CN114634913A - 肿瘤硬脑膜转移模型的构建方法 - Google Patents
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Abstract
本发明属于生物技术领域,具体为肿瘤硬脑膜转移模型的构建方法。本发明方法包括:乳腺癌细胞株(4T1)、肺癌细胞株(LLC)和人胚胎肾细胞株(293T)的培养;慢病毒包装;慢病毒感染;共表达荧光素酶和新霉素基因的乳腺癌细胞系和肺癌细胞系的获得;乳腺癌和肺癌硬脑膜转移小鼠模型的构建。硬脑膜转移是恶性肿瘤颅内转移的一种,可导致脑水肿、脑脊液循环障碍等多种神经系统症状。乳腺癌和肺癌是硬脑膜转移瘤常见的原发肿瘤类型,当前尚无肿瘤硬脑膜转移的小鼠模型。本发明建立乳腺癌和肺癌硬脑膜转移小鼠模型,为进一步研究其发病机制、筛选新药靶点提供重要的实验模型依据。
Description
技术领域
本发明属于生物技术领域,具体涉及硬脑膜转移瘤小鼠模型的构建方法。
背景技术
颅内转移是恶性肿瘤患者最常见的神经系统并发症,其中硬脑膜转移是指肿瘤细胞转移到硬脑膜或硬膜下隙,可引起多种严重神经系统症状,临床预后极差。约9%的晚期癌症患者在尸检中发现了硬脑膜转移,其中常见的原发肿瘤类型为乳腺癌、肺癌和前列腺癌[1]。
硬脑膜转移瘤(Dural Metastasis,DrM)模型由于受颅骨限制而向内压迫脑实质,引起脑水肿、脑脊液循环不畅等后果,还可导致多种神经系统症状,包括头痛、脑神经病变、视力下降、精神状态改变、颅内压升高等。此外,硬脑膜转移可同时合并脑实质转移或软脑膜转移;硬脑膜肿瘤新生血管破裂又可进一步引起硬膜下血肿[2]。随着肿瘤治疗的深入研究、转化应用以及神经影像学技术发展,癌症患者预期寿命逐渐增加,恶性肿瘤硬脑膜转移的发生率以及检出率随之增加,临床上应给予足够重视。目前迫切需要建立硬脑膜转移瘤动物模型来进一步探究其发病机制。
此外,硬脑膜本身除了保护脑实质、分隔颅腔等结构作用以外[3],还发挥着免疫监视的功能。自2015年发表在Nature的文章揭示了人硬脑膜的内层中存在功能性的淋巴管后[4],硬脑膜的免疫微环境受到广泛关注[5]。首先,近期研究发现硬脑膜窦是T细胞和B细胞发挥脑膜免疫功能的重要场所[6, 7]。其次,紧邻硬脑膜窦的硬脑膜淋巴管作为中枢神经系统与外周免疫系统连接的桥梁,可清除CNS来源的代谢废物、传送CNS来源的抗原、运输外周免疫细胞,从而对神经炎症、神经退行性疾病及脑肿瘤发挥重要的免疫监视功能[8-10]。除了淋巴管结构外,最新研究显示硬脑膜在正常生理情况下存在丰富的免疫细胞,主要为淋巴样细胞和髓样细胞,其中还包括B淋巴细胞祖细胞[11-13]。病理情况下,这些免疫细胞分化、成熟、迁移,构成硬脑膜动态的免疫微环境。然而,对于硬脑膜在肿瘤生成和转移过程中发挥的作用知之甚少。开发硬脑膜转移瘤小鼠模型有助于肿瘤发生发展与病因学全貌的动态分析,充分解析肿瘤微环境因素以及关键病理分子作用机制,从而填补该领域的认知缺口。
本发明提供了一种高效且稳定的硬脑膜转移瘤造模方法,建立乳腺癌和肺癌硬脑膜转移动物模型,为进一步研究硬脑膜转移瘤发病机制提供重要的实验模型依据。
发明内容
本发明的目的在于提供一种高效、稳定且能够广泛应用的肿瘤硬脑膜转移小鼠模型的构建方法,并将该模型用于乳腺癌和肺癌硬脑膜转移瘤的机制研究。
本发明提供的肿瘤硬脑膜转移模型的构建方法,包括:乳腺癌细胞株(4T1)、肺癌细胞株(LLC)和人胚胎肾细胞株(293T)的培养;慢病毒包装;慢病毒感染;共表达荧光素酶和新霉素基因的乳腺癌细胞系和肺癌细胞系的获得;乳腺癌和肺癌硬脑膜转移小鼠模型的构建;具体步骤如下。
(1)细胞培养
将本发明使用的乳腺癌细胞株(4T1)在细胞培养皿中用RPMI-1640完全培养基培养,肺癌细胞株(LLC)在细胞培养皿中用DMEM完全培养基培养;使用的包装慢病毒的人胚胎肾细胞株(293T)在细胞培养皿中用DMEM完全培养基培养;4T1、LLC和293T细胞放在无菌培养箱中培养,培养箱保持35℃-37℃的温度,CO2浓度维持在4%-6%,并且培养箱中需要始终含有无菌的灭菌水以保证培养箱内的湿度,使细胞更好地生长。
(2)病毒包装
本发明构建了可同时稳定表达荧光素酶(Luciferase,Luc)以及新霉素抗性基因的慢病毒转移质粒载体(pCDH-Luc-Neo);相关慢病毒转移质粒载体(pCDH-Luc-Neo)以及包装慢病毒载体(psPAX2、pMD2.G)经过高纯度无内毒素质粒提取试剂盒提取后,使用转染试剂共转染293T细胞,共转染时293T细胞融合度为30-50%,转染后6-10小时及时更换为完全培养基,36-72小时后,收集并过滤病毒上清液。
(3)病毒感染
将肿瘤细胞(4T1、LLC)平铺于细胞培养皿中,使细胞密度为30%-70%;将病毒上清加入培养皿中,病毒上清的体积和完全培养基的体积比在0.5-2之间;加入2-5 µg/mLpolybrene(聚凝胺), 充分摇匀后置于无菌培养箱中培养;病毒感染后12-24小时换液,待细胞长满后传代并用荧光素酶的底物(Luciferin)检测荧光素酶的表达。
(4)G418药物筛选
慢病毒感染后的肿瘤细胞通过扩增培养,以及G418药物筛选,获得表达新霉素抗性基因的肿瘤细胞系(称为parental细胞,简称Par细胞);该细胞系可以同时稳定表达荧光素酶以及新霉素抗性基因,其中荧光素酶的表达可用于生物发光成像,以便实时监测肿瘤位置和肿瘤负荷。
(5)硬脑膜转移模型构建
利用脑立体定位仪固定小鼠头部,正中剪开头皮暴露颅骨;用颅钻在颅骨矢状缝两侧顶骨中央磨出圆形颅窗,使硬脑膜充分暴露;使用微量进样针:
将2×104-1×105个Par细胞注射到硬脑膜,进行硬脑膜驯化;待种植的Par细胞适应硬脑膜环境,具备增殖能力导致小鼠出现行动迟缓、体重骤减的现象,对小鼠实施安乐死;收集硬脑膜上的肿瘤细胞,建立体外培养细胞系,该细胞称为硬脑膜驯化细胞1(adaptiveround 1),简称Adap-R1;
将2×104-1×105个Adap-R1细胞注射到硬脑膜上,待Adap-R1细胞适应硬脑膜环境,具备增殖能力导致小鼠出现行动迟缓、体重骤减的现象,对小鼠实施安乐死;收集硬脑膜上的肿瘤细胞,建立体外培养细胞系,该细胞称为硬脑膜驯化细胞2,简称Adap-R2;
将2×104-1×105个D-inject 2细胞注射到硬脑膜上,待Adap-R2细胞适应硬脑膜环境,具备增殖能力导致小鼠出现行动迟缓、体重骤减的现象,对小鼠实施安乐死;收集硬脑膜上的肿瘤细胞,建立体外培养细胞系,该细胞称为硬脑膜驯化细胞3,简称Adap-R3;Adap-R3细胞可较好的适应硬脑膜微环境;
将1×104-1×105个Adap-R3细胞和1×104-1×105个Par细胞分别注射到小鼠的颈动脉,细胞通过血液播散进入到颅内中,通过生物发光成像监测肿瘤进展和肿瘤位置,待小鼠出现硬脑膜转移现象,对小鼠实施安乐死;收集硬脑膜中生长的原代肿瘤细胞,建立体外培养细胞系,将该细胞系称为硬脑膜转移细胞系(duralmetastasis cell,简称DrM细胞);DrM细胞是可以通过血液播散稳定形成硬脑膜转移灶的细胞,将DrM细胞注射到小鼠的颈动脉后可获得硬脑膜转移瘤小鼠模型。
本发明提出的建模方法中,乳腺癌细胞以及肺癌细胞经过三轮的硬脑膜驯化,即可经颈动脉注射获得乳腺癌及肺癌硬脑膜转移瘤模型。
本发明具有以下优点:
(1)肿瘤细胞经过多轮硬脑膜驯化,对硬脑膜微环境的适应性高;
(2)肿瘤细胞带有抗性基因,便于通过药物进行筛选;
(3)模型构建成功率高;将DrM细胞注射到小鼠颈动脉,经过血液播散,80%以上的个体出现肿瘤硬脑膜转移。
附图说明
图1为本发明通过生物发光成像对乳腺癌细胞(4T1)在小鼠体内形成的肿瘤负荷进行定期监测。其中,A为第一轮硬脑膜筛选的小鼠不同时期的肿瘤进展情况,B为第二轮硬脑膜筛选的小鼠不同时期的肿瘤进展情况,C为第三轮硬脑膜筛选的小鼠不同时期的肿瘤进展情况,D为每轮筛选过程中小鼠的生长曲线。
图2为本发明通过生物发光成像对肺癌细胞(LLC)在小鼠体内形成的肿瘤负荷进行定期监测。其中,A为第一轮硬脑膜筛选小鼠不同时期的肿瘤进展情况,B为第二轮硬脑膜筛选小鼠不同时期的肿瘤进展情况,C为第三轮硬脑膜筛选小鼠不同时期的肿瘤进展情况,D为每轮筛选过程中小鼠的生长曲线。
图3为本发明将4T1-Par细胞和4T1- Adap-R3细胞注射到小鼠颈动脉后肿瘤细胞硬脑膜转移效果图。其中,A显示Par细胞转移到脑实质,B显示Adap-R3细胞可转移到硬脑膜。白色箭头代表硬脑膜位置。
图4为本发明将LLC-Par细胞和 LLC- Adap-R3细胞注射到小鼠颈动脉后肿瘤细胞硬脑膜转移效果图。其中,A显示Par细胞转移到脑实质,B显示Adap-R3细胞可转移到硬脑膜。白色箭头代表硬脑膜位置。
图5为DrM细胞硬脑膜转移的3D生物发光图和HE染色图。其中,A为3D成像显示肿瘤细胞位于硬脑膜,白色箭头指示硬脑膜位置。B为HE染色显示肿瘤细胞生长在硬脑膜。黑色箭头指示颅骨,红色箭头指示颅内硬脑膜,黑色三角形指示硬脑膜肿瘤。
具体实施方式
实施例1
步骤1:
在直径10cm细胞培养皿中,使用RPMI-1640完全培养基培养(含10% FBS以及1% 青霉素-链霉素溶液)培养4T1乳腺癌细胞株,用DMEM完全培养基培养(含10% FBS以及1% 青霉素-链霉素溶液)培养LLC肺癌细胞和293T细胞。细胞培养箱温度为37℃,CO2浓度为5%。
步骤2:
将第2代细胞293T细胞平铺到10cm细胞培养皿中,细胞密度为70%。使用Lipofectamine2000转染试剂将pCDH-Luc-Neo、psPAX2、pMD2.G三种慢病毒质粒按照质量比4:3:1共转染到293T细胞中。转染之后6小时换液,转染之后48小时收取病毒上清并且用0.45μm滤头过滤,放在4℃保存。
步骤3:
将乳腺癌细胞和肺癌细胞分别平铺到6孔板中,细胞密度为30%,将步骤2中纯化的病毒上清加入6孔板中,病毒体积和细胞培养基体积比为1:1。观察细胞的生长状态以及荧光情况,将病毒感染的细胞用G418进行筛选,观察细胞的生长状态。
步骤4:
用1×PBS重悬Par细胞,使其密度为4×106个/mL,置于冰上待用。将小鼠用气体麻醉箱麻醉并小心剃除头部的鼠毛。将小鼠头部固定于脑立体定位仪,正中剪开头皮暴露颅骨;用颅钻在颅骨矢状缝两侧顶骨中央顺时针磨出一个圆形颅窗,暴露硬脑膜。使用微量进样针将10 μL悬浮乳腺癌细胞或肺癌细胞的1×PBS缓慢注射到硬脑膜上,注射速度为1µL/分钟。注射完毕后停针10分钟,待细胞充分进入硬脑膜后移开进样针。缝合头皮,手术结束后将小鼠放在加热垫上,等待其苏醒。
通过生物发光成像(BLI)监测个体的肿瘤负荷,并每天观察小鼠健康状况。待小鼠头部出现明显肿瘤信号(如图1所示,为石蜡切片后经Hematoxylin&Eosin染色显示肿瘤在硬脑膜部位生长),体重骤减,行动迟缓,对小鼠实施安乐死。剥离硬脑膜,使用胶原酶消化硬脑膜细胞,充分收集硬脑膜转移的细胞。细胞以200 g的速度离心5分钟,4T1细胞重悬于RPMI-1640完全培养基,LLC细胞重悬于DMEM完全培养基,平铺到30 mm的细胞培养皿中,每两天更换含有G418的培养基。传代后获取稳定表达荧光素酶和新霉素抗性基因的乳腺癌细胞和肺癌细胞,命名为Adap-R。
如图1所示,为硬脑膜驯化4T1细胞的过程中通过生物发光成像定期监测小鼠的肿瘤负荷。如图2所示,为硬脑膜驯化LLC细胞的过程中通过生物发光成像定期监测小鼠的肿瘤负荷。
步骤5:
经过三轮的硬脑膜驯化,将5×104个4T1-Adap-R3细胞和5×104个LLC- Adap-R3细胞注射到小鼠颈动脉中,通过生物发光成像每周监测肿瘤负荷。当小鼠体重骤减,行动迟缓,小鼠头部出现明显肿瘤信号,按照步骤4中的方法收获硬脑膜上的肿瘤细胞,在RPMI-1640或DMEM完全培养基中培养,即得到4T1乳腺癌DrM细胞和LLC肺癌DrM细胞,称为4T1-DrM和LLC-DrM。
实施例2:
为了检验硬脑膜驯化后肿瘤细胞的转移效果,将5 x105个4T1-Par、4T1-Adap-R3细胞分别注射到小鼠颈动脉中,观察并比较两种细胞发生硬脑膜转移的能力。如图3所示,4T1-Adap-R3细胞可成功定植在硬脑膜,说明筛选到的4T1-Adap-R3细胞的硬脑膜转移能力强。
实施例3:
为了检验硬脑膜驯化肿瘤细胞的硬脑膜转移效果,将5 x105个LLC--Par、LLC-Adap-R3细胞分别注射到小鼠颈动脉中,观察并比较两种细胞发生硬脑膜转移的能力。如图4所示,LLC-Luc-Neo Adap-R3细胞可成功定植在硬脑膜,说明筛选到的LLC-Luc2-NeoAdap-R3细胞的硬脑膜转移能力强。
实施例4:
为了获得硬脑膜转移瘤小鼠模型,将DrM细胞注射到小鼠的颈动脉中,通过生物发光成像和HE染色确定肿瘤位置。如图5所示,A图为通过3D成像定位DrM细胞在硬脑膜分布,B图为HE染色显示DrM细胞生长在硬脑膜,说明使用的造模方法可成功获得硬脑膜转移瘤小鼠模型。
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Claims (1)
1.一种肿瘤硬脑膜转移模型的构建方法,其特征在于,具体步骤如下:
(1)细胞培养
将乳腺癌细胞株4T1在细胞培养皿中用RPMI-1640完全培养基培养,将肺癌细胞株LLC在细胞培养皿中用DMEM完全培养基培养;将包装慢病毒的人胚胎肾细胞株293T在细胞培养皿中用DMEM完全培养基培养;4T1、LLC和293T细胞放在无菌培养箱中培养,培养箱保持35℃-37℃的温度,CO2浓度维持在4%-6%;培养箱中始终含有无菌的灭菌水以保证培养箱内的湿度,使细胞更好地生长;
(2)病毒包装
构建可同时稳定表达荧光素酶以及新霉素抗性基因的慢病毒转移质粒载体pCDH-Luc-Neo;慢病毒转移质粒载体以及包装慢病毒的载体经过高纯度无内毒素质粒提取试剂盒提取后,使用转染试剂共转染293T细胞,共转染时293T细胞融合度为30-50%,转染后6-10小时及时更换为完全培养基,36-72小时后,收集并过滤病毒上清液;
(3)病毒感染
将肿瘤细胞4T1、LLC平铺于细胞培养皿中,使细胞密度为30%-70%;将病毒上清加入培养皿中,病毒上清的体积和完全培养基的体积比在0.5-2之间;加入2-5 µg/mL polybrene,充分摇匀后置于无菌培养箱中培养;病毒感染后12-24小时换液,待细胞长满后传代并用荧光素酶的底物检测荧光素酶的表达;
(4)G418药物筛选
慢病毒感染后的肿瘤细胞通过扩增培养,以及G418药物筛选,获得表达新霉素抗性基因的肿瘤细胞系,称为parental细胞,简称Par细胞;该细胞系可以同时稳定表达荧光素酶以及新霉素抗性基因,其中荧光素酶的表达用于生物发光成像,以便实时监测肿瘤位置和肿瘤负荷;
(5)硬脑膜转移模型构建
利用脑立体定位仪固定小鼠头部,正中剪开头皮暴露颅骨;用颅钻在颅骨矢状缝两侧顶骨中央磨出圆形颅窗,使硬脑膜充分暴露;使用微量进样针:
将2×104-1×105个Par细胞注射到硬脑膜,进行硬脑膜驯化;待种植的Par细胞适应硬脑膜环境,具备增殖能力导致小鼠出现行动迟缓、体重骤减的现象,对小鼠实施安乐死;收集硬脑膜上的肿瘤细胞,建立体外培养细胞系,该细胞称为硬脑膜驯化细胞1,简称Adap-R1;
将2×104-1×105个Adap-R1细胞注射到硬脑膜上,待Adap-R1细胞适应硬脑膜环境,具备增殖能力导致小鼠出现行动迟缓、体重骤减的现象,对小鼠实施安乐死;收集硬脑膜上的肿瘤细胞,建立体外培养细胞系,该细胞称为硬脑膜驯化细胞2,简称Adap-R2;
将2×104-1×105个D-inject 2细胞注射到硬脑膜上,待Adap-R2细胞适应硬脑膜环境,具备增殖能力导致小鼠出现行动迟缓、体重骤减的现象,对小鼠实施安乐死;收集硬脑膜上的肿瘤细胞,建立体外培养细胞系,该细胞称为硬脑膜驯化细胞3,简称Adap-R3;Adap-R3细胞可适应硬脑膜微环境;
将1×104-1×105个Adap-R3细胞和1×104-1×105个Par细胞分别注射到小鼠的颈动脉,细胞通过血液播散进入到颅内中,通过生物发光成像监测肿瘤进展和肿瘤位置,待小鼠出现硬脑膜转移现象,对小鼠实施安乐死;收集硬脑膜中生长的原代肿瘤细胞,建立体外培养细胞系,将该细胞系称为硬脑膜转移细胞系,简称DrM细胞;DrM细胞是通过血液播散稳定形成硬脑膜转移灶的细胞,将DrM细胞注射到小鼠的颈动脉后,即获得硬脑膜转移瘤小鼠模型。
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