CN114561360B - 一种小鼠肺癌脑转移细胞llc-bmt3及其构建方法和应用 - Google Patents
一种小鼠肺癌脑转移细胞llc-bmt3及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种小鼠肺癌脑转移细胞LLC‑BMT3及其构建方法和应用。本发明所述小鼠肺癌脑转移细胞LLC‑BMT3于2022年1月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202228,保藏地址为中国武汉大学。所述小鼠肺癌脑转移细胞LLC‑BMT3的脑转移发生率高,易于观察,不仅适于构建肺癌脑转移小鼠模型,用于筛选肺癌脑转移的分子标志物,鉴定肺癌脑转移的分子靶点,筛选或评估脑转移性肺癌的治疗药物,还能用于免疫健全小鼠的肺癌脑转移造模,开展肺癌细胞免疫逃逸及脑免疫微环境等相关研究,鉴定免疫治疗新靶点,评估免疫治疗效果,评估药物在改善免疫微环境中的效果等。
Description
技术领域
本发明属于生物技术领域。更具体地,涉及一种小鼠肺癌脑转移细胞LLC-BMT3及其构建方法和应用。
背景技术
脑转移瘤是指中枢神经系统外的组织肿瘤细胞经血液循环系统进入颅内浸润脑组织形成的转移性肿瘤,为恶性肿瘤的一种晚期表现。脑肿瘤患者中,脑转移瘤的数量大约是原发性脑肿瘤的10倍,是成年人最常见的颅内肿瘤。脑转移瘤的发生常意味着不良的预后,未治疗的患者中位生存期仅为1~2个月,经治疗后延长为4~5个月,患者死因多为脑水肿、颅内高压、脑疝、脑出血。临床上脑转移瘤的治疗方法主要为外科手术、全脑放射治疗、立体定向放射治疗以及化疗等,但总体来说治疗效果欠佳。而脑转移瘤的原发病灶以肺癌最为多见(40%~50%),因此,对肺癌脑转移机制的探索有着重要意义。
建立肺癌脑转移实验动物模型是进行肺癌脑转移机制研究以及药物筛选的基础。目前,国内外学者多采用在左心室、颈动脉、尾动脉、尾静脉等部位注射肿瘤细胞构建肿瘤转移模型,但由于血脑屏障的存在以及脑部功能和结构的特殊性,建立稳定的脑转移实验动物模型十分困难,关于成功构建稳定的肺癌脑转移动物模型的报道较少,且脑转移发生率也不高。例如,中国专利《一种高潜能脑转移中国人肺腺癌细胞株中》,通过取56岁女性肺腺癌患者十次化疗,放疗一次后产生的胸、腹水为细胞原代培养的材料,经培养获得人肺腺癌脑转移细胞株Yang2-脑M,通过裸鼠左心室种植试验后,分离脑转移灶经过三次筛选,获得脑转移发生率为60%的人肺腺癌脑转移亚株。此外,此方法虽成功获得了人肺腺癌脑转移亚株,但其只能应用于免疫缺陷裸鼠的肺癌脑转移模型构建,此种小鼠模型由于T淋巴细胞等免疫缺陷,无法开展肿瘤免疫逃逸及相关抗肿瘤药物的研究。
发明内容
本发明要解决的技术问题是克服现有技术中的肺癌脑转移细胞株脑转移发生率低、只能应用于免疫缺陷小鼠、筛选和示踪效果差等缺陷和不足,提供一种能应用于免疫健全小鼠且脑转移发生率高的小鼠肺癌脑转移细胞LLC-BMT3及其构建方法和应用。
本发明的第一个目的是提供一种小鼠肺癌脑转移细胞LLC-BMT3。
本发明的第二个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3的构建方法。
本发明的第三个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3在构建肺癌脑转移动物模型中的应用。
本发明的第四个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3在肺癌脑转移分子标志物筛选和分子靶点鉴定中的应用。
本发明的第五个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3在筛选治疗脑转移性的肺癌的药物中的应用。
本发明的第六个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3在构建肿瘤免疫逃逸小鼠模型中的应用。
本发明的第七个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3在构建肿瘤耐药模型中的应用。
本发明的第八个目的是提供所述小鼠肺癌脑转移细胞LLC-BMT3在开发肺癌脑转移风险评估检测试剂盒中的应用。
本发明上述目的通过以下技术方案实现:
本发明通过将携带嘌呤霉素抗性的绿色荧光蛋白和荧光素酶双基因标记的慢病毒转染小鼠肺癌LLC细胞,筛选获得了能稳定表达绿色荧光蛋白和荧光素酶的LLC-LUC/GFP细胞,再将所得细胞注射C57小鼠左心室,构建肺癌脑转移动物模型,通过活体成像确认发生脑转移后,取材收获脑组织并进行原代肺癌脑转移细胞培养,通过重复前述步骤并通过反复筛选获得了能应用于免疫健全小鼠且脑转移发生率高的小鼠肺癌脑转移细胞LLC-BMT3。
本发明提供了一种小鼠肺癌脑转移细胞LLC-BMT3,所述小鼠肺癌脑转移细胞LLC-BMT3于2022年1月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202228,保藏地址为中国武汉市武汉大学。
本发明还提供了所述小鼠肺癌脑转移细胞LLC-BMT3的构建方法,其包含以下步骤:
S1.将抗生素抗性基因、绿色荧光蛋白基因和荧光素酶基因转入小鼠肺癌细胞,筛选具有抗性且稳定表达绿色荧光蛋白和荧光素酶的细胞;
S2.取S1筛选的细胞注射小鼠左心室,小鼠饲养2周后经活体成像分析,选取发生脑转移的小鼠,取材脑组织,培养原代肺癌脑转移细胞;
S3.用所得肺癌脑转移细胞重复步骤S2再反复进行2轮筛选,即得小鼠肺癌脑转移细胞LLC-BMT3。
具体地,所用抗性基因为嘌呤霉素抗性基因。
具体地,步骤S1所述鼠肺癌细胞系为鼠肺癌细胞系LLC。
具体地,步骤S2中所用小鼠为C57小鼠。
具体地,步骤S2中注射的细胞量为2×105个。
本发明在构建小鼠肺癌脑转移细胞株时所用肺癌细胞系为鼠肺癌细胞系LLC,是小鼠来源的肺腺癌细胞,属于非小细胞癌,该细胞系增殖能力强,致瘤率高,但该细胞的脑转移潜能不高。一般来说,肿瘤细胞进入小鼠体内后,在体内微环境影响下,肿瘤细胞群会具备多种特性与不同部位的转移潜能。在进入心血管系统后,不同特性与转移潜能的细胞会转移到不同部位定植并存活,增殖后形成转移灶。本发明利用鼠肺癌细胞系LLC,通过对C57小鼠左心室注射稳定表达LUC/GFP的LLC细胞,发生脑转移后对脑转移灶进行原代培养,分离纯化脑转移的细胞群体,并通过多代重复筛选,最终获得了高潜能脑转移的LLC-BMT3细胞。本发明所述小鼠肺癌脑转移细胞LLC-BMT3的构建方法稳定可靠,可重复性好,成功率高。
本发明分别用LLC细胞和LLC-BMT3细胞注射小鼠后,小鼠活体成像发现LLC-BMT3细胞相比于LLC细胞具有更明显的定向脑转移倾向。用LLC-BMT3细胞构建小鼠肺癌脑转移模型,经脑组织HE染色可观察到明显的多部位散在脑转移灶;IF染色可观察到脑转移灶附近有大量小胶质细胞浸润,表明LLC-BMT3细胞具备更强的免疫逃逸能力,从而促进脑转移灶的生长。镜下观察细胞形态及westerning blot结果表明LLC-BMT3细胞更具间质型特征。CCK8及克隆形成表明BMT3细胞具备更强的增殖与集落形成能力。transwell实验表明,LLC-BMT3细胞具备更强的迁移与侵袭能力。综上,本发明通过反复在小鼠体内造模并取脑转移灶原代培养筛选纯化得到的LLC-BMT3细胞对建立定向脑转移的细胞系有极为重要的意义,可为肺癌脑转移研究提供可靠的动物模型,有利于寻找肺癌脑转移的关键通路及重要靶点,为临床治疗肺癌脑转移提供新的思路,具备重要的科学意义。
利用本发明所述小鼠肺癌脑转移细胞LLC-BMT3构建肺癌脑转移动物模型,小鼠发生脑转移的概率为80%。且小鼠肺癌脑转移细胞LLC-BMT3携带的绿色荧光蛋白和荧光素酶双标记,使该细胞易于活体成像和显微示踪,适于构建肺癌脑转移小鼠模型,筛选肺癌脑转移的分子标志物,鉴定肺癌脑转移的分子靶点,筛选或评估脑转移性肺癌的治疗药物等。本发明所述小鼠肺癌脑转移细胞LLC-BMT3还能用于免疫健全小鼠的肺癌脑转移造模,开展肺癌细胞免疫逃逸及脑免疫微环境相关研究,鉴定免疫治疗新靶点,评估免疫治疗效果等,极具实用价值。
因此,本发明还申请保护以下应用:
小鼠肺癌脑转移细胞LLC-BMT3在构建肺癌脑转移动物模型中的应用。
小鼠肺癌脑转移细胞LLC-BMT3在筛选肺癌脑转移的分子标志物中的应用。
小鼠肺癌脑转移细胞LLC-BMT3在筛选治疗脑转移性的肺癌的药物中的应用。
小鼠肺癌脑转移细胞LLC-BMT3在构建肿瘤免疫逃逸模型中的应用。
小鼠肺癌脑转移细胞LLC-BMT3在构建肿瘤耐药模型中的应用。
小鼠肺癌脑转移细胞LLC-BMT3在开发肺癌脑转移风险评估检测试剂盒中的应用。
本发明具有以下有益效果:
本发明提供了一种小鼠肺癌脑转移细胞LLC-BMT3,所述小鼠肺癌脑转移细胞LLC-BMT3于2022年1月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202228,保藏地址为中国武汉市武汉大学。本发明所述小鼠肺癌脑转移细胞LLC-BMT3的脑转移的概率为80%,不仅脑转移发生率高,其所携带的绿色荧光蛋白和荧光素酶双标记,使其易于进行活体成像和显微示踪,适于构建肺癌脑转移小鼠模型,用于筛选肺癌脑转移的分子标志物,鉴定肺癌脑转移的分子靶点,筛选或评估脑转移性肺癌的治疗药物等。本发明所述小鼠肺癌脑转移细胞LLC-BMT3还能用于免疫健全小鼠的肺癌脑转移造模,开展肺癌细胞免疫逃逸及脑免疫微环境相关研究,用于鉴定免疫治疗新靶点,评估免疫治疗效果,评估药物在改善免疫微环境中的效果等。此外,本发明所述小鼠肺癌脑转移细胞LLC-BMT3的构建方法稳定可靠,可重复性好,成功率高。
附图说明
图1为表达绿色荧光蛋白和荧光素酶的鼠肺癌细胞LLC-LUC/GFP的构建和鉴定;其中,图A为小鼠肺癌脑转移细胞的构建过程,图B为LLC-LUC/GFP细胞的形态比较图,图C为LLC-LUC/GFP细胞的生长曲线图,图D为克隆形成检测结果。
图2为脑转移活体的成像和病理切片;其中,图A为肿瘤细胞建立小鼠脑转移模型的活体成像对比示意图,图B为HE染色显示脑转移灶。
图3为肺癌脑转移组织中小胶质细胞标志物IBA1的表达情况检测结果。
图4为亲代肺癌细胞LLC-LUC/GFP和具有高潜能脑转移细胞LLC-BMT3细胞的细胞形态对比和间质性分子标记物表达结果;其中,图A为亲代肺癌细胞LLC-LUC/GFP和具有高潜能脑转移细胞LLC-BMT3细胞的细胞形态对比图,图B为亲代肺癌细胞LLC-LUC/GFP和具有高潜能脑转移细胞LLC-BMT3细胞中间质型分子标记相关蛋白的表达情况对比图。
图5为亲代肺癌细胞LLC-LUC/GFP和具有高潜能脑转移细胞LLC-BMT3细胞生物学特征对比结果;其中,图A为细胞生长曲线,图B为克隆形成检测结果。
图6为亲代肺癌细胞LLC-LUC/GFP和具有高潜能脑转移细胞LLC-BMT3的细胞体外迁移、侵袭能力比较结果;其中,图A为transwell细胞迁移实验结果,图A左侧为显微图,右侧为统计结果;图B为transwell实验结果,图B左侧为显微图,右侧为统计结果,“***”表示p<0.001。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
本发明所用肺癌细胞系为鼠肺癌细胞系LLC,购自中科院上海细胞库;所用携带嘌呤霉素抗性的绿色荧光蛋白和荧光素酶双基因标记的慢病毒购自上海吉凯基因化学技术有限公司;所用造模动物为C57小鼠,购自广东省医学实验动物中心。
实施例1绿色荧光蛋白和荧光素酶双基因标记的鼠肺癌细胞系的建立
本发明首先建立了能稳定表达绿色荧光蛋白和荧光素酶的亲代肺癌细胞LLC-LUC/GFP,具体过程如下:
1、将小鼠肺癌细胞系LLC细胞培养于37℃、5%CO2的细胞培养箱中,取生长状态良好的对数生长期细胞接种于六孔板中,做好细胞计数,待细胞融合度约30%时进行下一步处理;
2、在不同细胞株中分别加入MOI值为10~50(本实例中MOI值为25)的携带嘌呤霉素抗性的绿色荧光蛋白和荧光素酶双基因标记的慢病毒及助转剂(聚凝胺),24h后换液;待细胞融合度为80%时,加入4μg/mL的嘌呤霉素进行抗性筛选;待未处理的对照组全被杀死后,剩余细胞即为转染了绿色荧光蛋白和荧光素酶双基因标记的鼠肺癌细胞;将培养基中嘌呤霉素浓度调整为2μg/mL,继续培养至传代后,进行观察并进行后续实验。
将构建的绿色荧光蛋白和荧光素酶双基因标记的肺癌细胞置于倒置荧光显微镜下,观察细胞绿色荧光蛋白的表达情况,结果如图1所示。其中,图1A为小鼠肺癌脑转移细胞株构建过程,图1B为LLC-LUC/GFP细胞形态比较图,图1C为LLC-LUC/GFP细胞的生长曲线图,图1D为克隆形成检测结果。
由图1B可知,本发明所得能稳定表达绿色荧光蛋白和荧光素酶的亲代肺癌细胞LLC-LUC/GFP的细胞形态与LLC细胞相同,绿色荧光蛋白阳性率基本为100%,各细胞系在正常传代后绿色荧光蛋白仍稳定表达。由图1C可知,转染LUC/GFP对LLC细胞的增殖能力无影响。由图1D可知,转染LUC/GFP对LLC细胞集落形成能力无影响。
由上述结果可知,本发明所得亲代肺癌细胞LLC-LUC/GFP能稳定表达绿色荧光蛋白和荧光素酶,细胞传代后也能稳定表达,便于利用荧光显微镜观察细胞体外培养情况,利用小动物活体成像技术观察肿瘤脑转移情况。
实施例2定向脑转移的肺癌细胞的建立
利用实施例1所得能稳定表达绿色荧光蛋白和荧光素酶的亲代肺癌细胞LLC-LUC/GFP构建肺癌脑转移细胞。具体过程如下:
1、4~6周龄的C57小鼠适应性喂养一周后造模;
2、取生长状态良好的能稳定表达绿色荧光蛋白和荧光素酶的亲代肺癌细胞LLC-LUC/GFP,胰酶消化后,用含10%血清的完全培养基终止消化,800rpm/min离心并用PBS重悬,置于冰上待用;
3、实验小鼠用5%水合氯醛麻醉后,仰卧位固定于动物操作台上,胸前区备皮,酒精消毒;
4、用29G胰岛素注射器(BD公司)吸取100μL肺癌细胞悬液,细胞量为2×105个,在胸骨上切迹与剑突的中点偏左处寻找心间搏动点,做好标记,此外为左心室腔,从左心室腔正上方垂直进针4~6mm,轻微回抽可观察到血液喷射涌入注射器,缓慢将细胞悬液推注入左心室,为免细胞悬液随心脏搏动从出血点漏出,注射完成后应停留数秒,待肿瘤细胞进入全身循环系统后再拔针;
5、接种后的小鼠给予吸痰处理,以减少麻醉死亡率,保温垫中恢复,醒后于SPF环境内继续饲养,并每天观察小鼠的活动情况、神志、进食以及二便情况,有无消瘦乏力等恶病质状况及偏瘫等神经症状;
6、根据心室注射的细胞种类(LLC细胞:2周),进行每周小动物活体成像及磁共振影像学检查,发现小鼠发生脑转移概率为6.7%,确认脑转移后,进行取材和原代细胞培养;
7、小鼠断颈后泡入75%酒精缸中消毒,超净台内分离出大脑,置入含PBS的6cm无菌培养皿中;
8、PBS清洗多余血液,根据小动物活体成像及磁共振提示的肺癌脑转移部位,取对应的脑组织置于新的6cm无菌培养皿中,眼科剪将脑组织剪为约1mm的组织块,加入2mg/mL胶原酶,37℃下消化10分钟,离心后用含20%血清的1640或DMEM培养基重悬,使用75μm细胞过滤器处理细胞悬液,过滤后的细胞悬液接种于T25细胞培养瓶中,48h后用含2μg/mL嘌呤霉素的完全培养基换液培养;
9、倒置荧光显微镜观察脑转移瘤细胞(绿色荧光蛋白阳性)生长情况,脑转移细胞系扩增培养并作好记录和冻存保种;
10、重复步骤1~9两次,此时为第三轮筛选,将筛选所得细胞命名为BMT3细胞(第一轮筛选到的细胞命名为BMT1细胞,重复步骤1~9,继续筛选,第三轮筛选得到的细胞即为小鼠肺癌脑转移细胞LLC-BMT3)。
本发明所述小鼠肺癌脑转移细胞LLC-BMT3于2022年1月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202228,保藏地址为中国武汉市武汉大学。
用所建立的小鼠肺癌脑转移细胞LLC-BMT3造模并观察发现,小鼠发生脑转移的概率提升至了80%。造模后的小鼠脑转移活体成像和病理切片如图2所示,其中,图2A为肿瘤细胞建立小鼠脑转移模型的活体成像对比示意图,图2B为HE染色显示脑转移灶。由图2A可知,用建立的小鼠肺癌脑转移细胞LLC-BMT3与亲代肺癌细胞LLC-LUC/GFP相比,小鼠发生脑转移的概率大幅提升,脑转移率从6.6%(LLC-LUC/GFP)提高至了80%(LLC-BMT3)。由图2B可知,小鼠心室注射LLC-BMT3细胞两周后,脑组织切片HE染色可观察到多部位散在的脑转移灶。
实施例3 LLC-BMT3的表型观察
本发明对小鼠肺癌脑转移细胞LLC-BMT3及其构建所得脑转移模型的表型进行了观察,具体如下:
小鼠心室注射LLC-BMT3细胞两周后,对脑组织切片进行免疫荧光染色观察,并对脑转移组织中IBA1的表达情况进行了检测,结果如图3所示。由图3可知,小鼠肺癌脑转移细胞LLC-BMT3形成的脑转移灶附近存在大量microglia浸润。肿瘤相关小胶质细胞(Tumor-associated microglia,TAM)是浸润在肿瘤组织中的小胶质细胞(IBA1+),肿瘤细胞通过分泌特殊的细胞因子募集小胶质细胞并促进其M1/M2表型转化,并能导致免疫抑制。目前,越来越多的研究表明,TAM调控肿瘤细胞生长和侵袭转移,并抑制免疫微环境从而导致肿瘤免疫逃避。因此,本发明所述脑转移细胞LLC-BMT3可用于构建小鼠肺癌脑转移模型,用于研究肿瘤细胞免疫逃逸机制,进行相关抗肿瘤药物的筛选和评估。
本发明通过倒置显微镜对亲代肺癌细胞LLC-LUC/GFP和脑转移细胞LLC-BMT3的细胞形态进行了观察,同时通过western blot对二者细胞中间质型分子标记相关蛋白的表达情况进行了检测,结果如图4所示。其中,图4A为亲代肺癌细胞LLC-LUC/GFP和具有高潜能脑转移细胞LLC-BMT3细胞的细胞形态对比图,图4B为二者细胞中间质型分子标记相关蛋白的表达情况对比图。由图4A可知,相比于LLC细胞,LLC-BMT3细胞的形态明显改变,半悬浮细胞增加,贴壁细胞减少,偏向干细胞特点。由图4B可知,相比于LLC,LLC-BMT3细胞中间质型标志物N-cadherin、Twist、Vimentin表达上调,而表皮型标志物E-cadherin表达下调,表明LLC-BMT3细胞具有间质型细胞的表型。
本发明对亲代肺癌细胞LLC-LUC/GFP和脑转移细胞LLC-BMT3的细胞生物学特征进行了比较,结果如图5所示。其中,图5A为细胞生长曲线图,图5B为克隆形成检测结果。由图5可知,LLC-BMT3细胞的增殖能力更强。
本发明还通过transwell细胞迁移与侵袭实验对LLC-LUC/GFP和脑转移细胞LLC-BMT3的细胞体外迁移以及侵袭能力进行了比较,结果如图6所示。其中,图6A为transwell细胞迁移实验结果,图6A左侧为显微图,右侧为统计结果;图6B为transwell细胞侵袭实验结果,图6B左侧为显微图,右侧为统计结果,“***”表示p<0.001。由图6可知,相比于LLC细胞,LLC-BMT3细胞的迁移与侵袭能力更强。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (3)
1. 一种小鼠肺癌脑转移细胞株LLC-BMT3,其特征在于,所述细胞株LLC-BMT3于2022年1月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202228,保藏地址为中国武汉市武汉大学。
2.权利要求1所述小鼠肺癌脑转移细胞株LLC-BMT3在构建肺癌脑转移小鼠模型中的应用。
3.权利要求1所述小鼠肺癌脑转移细胞株LLC-BMT3在构建肿瘤免疫逃逸小鼠模型中的应用。
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