CN117402828A - 一种人肺癌脑转移瘤细胞系及其应用 - Google Patents
一种人肺癌脑转移瘤细胞系及其应用 Download PDFInfo
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Abstract
本发明提供了一种人肺癌脑转移瘤细胞系及其应用，属于细胞技术领域。本发明提供一种人肺癌脑转移瘤细胞系，该细胞系命名为人源肺腺癌脑转移瘤细胞系A549‑BM3，于2023年03月20日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO:63284。本发明的人源肺腺癌脑转移瘤细胞系A549‑BM3由A549细胞系构建得到，相对于由原代肺癌细胞构建得到的细胞系，在肿瘤基础研究中的应用上更具有普适性；本发明的A549‑BM3可应用于具有KRAS基因突变的肺癌脑转移瘤的相关研究。
Description
技术领域
本发明属于细胞技术领域，具体涉及一种人肺癌脑转移瘤细胞系及其应用。
背景技术
世界卫生组织(WHO)下属的国际癌症研究机构发布的最新数据表明，乳腺癌首次超过肺癌成为全球发病率最高的癌症。尽管如此，肺癌的全球发病率仍然高居全球第二，也是导致癌症死亡的首要原因。RAS基因是最早发现的肺癌驱动基因。在肺腺癌中，KRAS基因突变率最高，西方国家人群的KRAS基因突变率接近30％，东亚国家人群的KRAS基因突变率约15％。KRAS基因突变的肺癌患者预后较差，目前仍然缺乏有效的靶向药物。直到2021年，才开始有针对肺癌KRAS突变的靶向药物上市。脑部是肺癌最常见的转移部位，高达20％的肺癌患者在病程中发生脑转移。而对于KRAS基因突变的肺癌脑转移，目前也仍然缺乏有效的靶向药物。
人源肺腺癌细胞系A549是研究肺癌最常用的细胞系之一，并已知A549细胞系本身存在KRAS基因突变。专利CN111187753A公开了一种高潜能脑转移肺癌细胞株H1975luc-BM4。该发明采用裸鼠左心室反复接种稳定表达荧光素酶的肺癌EGFR突变细胞H1975-luc，并通过体外培养，将脑组织中的H1975-luc筛选出来，最终获得具有高度脑转移性的肺癌细胞株H1975luc-BM4。但H1975luc-BM4为EGFR突变，缺乏肺癌中的其他突变位点且没有标记绿色荧光蛋白(GFP)，限制了其在免疫荧光中的应用。目前尚未有基于A549细胞系来构建肺癌脑转移瘤细胞系的相关报道。
发明内容
针对上述问题，本发明的目的在于提供一种人肺癌脑转移瘤细胞系及其应用，本发明首次采用以A549细胞系为亲本，构建了人源肺腺癌脑转移瘤细胞系A549-BM3。
为实现上述目的，本发明采取的技术方案为：一种人肺癌脑转移瘤细胞系，该细胞系命名为人源肺腺癌脑转移瘤细胞系A549-BM3，于2023年03月20日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC NO:63284。本发明首次以A549细胞系为亲本，构建了人源肺腺癌脑转移瘤细胞系A549-BM3，A549细胞系相对于原代的肺癌细胞在肿瘤基础研究中的应用更加广泛，也更被广大研究者认可。本发明由A549细胞系构建的人源肺腺癌脑转移瘤细胞系A549-BM3在肿瘤基础研究中的应用上更具有普适性，且A549细胞系本身携带KRAS基因突变，因此本发明的A549-BM3可应用于具有KRAS基因突变的肺癌脑转移瘤的相关研究中。
本发明还提供所述的人肺癌脑转移瘤细胞系在制备具有KRAS基因突变的肺癌脑转移模型中的应用。
作为本发明所述应用的优选实施方式，所述模型包括体内模型和体外模型。
本发明还提供所述的人肺癌脑转移瘤细胞系在肺癌脑转移发生发展机制研究中的应用。
本发明还提供所述的人肺癌脑转移瘤细胞系在制备治疗肺癌脑转移瘤药物中的应用。本申请发明人研究发现，RNA测序显示A549-BM3在基因表达及信号通路的富集上与亲本的A549-LUC-GFP存在差异，因此本发明的A549-BM3可以作为研究肺癌脑转移瘤发生发展机制的工具。
本发明还提供所述的人肺癌脑转移瘤细胞系在顺铂耐药机制研究中的应用。本申请发明人研究发现，A549-BM3对顺铂产生耐药性，可用于探索肺癌脑转移瘤对顺铂耐药的机制研究。
本发明的有益效果为：本发明提供一种人肺癌脑转移瘤细胞系及其构建方法，(1)本发明的人源肺腺癌脑转移瘤细胞系A549-BM3由A549细胞系构建得到，相对于由原代肺癌细胞构建得到的细胞系，在肿瘤基础研究中的应用上更具有普适性；(2)由于A549细胞系本身携带KRAS基因突变，本发明的A549-BM3可应用于具有KRAS基因突变的肺癌脑转移瘤的相关研究；(3)本发明的A549-BM3既能标记荧光素酶，又能标记绿色荧光蛋白。在体内实验中，可以使用活体成像技术显示A549-BM3在体内的生长状态；在体外实验中，A549-BM3因携带GFP，可自发绿色荧光，可用于探索相关基因在A549-BM3中的表达定位；(4)本发明的心脏注射方法，包括进针的定位、角度、方向和深度，是经过多次实践总结出来，该方法的心脏注射成功率达到90％以上；(5)本发明使用的肺癌脑转移瘤细胞的分离和提取方法，使得肺癌脑转移瘤细胞A549-BM1/2/3成活率达到90％以上；(6)通过RNA Sequencing，本发明所构建的肺癌脑转移瘤细胞系A549-BM3较亲本肺癌细胞系A549-LUC-GFP在基因表达水平上有明显差异，主要体现在基因功能和信号通路上，可以作为研究肺癌脑转移瘤发生发展机制的工具。(7)药敏试验显示，A549-BM3较亲本肺癌细胞系A549-LUC-GFP对顺铂产生明显的耐药性。
附图说明
图1为A549-BM3构建的整体流程。
图2：A为A549-LUC-GFP在光镜下的细胞形态；B为A549-LUC-GFP在镜下GFP的表达情况；C为A549-LUC-GFP的Luciferase表达情况；D为A549-BM3在光镜下的细胞形态；E为A549-BM3在镜下GFP的表达情况；F为A549-BM3的Luciferase表达情况。
图3：A为采用A549-LUC-GFP进行心脏注射来构建的脑转移瘤动物模型第8周的活体成像图；B为采用A549-BM3进行心脏注射来构建的脑转移瘤动物模型第8周的活体成像图。
图4：A为A549-LUC-GFP差异分析中的基因功能富集分析图；B为A549-BM3差异分析中的基因功能富集分析图；C为A549-LUC-GFP差异分析中的KEGG通路富集分析图；D为A549-BM3差异分析中的KEGG通路富集分析图。
图5：A为A549-BM3与亲本A549-LUC-GFP对顺铂的半抑制浓度(IC50)；B为A549-BM3与亲本A549-LUC-GFP在相同顺铂浓度下的CCK8增殖试验。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例和附图详细说明本发明的技术方案。
实施例1
本实施例提供了一种人肺癌脑转移瘤细胞系的构建方法，其构建流程图如图1所示，具体步骤如下：
1、构建A549-LUC-GFP：A549细胞系(具有KRAS突变)购于美国ATCC细胞库，用携带有荧光素酶(Luciferase)，绿色荧光蛋白(GFP)及嘌呤霉素(Puromycin)抗性基因的慢病毒转染A549。将A549按1X105个/ml的密度接种于24孔板，待24小时细胞贴壁后，按细胞数与病毒数1:400的比例加入慢病毒，并于转染24小时后更换培养基。转染48-72小时后于荧光显微镜下观察GFP荧光强度(如图2B)并加入5ug/ml的Puromycin。待细胞稳定表达GFP后将Puromycin浓度调整为2ug/ml。将荧光素酶检测试剂(Promega E1500 Luciferase AssaySystem)按说明书分别加入慢病毒转染前和转染后的A549细胞中。将制好的样品加入96孔板中且每组样品设置3个重复，并将96孔板置于LUMIstar Omega酶标仪中上机检测，结果如图2C所示。由图2可知，A549-LUC-GFP同时携带有Luciferase和GFP。
2、将A549-LUC-GFP通过心脏注射来构建脑转移瘤动物模型：
(1)取5-6周龄的裸鼠(BALB/C)，麻醉剂为异氟烷，麻醉机为RWD瑞沃德R500。将裸鼠麻醉后，取仰卧位，固定其四肢。
(2)心脏注射的进针点为胸骨切迹下5mm，水平向右旁开3mm处，于第二肋间隙进针。定位好后用黑色签字笔标记。
(3)将A549-LUC-GFP消化后用PBS重悬，细胞浓度为2X107个/ml。用配有26G针头的1ml注射器吸取100ul细胞悬液备用。
(4)注射前用碘伏消毒皮肤，消毒范围为以进针点为中心，周围约2cm。进针时针头与裸鼠身体长轴平行，呈45度斜行进针。当进针深度为6-8mm时，有鲜红血液回流入注射器，并且液平面随着心脏跳动而上下浮动，说明针头已进入左心室，此时缓慢推注细胞悬液。
(5)注射完后停止麻醉，给裸鼠打上耳标等待其复苏。
3、活体成像：
(1)完成心脏注射后分别于24小时后、第7天、第8周三个时间段对裸鼠进行活体成像，观察A549-LUC-GFP在脑部的转移率以及生长情况。
(2)用PBS配置15mg/ml的荧光素酶底物工作液(Promega E1650)后，用1ml注射器吸取100ul荧光素酶底物工作液对裸鼠进行腹腔注射。10分钟后于活体成像仪器中观察A549-LUC-GFP在裸鼠身体里的分布及活性。
4、获得A549-BM3：
(1)在第8周时，若活体成像显示裸鼠头部仍有A549-LUC-GFP的细胞信号，则说明脑转移瘤模型构建成功。将建模成功的裸鼠安乐死后，于无菌操作下分离整个大脑并研磨；
(2)配置消化液：用含有Mg2+/Ca2+的DPBS配置400U/ml IV胶原酶(Worthington,LS004186)，1.2U/ml分散酶(Dispase,Worthington,LS02100)和32U/ml DNA酶(DNaseI,Worthington,LS006344)的混合液；
(3)取5ml消化液加入研磨充分的大脑中，于37度培养箱中孵育45分钟，在孵育期间每隔15分钟用移液管吹打脑组织，使得消化液与脑组织充分混合；
(4)加入1ml血清(FBS，牛胎血清)和20ml的PBS终止消化，于40um的细胞筛中过滤，并将滤液在400g下离心5分钟；
(5)弃上清，加10ml 20％w/v的BSA(牛血清白蛋白)充分混合病在4度下1000g离心25分钟
(6)弃上清，加红细胞裂解液后重悬，400g离心5分钟。
(7)弃上清，加入PBS重悬后，400g离心5分钟。将所获得细胞接种于6孔板中，培养基为DMEM+10％胎牛血清+1％双抗(青霉素和链霉素)。
(8)接种24小时后换液，加入5ug/ml的Puromycin。每天镜下观察细胞，待细胞稳定表达GFP后将Puromycin浓度调整为2ug/ml。
(9)将获得的第一代脑转移瘤细胞A549-BM1进行体外扩增，重复2次心脏注射，最终历经3个循环的体内选择，获得肺癌脑转移瘤细胞系A549-BM3(如图2D、E、F)。
实施例2
本实施例采用活体成像检测A549-LUC-GFP和A549-BM3的脑转移率，结果如图3所示。活体成像显示，建模第8周后A549-BM3脑转移率达到80％，而亲本A549-LUC-GFP脑转移率只有40％。
实施例3
本实施例将A549-BM3与亲本A549-LUC-GFP用Triso裂解后送公司进行RNASequencing。结果如图4所示。
由图4A可知，基因功能富集分析(Go term)显示A549-BM3相对于A549-LUC-GFP有差异表达的基因在生物学过程(Biological process)、细胞组成(Cellular Component)和分子功能(Molecular Function)中的分类；同时也显示了有显著差异的前20个基因集合(图4B)；KEGG富集分析显示了A549-BM3相对于A549-LUC-GFP有差异表达的通路。常见的信号通路如IL-17、MAPK、TNF等为有显著差异的前20个通路(图4C)，另外这前20个通路中上调和下调的基因所占的比列也显示在饼图中(图4D)；差异分析结果显示A549-BM3与亲本A549-LUC-GFP之间在基因功能和KEGG通路之间的差异，为进一步研究肺癌脑转移发生发展的机制提供了新的思路。
实施例4
本实施例将A549-BM3与亲本A549-LUC-GFP进行了顺铂的药敏试验。
结果如图5所示，由图5A可知，A549-BM3的顺铂半抑制浓度(IC50)是亲本A549-LUC-GFP的3.15倍。由图5B可知，CCK8增殖试验显示A549-BM3对顺铂的敏感性下降。以上结果提示相对于亲本的A549-LUC-GFP，发生脑转移后的A549-BM3产生对顺铂的耐药。这也解释了顺铂用于肺癌脑转移瘤患者治疗效果不佳的原因，因此，本发明的A549-BM3细胞可用于研究肺癌脑转移瘤对顺铂耐药的机制。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (6)
1.一种人肺癌脑转移瘤细胞系，其特征在于，该细胞系命名为人源肺腺癌脑转移瘤细胞系A549-BM3，于2023年03月20日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCCNO:63284。
2.权利要求1所述的人肺癌脑转移瘤细胞系在制备具有KRAS基因突变的肺癌脑转移模型中的应用。
3.根据权利要求2所述的应用，其特征在于，所述模型包括体内模型和体外模型。
4.权利要求1所述的人肺癌脑转移瘤细胞系在肺癌脑转移发生发展机制研究中的应用。
5.权利要求1所述的人肺癌脑转移瘤细胞系在制备治疗肺癌脑转移瘤药物中的应用。
6.权利要求1所述的人肺癌脑转移瘤细胞系在顺铂耐药机制研究中的应用。
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