【作者】 刘梅；

【导师】 何农跃；

【作者基本信息】 东南大学 ， 生物物理学， 2017， 硕士

【摘要】 乳腺癌是一种高度异质化的肿瘤,具有四种主要的分子亚型。不同分子亚型的乳腺癌对各种药物的治疗反应和预后差异较大。目前,临床乳腺癌分子分型主要依赖于病理检测的“金标准”——免疫组织化学技术(IHC),其操作费时、费力、成本高昂,已不能满足当前临床诊断的需要。所以,开发高效、快速、灵敏的乳腺癌分子分型新方法对提高乳腺癌的分子分型效率和准确率以及及时作出高效正确的乳腺癌治疗策略,降低乳腺癌病人的死亡率具有十分重要的现实意义。核酸适配体是在体外利用SELEX技术经过多轮筛选得到的能够和相应靶标特异性结合的短链RNA或者ssDNA。和抗体相比,核酸适配体具有特异性强、亲和性高、稳定性好、易修饰、成本低、靶标范围广等优点,被人们形象地称为“化学抗体”。近年来,核酸适配体在生物检测、分子诊断、生物成像、肿瘤分子分型以及药物输送等生物医学领域表现出巨大的应用潜力。在SELEX技术的基础上发展起来的以活细胞为筛选目标的Cell-SELEX技术可以在具体靶标未知的情况下筛选得到与特定肿瘤细胞特异性结合的核酸适配体。这种核酸适配体具有在一定条件下与天然构象的靶标高效结合的能力,克服了通过纯化靶标的SELEX技术获得的核酸适配体对天然靶标识别不灵敏的缺陷。本论文利用Cell-SELEX技术在体外经过连续21轮筛选得到了六条能够与人乳腺癌细胞SK-BR-3特异性结合的核酸适配体。系统考察了这六条核酸适配体的性质,然后经过序列优化后得到了具有人乳腺癌分子分型应用潜力的核酸适配体sk6Ea。主要内容如下:1.人乳腺癌细胞SK-BR-3特异性核酸适配体的筛选以人乳腺癌细胞SK-BR-3为靶细胞,乳腺癌细胞MCF-7、MDA-MB-231和人正常乳腺上皮细胞MCF-10A为对照细胞,利用Cell-SELEX技术在体外经过连续21轮筛选后,克隆次级文库,随机挑取30个克隆子进行测序,最终得到了能够和人乳腺癌细胞SK-BR-3特异性结合的六条核酸适配体,并对其二级结构进行了预测,这六条核酸适配体的二级结构中均具有明显的茎环结构。2.核酸适配体的性质考察及其序列优化系统地考察了所得六条核酸适配体的具体性质,重点考察了其特异性和亲和力大小。对其中特异性最强、亲和力最大的核酸适配体序列6进行了序列优化,得到新的长度为53 nt的核酸适配体sk6Ea。随后,进一步研究了核酸适配体sk6Ea的特异性和亲和力大小以及不同温度和不同酶消化对核酸适配体sk6Ea与靶细胞SK-BR-3结合能力的影响。结果表明:核酸适配体sk6Ea特异性强,能够区分靶细胞SK-BR-3和多种其他肿瘤细胞与正常细胞,其与靶细胞的结合解离常数Kd值大小为49.32 ± 14.53 nM;在4 ℃、25 ℃和37 ℃三个不同温度条件下,sk6Ea均能与靶细胞SK-BR-3结合良好,差异不明显。用胰酶和蛋白酶K消化靶细胞后,核酸适配体sk6Ea与靶细胞的结合能力显著降低,这说明核酸适配体sk6Ea与靶细胞膜上结合的具体靶标可能为一种膜蛋白,为进一步鉴定sk6Ea的靶标提供了参考依据。3.核酸适配体在人乳腺癌分子分型中的应用分别在细胞水平、组织水平和动物活体成像三个不同层面考察了核酸适配体sk6Ea应用于人乳腺癌分子分型的可能性。结果表明:核酸适配体sk6Ea能够显著鉴别人乳腺癌细胞SK-BR-3和MCF-7、MDA-MB-231以及人正常乳腺上皮细胞MCF-10A;在组织水平上能够鉴别HER2过表达型乳腺癌与Luminal A型、Luminal B型和三阴性乳腺癌四种乳腺癌分子亚型以及人正常乳腺组织。在动物活体内成像研究中,核酸适配体sk6Ea能够在体内成功鉴别SK-BR-3移植瘤和MCF-7移植瘤。同时,核酸适配体sk6Ea在SK-BR-3移植瘤小鼠体内的生物分布研究结果显示,核酸适配体在瘤体部位的富集明显强于肾脏、肝脏、肺脏、脾脏和心脏。以上结果初步表明,核酸适配体sk6Ea具有人乳腺癌分子分型和体内应用研究的潜力。更多还原

【Abstract】 Breast cancer is a highly heterogeneous tumor with four major molecular subtypes.Different breast cancer subtypes show distinct response to therapy and prognosis.Most currently,the commonly used methods for breast cancer molecular classification is immunohistochemistry(IHC),which is recommended as the gold standard for the pathogenic detection of breast cancer.However,IHC is always fussy,time-consuming and also very expensive,and cannot meet the requirement of fast detection in clinics.Therefore,it is in urgent need to develop effective,fast and more sensitive methods for breast cancer molecular classification,which is of great significance for decreasing the mortality and improving the therapeutic effect of breast cancer.Aptamer is a short RNA or ssDNA that is derived from SELEX(Systematic Evolution of Ligands by Exponential enrichment)method and can specifically bind with varieties of targets.Compared with antibodies,aptamer is more specific with high binding affinity,good stability,easy modification,low cost and a wide arrange of targets,which is known as "chemical antibodies".Recently,the biomedical application of aptamers for biological detection,molecular diagnosis,biological imaging,tumor molecular classification and drug delivery has attracted the most attention.Moreover,the Cell-SELEX,which is evolved from general SELEX,can help select aptamers against various tumor cells without knowing the targets.What is more,aptamers evolved from Cell-SELEX can sensitively bind with the targets with native conformation,surmounting the drawback of aptamers selected by purified protein based SELEX that they are not sensitive enough to native targets.In this study,six specific aptamers of human breast cancer cell SK-BR-3 were selected by Cell-SELEX in vitro.Then,the characteristics and optimization of the aptamers were analyzed systematically.Finally,the optimized aptamer sk6Ea was identified,which shows great potential in breast cancer classification.The main points of this study are as follows:1.Selection of aptamers against human breast cancer cell SK-BR-3In this detailed Cell-SELEX procedure,human breast cancer cell SK-BR-3 was set as the target cell,human breast cancer cell MCF-7,MDA-MB-231 and human normal breast epithelial cell MCF-10A were set as the negative cells.After successive selection of 21 cycles,the library was cloned and 30 randomly picked clones were sequenced.Finally,six aptamers were identified and the secondary structures of the aptamers were simulated.All the aptamers had typical stem-loop structures.2.Study of the characteristics and optimization of aptamersThe characteristics of the six aptamers were systematically studied,all the aptamers showed high specificity and affinity toward breast cancer cell SK-BR-3,among which sequence 6 owned both the optimal specificity and affinity.Then,the length of sequence 6 was optimized and a new aptamer sk6Ea of 53 nt was identified.Finally,the specificity,binding affinity,the influence of different temperatures and enzymatic dissociations for the binding ability of sk6Ea were tested.The results indicated that the aptamer sk6Ea had high specificity and its Kd value is 49.32±14.53 nM.Aptamer sk6Ea could bind with SK-BR-3 cells very well at any temperature of 4 ℃,25℃ and 37 ℃ without significant difference.When SK-BR-3 cells were dissociated by trypsin and proteinase K,aptamer sk6Ea showed no binding with the target cell SK-BR-3,indicating the target of sk6Ea was likely to be a protein.3.Application of aptamers for breast cancer classificationAptamer sk6Ea was utilized for breast cancer classification including breast cancer cells classification,breast cancer tissue classification and in vivo imaging.The results showed that the aptamer sk6Ea could distinguish breast cancer cell SK-BR-3 from MCF-7,MDA-MB-231 and normal breast epithelial cell MCF-10A.As for breast cancer tissue fluorescence staining,aptamer sk6Ea could distinguish HER2 overexpressed breast cancer tissue from Luminal A,Luminal B,triple negative breast cancer tissue and normal breast tissue.For in vivo imaging,aptamer sk6Ea could distinguish SK-BR-3 xenografted tumor from MCF-7 xenografted tumor.The biodistribution of aptamer sk6Ea in vivo was also examined at 1 h post-injection.The results showed that the tumor site had a stronger fluorescence singal than kinder,liver,lung,spleen and heart.All the results indicated that aptamer sk6Ea was very promising for breast cancer classification and in vivo applications.更多还原