CN110747170A - 敲减cas的乳腺癌细胞模型及利用其的细胞学实验 - Google Patents
敲减cas的乳腺癌细胞模型及利用其的细胞学实验 Download PDFInfo
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Abstract
本发明提供一种敲减CAS的乳腺癌细胞模型及敲减CAS后发生显著改变的基因组合。该细胞模型和基因组合能够应用于制备Luminal A型乳腺癌的诊断和治疗制剂中。敲减CAS的乳腺癌细胞模型利用慢病毒转染技术敲减Luminal A型乳腺癌细胞系MCF‑7的CAS构建而成。构建方法包括如下步骤：将乳腺癌细胞MCF‑7以2×105种于6孔板中，次日细胞贴壁后，按照病毒使用说明，每孔加入4uL慢病毒和终浓度5ug/mL polybrene，48h后加入嘌呤霉素筛选，一周后构建成稳定敲减CAS的乳腺癌细胞模型。本发明利用慢病毒转染技术敲减乳腺癌细胞的CAS，建立敲减CAS的细胞模型，通过细胞活力实验表明敲减CAS显著抑制细胞的活力，深入探讨CAS调控乳腺癌增殖和存活的分子机制，为寻找Luminal A型乳腺癌发病机理提供新的研究方法。
Description
技术领域
本发明涉及生物医学技术领域，具体涉及一种敲减CAS的乳腺癌细胞模型及利用其的细胞学实验，本发明用于非治疗目的。
背景技术
乳腺癌是最常见的女性恶性肿瘤，严重威胁着女性的健康。乳腺癌是一种高度异质性的恶性肿瘤，在组织形态、免疫表型、生物学行为、治疗反应上都存在着极大的差异。1999年美国国立癌症研究所提出肿瘤分子分型的概念，即通过综合的分子分析技术，使肿瘤的分类基础由形态学转向以分子特征为基础的新的肿瘤分类系统（Weili H & WNingxia . J Clin Exp Pathol 2012 May; 28(5).）。Perou（Pemu CM, Sorlie T, EisenMB, et a1. Comprehensive molecular portraits of human breast tumorsl l[J].Nature, 2000, 406(6797): 747~752.）等在2000年提出了乳腺癌的分子分型这一学说，通过对3种免疫表型ER、PR、Her-2，将乳腺癌分为5型：管腔A型、管腔B型、（Her-2）过表达型、基底细胞样型及正常乳腺样型。2011年在St．Gallen会议上专家组达成了共识：可根据IHC对ER、PR、Her-2和低表达增殖细胞核抗原-67（Ki-67）指标的检测结果，将乳腺癌分为Luminal A型、Luminal B型、Her-2阳性和三阴性乳腺（TNBC）4个类型，作为一种简单的近似替代方法，特称为“临床病理分型”（Goldhirseh A, Wood WC, Coates AS, et a1.Strategies for subtypes dealing with the diversity of breast cancer：highlights of the St. Gallen International Expert Consensus on the PrimaryTherapy of Early Breast Cancer 2011[J]. Ann Oncol, 2011，22(8)：1736—1747．）。2013年St．Gallen会议提出了临床病理替代分子分型分为Luminal A型、Luminal B型、Her-2过表达型、基底细胞型（三阴型乳腺癌属于此型）4个类型，各分子亚型间在基因特征、发病年龄、临床特征、恶性程度、治疗敏感性及预后等方面均存在差异。在4种分子分型中，Luminal A型乳腺癌最常见的分子亚型，Ihemelandu等（Rouzier R，Perou CM，Symmans WF，et a1．Breast cancer molecular subtypes respond differently to preoperativechemotherapy[J]，Clin Cancer Res，2005，11(16)：5678-5685）的研究结果显示，乳腺癌患者中Luminal A型占50％，Luminal B型、Her-2(+)型及基底细胞型分别占14.1％、12.7％和23.2％。Luminal A型乳腺癌病理IHC表达情况为：ER/PR阳性，且PR高表达（≥20%）；HER2阴性；Ki-67低表达，高表达CK18、CK8及AR，研究其治疗手段对降低乳腺癌的死亡率具有重要意义。
乳腺癌的治疗方法除了传统的三大治疗手段：手术治疗、放疗和化疗，还包括内分泌治疗和靶向治疗。Luminal A型也称激素依赖型乳腺癌，目前多采用内分泌治疗手段，虽然其对内分泌治疗敏感，但内分泌治疗的周期长，患者需要面对长期服药的痛苦。靶向疗法具有特异性强、疗效显著、毒副反应小等优点。目前乳腺癌靶向治疗所针对的靶点和通路主要包括HER-2、VEGF、EGFR、PARP、PI3K/Akt/mTOR、CDK4/6等。因此，研究靶向治疗Luminal A型乳腺癌的靶点和通路具有重要意义。
细胞凋亡易感基因(cellular apoptosis susceptibility gene, CAS/CSE1L)，与酵母染色体分离基因(CSE1)同源，最初在乳腺癌细胞MCF-7细胞中发现，它位于人的染色体20q13，包含25个外显子，CAS蛋白分子量约为110 KD，共由971个氨基酸组成。CAS除了作为核转录因子介导转入蛋白的核转运，参与调节细胞凋亡、增殖和有丝分裂，还参与微泡形成以及癌转移，并在早期胚胎生长发育过程中发挥关键作用。CAS被报道在多种癌症中高表达，例如乳腺癌、精原细胞瘤、黑色素瘤、骨肉瘤、肝癌、大肠癌、少突神经胶质瘤、卵巢癌等。虽然CAS最初是在乳腺癌细胞系中发现的，但关于CAS 如何调控MCF-7细胞的增殖和凋亡的机制，迄今为止仍不清楚。
发明内容
本发明要解决的技术问题是提供一种敲减CAS的乳腺癌细胞模型及利用其的细胞学实验，利用慢病毒介导的CAS基因沉默技术建立敲减CAS的乳腺癌细胞模型，为寻找Luminal A 型乳腺癌发病机理提供新的研究方法。
为解决上述技术问题，本发明的实施例提供一种敲减CAS的乳腺癌细胞模型，利用慢病毒转染技术敲减Luminal A型乳腺癌细胞系MCF-7的CAS构建而成。
本发明还提供一种敲减CAS的乳腺癌细胞模型的构建方法，包括如下步骤：将乳腺癌细胞MCF-7以2×105种于6孔板中，次日细胞贴壁后，按照病毒使用说明，每孔加入4uL慢病毒和终浓度5ug/mL polybrene，48h后加入嘌呤霉素筛选，一周后构建成稳定敲减CAS的乳腺癌细胞模型。
本发明还提供一种基于敲减CAS的乳腺癌细胞模型的细胞学实验，包括细胞迁移和侵袭实验、细胞活力实验，其中，所述细胞迁移和侵袭实验包括如下步骤：
（1-1）将上述的细胞模型用无血清培养基重悬后以一定细胞数量接种于Corning CellCulture Insert/Corning Matrigel Matrix 的Transwell中，24孔板下室添加含10%FBS的培养基；
（1-2）放置于37℃、5% CO2培养箱中培养24～48小时后，4%PFA固定10分钟，PBS清洗两遍后，苏木精染色10分钟，洗去Insert /Transwell内侧细胞，于显微镜下拍照并统计穿过Insert /Transwell的细胞数量，完成细胞迁移和侵袭实验。
其中，所述细胞活力实验包括如下步骤：
（2-1）上述的细胞模型以2000个细胞/孔的密度，接种于培养板中，于37℃、5% CO2 培养箱中培养24小时后，加入CCK8工作液，CO2 箱温育1-2小时；
（2-2）酶标仪检测OD值，比较细胞的活力情况，完成细胞活力实验。
本发明还提供一种敲减CAS的乳腺癌细胞模型的转录组测序，得到基因改变的组合；转录组测序包括如下步骤：收集敲减CAS 后6天的对照组细胞系MCF-7-LV-U6和实验组细胞系MCF-7-shCAS，保存于TRIZOL 置于-80度冰箱，提总RNA进行质量检测， 选取260/280在1.8-2.0之间且琼脂糖凝胶电泳检测28S:18S至少大于1.8的样本，外送至测序公司，由测序公司进行构建文库、上机测序等步骤，测序完成后将获取的测序数据发回，进行解读及后续分析。
本发明的上述技术方案的有益效果如下：
本发明利用慢病毒转染技术敲减细胞的CAS，建立敲减CAS的细胞模型，通过细胞活力实验表明敲减CAS显著抑制细胞的活力，深入探讨CAS调控乳腺癌增殖和存活的分子机制，为寻找Luminal A 型乳腺癌发病机理提供新的研究方法。
附图说明
图1为本发明中细胞迁移和侵袭实验结果图；
图2为本发明中细胞活力实验结果图；
图3为本发明中转录组测序结果图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
本发明提供一种敲减CAS的乳腺癌细胞模型，利用慢病毒转染技术敲减Luminal A型乳腺癌细胞系MCF-7的CAS构建而成。
上述的敲减CAS的乳腺癌细胞模型的构建方法包括如下步骤：将乳腺癌细胞MCF-7以2×105种于6孔板中，次日细胞贴壁后，按照病毒使用说明，每孔加入4uL慢病毒和终浓度5ug/mL polybrene，48h后加入嘌呤霉素筛选，一周后构建成稳定敲减CAS的乳腺癌细胞模型。
本发明还提供一种基于敲减CAS的乳腺癌细胞模型的细胞学实验，包括细胞迁移和侵袭实验、细胞活力实验，其中，所述细胞迁移和侵袭实验包括如下步骤：
（1-1）将上述的细胞模型用无血清培养基重悬后以一定细胞数量接种于Corning CellCulture Insert (Transparent PET Membrane 24 Well 8.0 um pore size)/CorningMatrigel Matrix 的Transwell中，24孔板下室添加含10%FBS的培养基；
（1-2）放置于37℃、5% CO2培养箱中培养24～48小时后，4%PFA固定10分钟，PBS清洗两遍后，苏木精染色10分钟，洗去Insert /Transwell内侧细胞，于显微镜下拍照并统计穿过Insert /Transwell的细胞数量，完成细胞迁移和侵袭实验。
图1所示为细胞迁移和侵袭实验结果图，数据表明敲减CAS 对MCF-7细胞的迁移和侵袭无影响。
所述细胞活力实验包括如下步骤：
（2-1）上述的细胞模型以2000个细胞/孔的密度，接种于培养板中，于37℃、5% CO2 培养箱中培养24小时后，加入CCK8工作液，CO2 箱温育1-2小时；
（2-2）酶标仪检测OD值，比较细胞的活力情况，完成细胞活力实验。
图2所示为细胞活力实验结果图，表明敲减CAS 显著抑制细胞的活力。
本发明还提供一种敲减CAS的乳腺癌细胞模型的转录组测序 （RNA-seq）后的基因组合。包括如下步骤：收集敲减CAS 后6天的对照组细胞系MCF-7-LV-U6和实验组细胞系MCF-7-shCAS，保存于TRIZOL 置于-80度冰箱，提总RNA然后进行转录组测序。转录组测序数据显示：敲减CAS后，Luminal A型乳腺癌细胞MCF-7中CYP24A1、INHA、TFPI2、NR4A1、FOSB和MC4R的表达量显著上调。同时，SELENBP1、NECAB1、TAF13、PMP22和ADM2的表达显著下调。
图3所示为RNA-seq 结果图，在MCF-7细胞系中敲减CAS后差异基因热图。(MCF_NC_1和MCF_NC_2和MCF_NC_3为MCF-7-LV-U6 的三个独立重复样本，MCF_sh1、MCF_sh2和MCF_sh3为MCF-7-ShCAS 的三个独立重复样本。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种敲减CAS的乳腺癌细胞模型，其特征在于，利用慢病毒转染技术敲减Luminal A型乳腺癌细胞系MCF-7的CAS构建而成。
2.一种如权利要求1所述的敲减CAS的乳腺癌细胞模型的构建方法，其特征在于，包括如下步骤：将乳腺癌细胞MCF-7以2×105种于6孔板中，次日细胞贴壁后，按照病毒使用说明，每孔加入4uL慢病毒和终浓度5ug/mL polybrene，48h后加入嘌呤霉素筛选，一周后构建成稳定敲减CAS的乳腺癌细胞模型。
3.一种基于如权利要求1所述的敲减CAS的乳腺癌细胞模型的细胞学实验，其特征在于，包括细胞迁移和侵袭实验、细胞活力实验，其中，所述细胞迁移和侵袭实验包括如下步骤：
（1-1）将权利要求1所述的细胞用无血清培养基重悬后以一定细胞数量接种于CorningCell Culture Insert/Corning Matrigel Matrix 的Transwell中，24孔板下室添加含10%FBS的培养基；
（1-2）放置于37℃、5% CO2培养箱中培养24～48小时后，4%PFA固定10分钟，PBS清洗两遍后，苏木精染色10分钟，洗去Insert /Transwell内侧细胞，于显微镜下拍照并统计穿过Insert /Transwell的细胞数量，完成细胞迁移和侵袭实验。
4.根据权利要求3所述的细胞学实验，其特征在于，所述细胞活力实验包括如下步骤：
（2-1）权利要求1所述的细胞以2000个细胞/孔的密度，接种于培养板中，于37℃、5%CO2 培养箱中培养24小时后，加入CCK8工作液，CO2 箱温育1-2小时；
（2-2）酶标仪检测OD值，比较细胞的活力情况，完成细胞活力实验。
5.一种敲减CAS的乳腺癌细胞模型的转录组测序，其特征在于，包括如下步骤：收集敲减CAS 后6天的对照组细胞系MCF-7-LV-U6和实验组细胞系MCF-7-shCAS，保存于TRIZOL 置于-80度冰箱，提总RNA进行质量检测，选取260/280在1.8-2.0之间且琼脂糖凝胶电泳检测28S:18S至少大于1.8的样本，外送至测序公司，由测序公司进行构建文库、上机测序等步骤，测序完成后将获取的测序数据发回，进行解读及后续分析。
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