氧核苷酸,所述寡聚脱氧核苷酸由N个5’-CATT-3’碱基序列组成,N大于等于2。
优选的,所述寡聚脱氧核苷酸还进行全硫代磷酸化修饰;所述全硫代磷酸化修饰具体为:核苷酸链中磷酸上带双键的氧原子被硫原子取代。
优选的,N大于等于2且小于等于30;进一步优选的,N大于等于5且小于等于15。
优选的,所述寡聚脱氧核苷酸的碱基序列分别如下所示:5’-CATTCATTCATTCATTCATTCATT-3’(SEQ ID NO:1)、5’-CATTCATTCATTCATTCATTCATTCATT-3’(SEQ ID NO:2)、5’-CATTCATTCATTCATTCATTCATTCATTCATT-3’(SEQ ID NO:3)、5’-CATTCATTCATTCATTCATTCATTCATTCATTCATT-3’(SEQ ID NO:4)。
相对于现有技术,本发明第一方面提供的一种寡聚脱氧核苷酸的有益效果如下:本发明提供了新靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸能够抑制肿瘤细胞MIF的表达,从而抑制肿瘤恶性进展。这开拓了核酸类药物在抗肿瘤防治中的新领域,从而为肿瘤靶向MIF药物的开发提供新的思路及方向,并为临床抗肿瘤治疗提供新思路。
本发明的第二方面提供一种具有所述寡聚脱氧核苷酸的重组表达载体、转基因细胞系或重组菌。
本发明的第三方面提供一种所述寡聚脱氧核苷酸、含有所述寡聚脱氧核苷酸的重组表达载体、转基因细胞系或重组菌在研究肿瘤细胞/癌细胞的发生机理上的应用。
本发明的第四方面提供一种具有所述寡聚脱氧核苷酸、含有所述寡聚脱氧核苷酸的重组表达载体、转基因细胞系或重组菌在在制备预防或治疗肿瘤/癌症药物中的应用。
优选的,所述肿瘤/癌症包括肺肿瘤、鼻咽癌、膀胱癌、神经母细胞瘤、前列腺癌、结肠癌、食管鳞状细胞癌、胃癌、肝细胞癌、颅咽管瘤中的至少一种。
优选的,所述预防或治疗肿瘤/癌症药物能够对肿瘤细胞/癌细胞的MIF表达具有抑制作用;进一步优选的,肿瘤细胞/癌细胞可因MIF表达被抑制而减慢或停止生长、缩小或消失。
优选的,所述抑制作用为:使得所述肿瘤细胞/所述癌细胞的MIF相对表达量(relative expression)下降至小于0.4;进一步优选的,所述抑制作用为:使得所述肿瘤细胞/所述癌细胞的MIF相对表达量(relative expression)下降至小于0.3。
优选的,所述抑制作用为:使得所述肿瘤细胞/所述癌细胞的MIF相对表达量(relative expression)在用药48小时内下降至小于0.4;进一步优选的,所述抑制作用为:使得所述肿瘤细胞/所述癌细胞的MIF相对表达量(relative expression)在用药60小时内下降至小于0.3。
优选的,所述寡聚脱氧核苷酸的浓度为1-150nM;进一步优选的,所述寡聚脱氧核苷酸的浓度为10-100nM;更进一步优选的,所述寡聚脱氧核苷酸的浓度为25-75nM。
优选的,所述肿瘤细胞包括肺肿瘤细胞;进一步优选的,所述肿瘤细胞包括A549细胞。
优选的,上述所述药物,还包括:药学上可接受的载体;预防或治疗肿瘤的其它活性成分。
优选的,药学上可接受的载体和/或辅料包括但不限于缓冲剂、乳化剂、悬浮剂、稳定剂、防腐剂、生理食盐、赋形剂、填充剂、凝结剂与调和剂、界面活性剂、扩散剂、消泡剂。
优选的,所述预防或治疗肿瘤的其他活性成分包括:化疗剂、放疗剂或抗体药物。
优选的,所述药物的剂型为片剂、胶囊剂、颗粒剂、注射剂、粉针剂、滴眼剂、搽剂、栓剂、软膏剂、气雾剂、散剂、滴丸剂、乳剂、凝胶剂、膜剂、透皮吸收贴剂、控释制剂或纳米制剂中的任意一种。
相对于现有技术,本发明的有益效果如下:
本发明提供了新靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸能够抑制肿瘤细胞MIF的表达,从而抑制肿瘤恶性进展。这开拓了核酸类药物在抗肿瘤防治中的新领域,从而为肿瘤靶向MIF药物的开发提供新的思路及方向,并为临床抗肿瘤治疗提供新思路。
附图说明
图1为Blank Control组、CATT5-8 Decoy ODN组MIF的mRNA的相对表达量;
图2为Blank Control组、CATT5-8 Decoy ODN组的MIF蛋白质免疫印迹分析结果。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案,现列举以下实施例进行说明。需要指出的是,以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明,均可从常规商业途径得到,或者可以通过现有已知方法得到。
本发明中Blank Control、CATT5-8 Decoy ODN、MIF及GAPDH(甘油醛-3-磷酸脱氢酶)的引物由苏州金唯智生物科技有限公司合成。
本发明中所用到的A549细胞购自:American Type Culture Collection(ATCC;Manassas,VA,USA)。
实施例1
一种靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸(Decoy ODN)
该寡聚脱氧核苷酸的碱基序列如下:5’-CATTCATTCATTCATTCATTCATT-3’(SEQ IDNO:1),命名为CATT5 Decoy ODN。
实施例2
一种靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸(Decoy ODN)
该寡聚脱氧核苷酸的碱基序列如下:5’-CATTCATTCATTCATTCATTCATTCATT-3’(SEQID NO:2),命名为CATT6 Decoy ODN。
实施例3
一种靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸(Decoy ODN)
该寡聚脱氧核苷酸的碱基序列如下:5’-CATTCATTCATTCATTCATTCATTCATTCATT-3’(SEQ ID NO:3),命名为CATT7 Decoy ODN。
实施例4
一种靶向MIF启动子区域多拷贝CATT元件的诱捕寡聚脱氧核苷酸(Decoy ODN)
该寡聚脱氧核苷酸的碱基序列如下:5’-CATTCATTCATTCATTCATTCATTCATTCATTCATT-3’(SEQ ID NO:4),命名为CATT8Decoy ODN。
实施例5
实施例1-4中的CATT5-8 decoy ODN抑制肿瘤细胞MIF的mRNA合成实验
Blank Control的碱基序列信息为:5’-TCACATATCTACATATTACTATCTATCTTATCTTAT-3’(SEQ ID NO:9),命名为Blank Control。
1.根据相应的Blank Control、CATT5-8 decoy ODN碱基序列信息进行合成、纯化、全硫代磷酸化修饰,制得寡聚核苷酸。
2.序列稀释与退火:合成、纯化得到的寡聚核苷酸稀释于无酶无菌的TE溶液(Tris-EDTA buffer solution,TE缓冲溶液),浓度分别为100μM,将稀释好的各对应互补寡聚核苷酸链按照1:1混合进行退火、退火温度为95℃,升温程序为在梯度PCR仪进行升温降温、升温由4℃快速加到95℃,退火结束后按照2℃/min的速度降至25℃,制得相应的BlankControl、CATT5-8decoy ODN,产物保存于-80℃,待用。
3.转染肿瘤细胞:
(1)以A549细胞为细胞模型,转染前24h,将1×105个细胞接种于12孔板培养,培养基为含10%FBS(Bovogen,Keilor East,Australian)的DMEM(GIBCO,Carlsbad,CA,USA),)1ml;
(2)在50μL Opti-MEM培养基中分别加入对应的0.5μL Blank Control、CATT5Decoy ODN、CATT6 Decoy ODN、CATT7 Decoy ODN、CATT8 Decoy ODN,并用枪轻轻吹打混匀、混匀,分别记为Blank Control组、CATT5 Decoy ODN组、CATT6 Decoy ODN组、CATT7 DecoyODN组、CATT8 Decoy ODN组;再加入10μL Lipo8000TM转染试剂,用枪轻轻吹打混匀、混匀,制得混合液;混合液加入到接种有A549细胞的培养基内,此时CATT5-8 Decoy ODN的浓度分别为50nM,置于37℃培养箱中培养,5h后换液,各组分别继续培养24、36、48、60h,并在各时间点的转染肿瘤细胞收集RNA。
4.qPCR分析肿瘤细胞MIF的mRNA含量:
(1)将各组转染肿瘤细胞所在的原培养基移弃,用4℃预冷1×PBS洗涤细胞2次,加入1mL Trizol试剂收集细胞,移入1.5mL无酶离心管;
(2)加入500μL氯仿,涡旋混匀30s后,于4℃静置5min,然后于4℃离心机10000rpm离心10min;
(3)取上层水相,加到1.5ml另一无酶离心管中,加入等体积的异丙醇,涡旋混匀后,-40℃静置30min;
(4)-40℃静置后的样品于4℃离心机10000rpm离心10min,移弃上清液,并依次用75%的乙醇和无水乙醇洗涤沉淀;洗涤方法为添加适量75%的乙醇或无水乙醇经涡旋、4℃离心机10000rpm离心10min、然后移弃上清液。
(5)静置待乙醇充分挥发,用无RNA酶水(无核酸酶水)收集RNA。
(6)使用Nano Drop1000核酸蛋白定量仪,依次记录所测得的浓度,用NRA freewater按比例将收集到的RNA稀释成1μg/μL,-80℃保存。
(7)于无RNA酶的0.2mL EP管中依次加入:gDNA clean buffer 2μL、RNA(1μg/μL)2μL、gDNA regent 1μL、DEPC-ddH2O 5μL,离心混匀,置于PCR仪上,按照仪器操作说明:42℃运行反应2min;
(8)反转录制cDNA:反应完后依次在同样的0.2ml EP管(聚乙烯管)中依次加入:4μLDEPC-ddH2O、1μL Evo M-MLV RTase Enzyme Mix、4μL RT Buffer,1μL Random 6mersPrimer,离心混匀,置于PCR仪上,按照仪器操作说明:37℃运行反应15min,85℃运行反应5s,4℃保存,分别制得各cDNA。
(9)根据下表1设计并合成引物:
表1 MIF及GAPDH(甘油醛-3-磷酸脱氢酶)的引物序列
(10)按照Green Premix Pro Taq HS qPCR Kit(Code.AG11701)试剂盒说明书依次在8连管中加入:2XGreen Pro Taq HS Premix 10μL,MIF Primer F 1μl,MIF Primer R 1μl(或者GAPDH Primer F 1μL,GAPDH Primer R 1μl),RNase free water6μL,cDNA2μL,离心混匀。
(11)PCR反应反应条件:95℃反应30s,95℃反应5s,60℃反应30s,35个重复循环,最后PCR仪自动收集荧光信息。以Blank Control组为参照组,各组mRNA的相对表达量作图。
图1为Blank Control组、CATT5 Decoy ODN组、CATT6 Decoy ODN组、CATT7 DecoyODN组、CATT8 Decoy ODN组MIF的mRNA的相对表达量,横坐标为时间(h)、纵坐标为MIF的mRNA相对表达量(MIF mRNA relative expression);qPCR分析证明CATT5 Decoy ODN、CATT6 Decoy ODN、CATT7 Decoy ODN、CATT8 Decoy ODN具有抑制肿瘤细胞MIF的mRNA合成的作用。当聚脱氧核苷酸(Decoy ODN)的5’-CATT-3’碱基序列重复数量N=6-9时,随着N的增大其抑制肿瘤细胞MIF的mRNA合成的作用增大。
实施例6
实施例1-4中CATT5-8 decoy ODN降低肿瘤细胞MIF的蛋白水平的实验
Blank Control的碱基序列信息为:5’-TCACATATCTACATATTACTATCTATCTTATCTTAT-3’(SEQ ID NO:9),命名为Blank Control。
1.根据相应的Blank Control、CATT5-8 decoy ODN碱基序列信息进行合成、纯化、全硫代磷酸化修饰,制得寡聚核苷酸。
2.序列稀释与退火:合成、纯化得到的寡聚核苷酸稀释于无酶无菌的TE溶液(Tris-EDTA buffer solution,TE缓冲溶液),浓度分别为100μM,将稀释好的各对应互补寡聚核苷酸链按照1:1混合进行退火、退火温度为95℃,升温程序为在梯度PCR仪进行升温降温、升温由4℃快速加到95℃,退火结束后按照2℃/min的速度降至25℃,制得相应的BlankControl、CATT5-8decoy ODN,产物保存于-80℃,待用。
3.转染肿瘤细胞:
(1)以A549细胞为细胞模型,转染前24h,将1×105个细胞接种于12孔板培养;
(2)在50μL Opti-MEM培养基中分别加入对应的0.5μL Blank Control、CATT5Decoy ODN、CATT6 Decoy ODN、CATT7 Decoy ODN、CATT8 Decoy ODN,并用枪轻轻吹打混匀、混匀;再加入10μL Lipo8000TM转染试剂,用枪轻轻吹打混匀、混匀,制得混合液;混合液加入到接种有A549细胞的培养基内,此时CATT5-8 Decoy ODN的浓度分别为50nM,置于37℃培养箱中培养,5h后换液,继续培养,24h后收集细胞蛋白,分别记为Blank Control组、CATT5Decoy ODN组、CATT6 Decoy ODN组、CATT7 Decoy ODN组、CATT8 Decoy ODN组。
4.配制聚丙烯酰胺凝胶(SDS-PAEG)
(1)分离胶的配制:依次按比例加入各种试剂:ddH2O,30%丙烯酰胺,4×LowerTris Buffer,10%APS和TEMED;
(2)加入TEMED后立即晃动混匀,并缓慢的在玻璃板间加入配制好的分离胶,并用2ml无水乙醇封胶,常温静置至分离胶充分凝固后,移去无水乙醇;
(3)浓缩胶的制备:依次按比例加入各种试剂:ddH2O,30%丙烯酰胺,4×UpperTris Buffer,10%APS和TEMED;
(4)加入TEMED后立即均匀晃动,在两块玻璃板间添加配制好的浓缩胶,水平插入梳子。室温静置至浓缩胶凝固后,可用于电泳。
5.垂直电泳
(1)根据所需蛋白总量,换算出各组蛋白样品的体积,用1×sampling Buffer补齐,并按1:10的比例加入10×loading Buffer,瞬时离心,于95℃干浴锅加热处理10min后,瞬时离心;
(2)将预先配制的聚丙烯酰胺凝胶装配到垂直电泳装置,加入新配制的1×running Buffer,根据实验设计顺序依次加入预处理好的蛋白样品,按照电泳仪操作说明进行电泳。
6.湿法转膜
(1)准备9×6cm的3mm滤纸4张和8×5cm PVDF膜(polyvinylidene fluoride,聚偏二氟乙烯膜)1张,PVDF膜作好对应的标记,并用甲醇中浸泡PVDF膜5min,用1×TransferBuffer将海绵和滤纸预先泡湿中;
(2)按照电转印装置操作要求依次摆放转移夹、海绵、滤纸、滤纸和PVDF膜,注意凝胶、PVDF膜对齐,而且居于滤纸中间,全过程要避免产生气泡;
(3)将含有凝胶的转移夹按照要求操作要求放到电转槽中,加入预冷(4℃)的1×Transfer Buffer,350mA恒流,转膜1.5h,全过程保持Transfer Buffer温度不高于25℃;
(4)电转结束,关闭电源,小心取出PVDF膜,并迅速用5%牛奶封闭1h。
7.抗体孵育和显影检测
(1)用5%脱脂牛奶将第一抗体按比例稀释,并与相应的PVDF膜混合4℃孵育过夜;
(2)回收第一抗体溶液,用0.1%TBST缓冲液洗去PVDF膜上的非特异结合;
(3)用5%脱脂牛奶将第二抗体按比例稀释并与PVDF膜混匀,于摇床上常温孵育1h;
(4)回收第二抗体溶液,用0.1%TBST洗去PVDF膜上的非特异结合;
(5)化学发光显影检测:将预先配制好的A液和B液按照1:1比例混合形成工作液,将PVDF膜充分泡在工作液上3-5s,曝光拍照,保存结果。
图2为Blank Control组、CATT5 Decoy ODN组、CATT6 Decoy ODN组、CATT7 DecoyODN组、CATT8 Decoy ODN组的MIF蛋白质免疫印迹分析结果,内参表达抗体为β-actin(β-肌动蛋白);从图2可知,CATT5 Decoy ODN、CATT6 Decoy ODN、CATT7 Decoy ODN、CATT8 DecoyODN具有促使肿瘤细胞MIF的蛋白水平降低的作用。当聚脱氧核苷酸(Decoy ODN)的5’-CATT-3’碱基序列重复数量N=6-9时,随着N的增大其促使肿瘤细胞MIF的蛋白水平降低的作用增大。
SEQUENCE LISTING
<110> 南方医科大学顺德医院
<120> 一种寡聚脱氧核苷酸及其在制备抗肿瘤药物中的应用
<130> 2022
<160> 9
<170> PatentIn version 3.5
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Claims (10)
1.一种寡聚脱氧核苷酸,其特征在于,所述寡聚脱氧核苷酸由N个5’-CATT-3’碱基序列组成,N大于等于2。
2.根据权利要求1所述的寡聚脱氧核苷酸,其特征在于,所述寡聚脱氧核苷酸还进行全硫代磷酸化修饰。
3.根据权利要求1所述的寡聚脱氧核苷酸,其特征在于,所述寡聚脱氧核苷酸中,N大于等于2且小于等于30。
4.根据权利要求1所述的寡聚脱氧核苷酸,其特征在于,所述寡聚脱氧核苷酸的碱基序列包括如下所示的至少一种:
5’-CATTCATTCATTCATTCATTCATT-3’、
5’-CATTCATTCATTCATTCATTCATTCATT-3’、
5’-CATTCATTCATTCATTCATTCATTCATTCATT-3’、
5’-CATTCATTCATTCATTCATTCATTCATTCATTCATT-3’。
5.具有权利要求1-4任一项所述的寡聚脱氧核苷酸的重组表达载体、转基因细胞系或重组菌。
6.权利要求1-4任一项所述的寡聚脱氧核苷酸,或权利要求5所述的重组表达载体、转基因细胞系或重组菌在制备预防或治疗肿瘤/癌症药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述肿瘤/癌症包括肺肿瘤、鼻咽癌、膀胱癌、神经母细胞瘤、前列腺癌、结肠癌、食管鳞状细胞癌、胃癌、肝细胞癌、颅咽管瘤中的至少一种。
8.根据权利要求6所述的应用,其特征在于,所述预防或治疗肿瘤/癌症药物能够对肿瘤细胞/癌细胞的巨噬细胞移动抑制因子表达具有抑制作用。
9.根据权利要求6所述的应用,其特征在于,所述药物还包括:药学上可接受的载体、预防或治疗肿瘤的其它活性成分;
所述预防或治疗肿瘤的其他活性成分包括:化疗剂、放疗剂或抗体药物。
10.根据权利要求9所述的应用,其特征在于,药学上可接受的载体和/或辅料包括缓冲剂、乳化剂、悬浮剂、稳定剂、防腐剂、生理食盐、赋形剂、填充剂、凝结剂与调和剂、界面活性剂、扩散剂、消泡剂中的至少一种。
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US20030215446A1 (en) * | 2001-12-21 | 2003-11-20 | Baugh John A. | Macrophage migration inhibitory factor (MIF) promoter polymorphism in inflammatory disease |
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