CN100572557C - 基于组织培养的表达图谱 - Google Patents

基于组织培养的表达图谱 Download PDF

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CN100572557C
CN100572557C CNB2003801086762A CN200380108676A CN100572557C CN 100572557 C CN100572557 C CN 100572557C CN B2003801086762 A CNB2003801086762 A CN B2003801086762A CN 200380108676 A CN200380108676 A CN 200380108676A CN 100572557 C CN100572557 C CN 100572557C
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姜平
徐明旭
谭玉英
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Abstract

从组织样品获得可靠表达文库的方法，包括从完整组织中提取RNA，所述组织是基于组培的在三维海绵凝胶中培养的。

Description

基于组织培养的表达图谱
相关申请的交叉引用
本申请依据35U.S.C.§119(e)要求2002年11月12日申请的美国系列(Serial)号No60/425,945的权益。该申请的内容在此引入作为参考。
技术领域
本发明涉及样品尤其是肿瘤样品的制备，作为表达分析的mRNA来源。
背景技术
组织培养的肿瘤样品用于肿瘤发展的预后和作为预测药物应答性的工具已经有很长的历史了。作为组织培养药物应答试验(HDRATM)基础的这些组织化学技术有很多文献。如这样应用，将描述三维组织培养用途的参考书目附于本申请作为附录。例如，在Singh，B.，等最近的论文Head and Neck(2002)24：437-442中，该技术的总的特征得到了描述。如该论文中所述的，简要地，洗涤活体检查的组织并切成1至2mm3的碎块，等量放置在0.5cm3的胶原海绵凝胶(Gel Foam，Pharmacia & Upjohn，Inc.)块上。然后将海绵凝胶培养物放入含有10％胎牛血清和庆大霉素(50μg/ml)的DMEM/Ham’s F12培养基中。接着，将培养物在37℃和5％CO2下孵育24小时。对该技术进行改造也是容许的，提供的样品三维特征得到保存。
已经发现除了作为预后和药物筛选工具的用途以外，这样的培养物也可以用作表达图谱(expression profiling)底物的信使RNA的来源。考虑提供可靠表达文库相关的问题是重要的，尤其是源自患者样品时，鉴于存在于肿瘤中的坏死区域和鉴于需要将肿瘤组织从治疗或诊断中心转移至能够进行图谱分析的实验室，其中高质量、无降解RNA的提取是困难的。通过将肿瘤非坏死部分维持在三维组织培养中，肿瘤原位表达图谱得到有效地保存。
发明公开
本发明提供了制备表征组织尤其是肿瘤组织表达图谱mRNA文库的方法，以便表征组织的特性。在肿瘤分析的情况中，该图谱有助于设计治疗，尤其是和具有相似表达模式的组织样品(historical sample)相比较，该组织样品对特定方案的应答性是已知的。如涉及特定性组织来源的特征表达图谱的其它用途对本领域技术人员是显而易见的。
因此，一方面，本发明涉及制备表征组织表达RNA的方法，该方法包括，在海绵凝胶三维培养中培养完整组织样品后，从所述培养物中提取RNA。
然后使用任何本领域已知技术如Northern印迹来分析提取的信使RNA。然而，优选将提取的mRNA用作模板来制备cDNA文库，接着使用已知的如那些基于基因芯片的矩阵技术来分析。另一方面，本发明涉及通过本发明方法制得的mRNA、cDNA和cRNA文库和将这些文库用于预后和治疗选择的方法。
实施发明的方式
本发明解决了用于表达图谱的恰当保存的组织样品，尤其是肿瘤组织样品的问题。目前，在活组织检查和提取用于分析的RNA的间隔中，将活组织检查样品进行RNA降解并改变了表达模式。通过在三维组织培养中维持组织完整性，保存了表达图谱的准确度并最小化了RNA的降解。
本发明方法中，使用常规技术将组织活体检查，然后分成约1mm3大小的完整部分。当然允许样品大小有一些改变，例如，使用0.25mm3-5mm3、0.5-3mm3的块，优选1-2mm3。然后将完整的组织块放入三维组织培养中，通常将完整样品和胶原海绵凝胶(collagen sponge-gel)结合，如Singh，B.，等(上文)描述的和许多其它论文评论的那些，例如Hoffman，R.M.，等，Int’lJ.Oncol.(1992)1：467-474；Hoffman，R.M.，Encyclopedia of Life Sciences(生 命科学百科全书)(2001)Nature Publishing Group，London.，以及本领域通常所知的HDRATM实验。用于本发明方法的胶原型海绵凝胶是用作组织的支持物，因此海绵凝胶的大小基本上大于碎块的大小；通常，海绵凝胶的表面积大概是完整组织样品直径的两倍。然后在合适的培养基如所附出版物中所述的培养基中维持三维培养。按需要保持保存用于RNA提取的样品的时间来保持培养。
使用本领域的标准技术进行RNA提取。
为表达文库来源的组织样品通常是肿瘤组织。因此，本发明方法特别地用于评价乳房、肺、结肠、肝脏、胃、胰腺、前列腺、头和颈、卵巢，和脑肿瘤的表达图谱。该列举不是穷举，如任何固体肿瘤或，实际上，任何组织可以用作文库的来源。
然后根据研究者的需要分析提取的RNA。可以使用Northern印迹技术，但是使用商业上可获得的表达矩阵例如目前从Affymetrix可获得的表达矩阵可以获得另外的信息。需要大约15μg标记的cDNA。通常，通过反转录将提取的RNA转化成cDNA，最常规通过寡-dT引物和T7RNA聚合酶启动子来引发。将所得到的单链cDNA转化成双链DNA，其因此产生适于T7聚合酶启动的体外转录(IVT)模板。在体内转录过程中整合标记的核苷酸，来许可所得到cRNA随后的检测。然后，将所得到的转录产物cRNA用于提供根据芯片上的DNA矩阵检测到的图谱。
在随后的检测中，通过在含有Mg++缓冲液中加热将标记的cRNA片断化，并在含有鲑鱼精子DNA、BSA和有标记的(spiked)对照RNA的杂交混合物中结合。如Affymetrix Gene Chip Expression Analysis Technical Manual(Affymetix基因芯片表达技术手册)中所述的(2001)，将大约250μl混合物运用至GeneChipTM并在50℃杂交16小时。如所述的，杂交后，使芯片通过高度和低严谨性洗涤，接着用生物素化的抗-链霉抗生物素蛋白抗体(Vector Labs)放大(amplification)的藻红蛋白标记的链霉抗生物素蛋白(分子探针)抗体染色，和用藻红蛋白标记的链霉抗生物素蛋白另外进行染色。进一步洗涤后，将矩阵在Affeymetrix扫描仪中数字化并使用MicroarraySuite 5TM软件评价图像。
之前所述的仅仅是用以分析从根据本发明方法培养物中提取的RNA的多种技术的实例。
从文库成分测定获得的数据可以有效地用于预测切除肿瘤患者的结果，根据本发明方法分析，还可以用于设计治疗方案，以及预测化学敏感性。

Claims (7)

1.制备组织表达谱的方法，该方法包括下述步骤：从所述组织的完整样品中提取RNA，所述组织是使用三维胶原海绵凝胶培养进行培养的。
2.权利要求1的方法，其进一步包括将所述RNA进行分析以获得表达数据。
3.权利要求1的方法，其进一步包括将提取的RNA转化成cDNA。
4.权利要求3的方法，其进一步包括从所述cDNA制备标记的cRNA并使用微矩阵分析来分析所述cRNA。
5.权利要求1的方法，其中所述组织是肿瘤组织。
6.权利要求5的方法，其中所述肿瘤是乳房、肺、结肠、肝脏、胃、胰腺、前列腺、头、颈、卵巢，或脑的肿瘤。
7.根据权利要求2的方法，其进一步包括基于所述数据制定预后。
CNB2003801086762A 2002-11-12 2003-11-12 基于组织培养的表达图谱 Expired - Fee Related CN100572557C (zh)

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Publication number Priority date Publication date Assignee Title
US5726009A (en) 1989-03-20 1998-03-10 Anticancer, Inc. Native-state method and system for determining viability and proliferative capacity of tissues in vitro
DE69228055T2 (de) 1991-02-28 1999-05-20 Anticancer Inc., San Diego, Calif. Ein gewebekulturverfahren für haut im natürlichen zustand
US5474909A (en) * 1992-08-07 1995-12-12 Anticancer, Inc. Noncolorimetric histoculture method for predicting drug response of tumors
AU7357894A (en) * 1993-06-29 1995-01-24 Anticancer, Inc. Native-state method and system for determining viability and proliferative capacity of tissues (in vitro)
US6203984B1 (en) * 1998-07-02 2001-03-20 Pacron, Inc. Proportional amplification of mRNA from a linear template in vitro

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Early Shifts in Gene Expressionduring Chondroinduction of Human Dermal Fibroblasts. Karen E. Yates.Experimental Cell Research,Vol.265 . 2001
Early Shifts in Gene Expressionduring Chondroinduction of Human Dermal Fibroblasts. Karen E. Yates.Experimental Cell Research,Vol.265 . 2001 *
In Vivo Gene Expression Profile Analysisof Human Breast Cancer Progression. Dennis C. Sgroi.CANCER RESEARCH,Vol.59 . 1999
In Vivo Gene Expression Profile Analysisof Human Breast Cancer Progression. Dennis C. Sgroi.CANCER RESEARCH,Vol.59 . 1999 *

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AU2003294267A1 (en) 2004-06-03
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ATE429514T1 (de) 2009-05-15
CN1738910A (zh) 2006-02-22
KR20050063805A (ko) 2005-06-28
CA2505895A1 (en) 2004-05-27
US7252941B2 (en) 2007-08-07
WO2004044177A2 (en) 2004-05-27
WO2004044177A3 (en) 2005-03-17
CA2505895C (en) 2013-10-22
US20080058222A1 (en) 2008-03-06
KR101142716B1 (ko) 2012-05-04

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