CN103343128B - 乳腺癌细胞mda-mb-231的核酸适体lxl-3及其应用 - Google Patents
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Abstract
乳腺癌细胞MDA-MB-231的核酸适体LXL-3及其应用,涉及乳腺癌细胞。所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在0℃,0.157M/L Na+,0.005M/L Mg2+条件下具有独特的茎环结构。所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在制备乳腺癌诊断试剂、治疗乳腺癌药物、乳腺癌的肿瘤标志物等中,以及在研究乳腺癌与正常组织的差异性、乳腺肿瘤组织切片成像、乳腺肿瘤相关的活体成像、乳腺癌的靶向治疗等中的应用。
Description
技术领域
本发明涉及乳腺癌细胞,尤其是涉及乳腺癌细胞MDA-MB-231的核酸适体LXL-3及其应用。
背景技术
乳腺癌是女性最常见的癌症,主要包括导管癌和小叶癌。2011年美国CA(A Cancer Journalfor Clinicians)公布的最新统计数据显示,美国2011年预计将有230480例女性患乳腺癌,占女性新发恶性肿瘤的30%,居女性恶性肿瘤发病率第一位,并且死亡人数将达39520例。更为严重的是全球的乳腺癌发病率正在逐年增长。乳腺癌的诊断方法主要包括乳腺钼靶X线摄影、乳腺彩色多谱勒超声检查、乳腺导管内视镜检查、乳腺导管灌洗、CT和磁共振。但是这些检测方法主要是基于形态学标准,依然缺乏与乳腺癌相关的特异性标志。乳腺癌的治疗主要包括手术切除、化疗以及放疗或者几者综合治疗等方式。但是传统化疗、放疗的方式由于缺乏肿瘤细胞的靶向性导致非常严重的副作用。近年来发展的单克隆抗体虽然具有较高的特异性和亲和力,但是抗体本身所具有的一些缺陷,如免疫原性、不稳定性、批次间的差异性等使其在临床上的实际应用中具有一定的局限性。因此,找到乳腺肿瘤细胞的特异性靶标,在肿瘤的诊断和治疗中都具有重要意义。近些年发展的核酸适体探针,尤其是细胞靶向的核酸适体,能够特异性识别包括全血样品的复杂样品中的癌细胞,将给肿瘤的早期特异性快速诊断和靶向治疗带来新的希望和解决途径。
核酸适体(Tuerk,C.;Gold,L.,Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4DNA polymerase.Science1990,249(4968),505-510;Ellington,A.D.;Szostak,J.W.,In vitro selection of RNA molecules that bind specific ligands.Nature1990,346(6287),818-822)是指从人工合成的单链DNA/RNA文库中筛选得到的能够高亲和性和高特异性地与靶标分子结合的单链寡核苷酸。核酸适体借助氢键、范德华力、疏水作用等分子间作用力形成特殊的三维结构,如发夹、假结、凸环、G-四聚体等,从而特异地识别靶物质并影响其生物活性。核酸适体本身特有的生化特性使其在生物医学应用领域具有诸多优势,如靶分子范围广、亲和力与特异性强、合成修饰方便快速、良好的生物兼容性、无毒、体外稳定性好等。核酸适体可以通过配体指数富集系统进化技术(systematic evolution of ligands byexponential enrichment,SELEX)(Sefah,K.;Shangguan,D.;Xiong,X.;O′Donoghue,M.B.;Tan,W.,Development of DNA aptamers using Cell-SELEX.Nat.Protocols2010,5(6),1169-1185)筛选获得,其基本原理是在体外人工化学合成一个寡核苷酸文库,包括RNA、ssDNA或修饰的RNA和ssDNA,通过寡核苷酸文库与目标分子的相互作用,结合PCR技术指数级放大与靶分子特异结合的寡核苷酸,经过几轮或数十轮筛选过程,获得高亲和力和高特异性的核酸适体。
发明内容
本发明的第一目的在于提供具有高特异性和高亲和力的乳腺癌细胞MDA-MB-231的核酸适体LXL-3。
本发明的第二目的在于提供乳腺癌细胞MDA-MB-231的核酸适体LXL-3在与乳腺癌相关方面的应用。
所述乳腺癌细胞MDA-MB-231的核酸适体命名为LXL-3,其序列如下:
gaattcagtc ggacagcgaa gtagttttcc ttctaaccta agaacccgcg gcagtttaat 60
gtagatggac gaa 73
所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在0℃,0.157M/L Na+,0.005M/L Mg2+条件下具有独特的茎环结构,其茎环结构如下:
所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3的应用如下:
1)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在制备乳腺癌诊断试剂中的应用。
2)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在制备治疗乳腺癌药物中的应用。
3)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在研究乳腺癌与正常组织的差异性中的应用。
4)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在制备乳腺癌的肿瘤标志物中的应用。
5)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在乳腺肿瘤组织切片成像中的应用。
6)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在乳腺肿瘤相关的活体成像中的应用。
7)所述乳腺癌细胞MDA-MB-231的核酸适体LXL-3在乳腺癌的靶向治疗中的应用。
本发明的优点在于:
首先,利用与乳腺癌细胞MDA-MB-231相对应的正常乳腺上皮细胞MCF-10A作为反筛细胞,筛选除去DNA随机寡核苷酸库中的乳腺癌细胞与正常乳腺上皮细胞共同分子的结合部分,保证筛选得到的核酸适配体可以识别乳腺癌细胞MDA-MB-231。
其次,基于细胞的筛选方法保证了细胞表面所表达的生物大分子更加接近于生物体内的分子构象。
而且,筛选获得的核酸适体无毒性,分子量小,渗透性好,易于合成与标记。通过SELEX方法筛选获得的核酸适体只特异性识别乳腺癌细胞MDA-MB-231,对其他乳腺癌细胞、正常乳腺上皮细胞、其他肿瘤细胞等不具有或者具有较弱的识别功能。
上述优点使得所述核酸适体在生物医学检测、肿瘤生物标志物发现以及靶向治疗方面将有重要的潜在应用价值。
本发明的优点在于:
首先,利用与乳腺癌细胞MDA-MB-231相对应的正常乳腺上皮细胞MCF-10A作为反筛细胞,筛选除去DNA随机寡核苷酸库中的乳腺癌细胞与正常乳腺上皮细胞共同分子的结合部分,保证筛选得到的核酸适配体可以识别乳腺癌细胞MDA-MB-231。
其次,基于细胞的筛选方法保证了细胞表面所表达的生物大分子更加接近于生物体内的分子构象。
而且,筛选获得的核酸适体无毒性,分子量小,渗透性好,易于合成与标记。通过SELEX方法筛选获得的核酸适体只特异性识别乳腺癌细胞MDA-MB-231,对正常乳腺上皮细胞不具有识别功能。
上述优点使得所述核酸适体在生物医学检测、肿瘤生物标志物发现以及靶向治疗方面将有重要的潜在应用价值。
附图说明
图1为流式细胞仪测定所得核酸适体LXL-3对乳腺癌细胞MDA-MB-231的偏移。在图1中,横坐标为荧光强度,纵坐标为细胞个数,实线为初始DNA随机寡核苷酸库,虚线为LXL-3。
图2为流式细胞仪测定所得核酸适体LXL-3对正常乳腺上皮细胞MCF-10A的偏移。在图2中,横坐标为荧光强度,纵坐标为细胞个数,实线为初始DNA随机寡核苷酸库,虚线为LXL-3。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
实施例 以流式分析法检测所得核酸适体与乳腺癌细胞MDA-MB-231结合的特异性
1)将合成好的0.5nmol单链FAM-DNA核酸溶于200μL结合缓冲液(12mmol/L PBS,5mmol/L MgCl2)中,进行热处理:95℃加热5min,在冰上放置10min;
2)将处理好的单链DNA核酸与10万个种于24孔板24h的细胞进行孵育,在冰上孵育30min。
3)孵育好后,缓冲溶液洗两次,再将细胞刮下来溶于200μL缓冲溶液,使用BD公司的FACSAria流式细胞仪对细胞进行荧光测定(参见图1和2)。
Claims (1)
1.乳腺癌细胞MDA-MB-231的核酸适体LXL-3,其特征在于其序列如下:
gaattcagtc ggacagcgaa gtagttttcc ttctaaccta agaacccgcg gcagtttaat 60
gtagatggac gaa 73;
在0℃,0.157mol/L Na+,0.005mol/L Mg2+条件下其茎环结构如下:
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