CN102053150B - 一种人成骨肉瘤干细胞相关抗原标记物及其应用 - Google Patents
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Abstract
本发明公开了一种人成骨肉瘤干细胞相关抗原标记物SSEA-4及其在成骨肉瘤靶向治疗中的应用。SSEA-4是一特异性的成骨肉瘤干细胞表面标记物，在各正常成体组织中几乎不表达。此表面标记物易于抗体标记，不影响细胞活力，适合分离后的功能实验，为进一步研究干细胞在成骨肉瘤的起源、发生和发展过程的作用提供了一可靠的分子示踪物，为解释临床成骨肉瘤耐药、转移和寻找靶向治疗的细胞和分子靶点奠定了基础。
Description
技术领域
本发明涉及肿瘤生物学领域，更具体地讲，涉及一种人成骨肉瘤干细胞相关抗原标记物及其应用。
背景技术
近年来，随着对肿瘤干细胞理论认识的不断深入，要求我们对肿瘤的起源和发生、发展有更加清楚的认识。尤其是对白血病、乳腺癌、人神经胶质瘤和大肠癌等肿瘤干细胞分离和鉴定工作的报道，不仅印证和巩固了肿瘤干细胞理论，也使对其他肿瘤干细胞的鉴定和内在分子机制特征的研究成为热点。目前，国内外关于成骨肉瘤干细胞的报道极少，2005年国外Gibbs等人采用体外培养方式，首次从细胞系中分离出含有干细胞样骨肉瘤细胞的肉瘤细胞球(sarcosphere)，表达多种间充质干细胞的表面标记物(如Stro-1、CD44和CD105)以及胚胎干细胞特征基因(Nanog和Oct-4)；2007年Colleen Wu等发现体外培养之成骨肉瘤细胞群体中存在SP细胞，但也未能分离鉴定具体的、特异性的成骨肉瘤干细胞表面标记物。最为重要的是，这些工作均不是基于人原代肿瘤组织的研究工作，未进行严格的细胞分离和体内接种试验。
2005年Gibbs等人首次分离出含有干细胞样骨肉瘤细胞的肉瘤细胞球以及2007年Colleen Wu等人从肉瘤病人标本中发现SP细胞。一方面，上述学者的工作主要局限于体外细胞培养水平，未能在体内建立成骨肉瘤细胞异种移植模型以鉴定成骨肉瘤干细胞的表型和功能(而这是肿瘤干细胞研究的金标准)；另一方面，SP细胞的分离方法存在hoechst33342染料毒性、细胞活力低下、仪器配置较高等局限性。因而分离和鉴定特异性的成骨肉瘤干细胞表面标记物是进行肿瘤干细胞针对性研究的技术基础。
SSEA-4的现有研究和应用：SSEA-4即stage specific embryonic antigen4，是属于阶段特异性胚胎抗原的一种，作为一种早期胚胎的糖脂类抗原，常用作为胚胎干细胞和间充质干细胞的表面标志物，但从未肿瘤干细胞的鉴定和筛选，本发明第一次将SSEA-4作为表面标志物，作为成骨肉瘤干细胞的鉴定。
发明内容
本发明的目的是提供一种人成骨肉瘤干细胞相关抗原标记物。
本发明的第二个目的是提供所述人成骨肉瘤干细胞相关抗原标记物SSEA-4在成骨肉瘤靶向治疗中的应用。
本发明利用前期建立的19个人成骨肉瘤跨物种移植模型和无血清培养技术，从体内和体外两方面，分离和鉴定出具有间充质干细胞和胚胎干细胞双重特征的成骨肉瘤表面标记物SSEA-4与肿瘤干细胞群体共存。表面标记物SSEA-4易于抗体标记，标记不影响细胞活力，适合分离后的功能实验；同时SSEA-4不仅提示成骨肉瘤可能起源于间充质干细胞阶段，为后续机制研究提供具体分子对象和模型平台。在临床上，在一部分病人中的表达程度，能够提示其恶性程度、转移及预后情况，是潜在的可作为靶向治疗的分子靶点。
由于成骨肉瘤病人原代标本跨物种移植成功率较低，所以我们尝试了大量的临床标本，最后得到3个动物模型。另外，我们检测了多个具有间充质干细胞和胚胎干细胞表面标记物，根据表达情况和文章报道的相关标记物，选择具有间充质干细胞和胚胎干细胞双重特征的SSEA-4作为骨肉瘤表面标记物后，遂加以证实，最后我们结合细胞系和病人临床标本，对标记物的特异性进行了验证。
SSEA-4是一特异性的成骨肉瘤干细胞表面标记物，在各正常成体组织中几乎不表达。此表面标记物易于抗体标记，不影响细胞活力，适合分离后的功能实验，为进一步研究干细胞在成骨肉瘤的起源、发生和发展过程的作用提供了一可靠的分子示踪物，为解释临床成骨肉瘤耐药、转移和寻找靶向治疗的细胞和分子靶点奠定了基础。
附图说明
下面结合说明书附图，对本发明进一步详细说明。
图1：成骨肉瘤细胞免疫表型的筛选。选择间充质干细胞和胚胎干细胞常用的表面标记物CD105、CD166、CD117、CD90、CD44、STRO-1、SSEA-4、SSEA-3等。
图2A：原代成骨肉瘤细胞表达SSEA-4和Oct-4；图2B：建立的成骨肉瘤细胞系5well表达SSEA-4和Oct-4。
图3：成骨肉瘤病理标本免疫组化显示SSEA-4、nanog、oct-4表达。
图4：成骨肉瘤原代细胞在体外无血清培养条件下形成sphere。
具体实施方式
I、我们在前期研究中，采用初发成骨肉瘤病人的原代标本，消化成单细胞悬液后，利用无血清培养技术和NOD/SCID小鼠成功建立了成骨肉瘤细胞球和跨物种移植模型。通过流式细胞分析技术，对取自成瘤小鼠的成骨肉瘤细胞进行表型分析，发现这些细胞经常表达SSEA-3、SSEA-4、CD184、CD105、CD166和CD49d等多个具有间充质干细胞和胚胎干细胞表面标记物。
II、对SSEA-4进行更加详尽的验证：利用细胞系，采用免疫荧光和免疫组化得方法，进行表型鉴定和验证，在原代标本中测试表达情况等。
III、通过对不同表面标记物之间更加细致的区分和比较，选取一小部分特异性最强的标记物，进行免疫荧光抗体标记，利用流式分选技术将不同表达情况的成骨肉瘤细胞分选出来，分别以梯度稀释的方式移植入NOD/SCID小鼠，通过对成瘤情况的比较分析，选定与成骨肉瘤致瘤能力最为相关的特异的表面标记物，即SSEA-4。
IV、将得到的成骨肉瘤原代细胞在体外无血清培养条件下，加入FGF、N2B27等细胞因子，能在体外形成成骨肉瘤细胞球(sphere)。
Claims (1)
1.SSEA-4在制备人成骨肉瘤干细胞相关抗原标记物中的应用。
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| CN110476892A (zh) * | 2019-09-09 | 2019-11-22 | 中南大学湘雅二医院 | 一种骨肉瘤异种移植小鼠模型的构建方法及应用 |
| CN110402895A (zh) * | 2019-09-09 | 2019-11-05 | 中南大学湘雅二医院 | 一种骨肉瘤异种移植小鼠模型的构建方法 |
| CN110495424B (zh) * | 2019-09-09 | 2022-09-27 | 中南大学湘雅二医院 | 一种滑膜肉瘤异种移植小鼠模型的构建方法 |
| CN110476891B (zh) * | 2019-09-09 | 2022-09-23 | 中南大学湘雅二医院 | 一种滑膜肉瘤异种移植小鼠模型的构建方法及其应用 |
| CN110495422B (zh) * | 2019-09-23 | 2022-12-20 | 中南大学湘雅二医院 | 一种恶性神经鞘瘤异种移植小鼠模型的构建方法及其应用 |
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| CA2013544A1 (en) * | 1989-03-31 | 1990-09-30 | Yuen Kai Fung | Retinoblastoma gene product antibodies and uses therefor |
| CN1331140A (zh) * | 2000-06-26 | 2002-01-16 | 上海博德基因开发有限公司 | 一种新的多肽——人成视网膜细胞瘤结合蛋白(rbbp9)25.52和编码这种多肽的多核苷酸 |
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| CA2013544A1 (en) * | 1989-03-31 | 1990-09-30 | Yuen Kai Fung | Retinoblastoma gene product antibodies and uses therefor |
| CN1331140A (zh) * | 2000-06-26 | 2002-01-16 | 上海博德基因开发有限公司 | 一种新的多肽——人成视网膜细胞瘤结合蛋白(rbbp9)25.52和编码这种多肽的多核苷酸 |
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