CN114703138A - 一种淋巴结来源淋巴瘤相关成纤维细胞肿瘤细胞株及其应用 - Google Patents
一种淋巴结来源淋巴瘤相关成纤维细胞肿瘤细胞株及其应用 Download PDFInfo
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Abstract
本发明公开了一种淋巴结来源淋巴瘤相关成纤维细胞肿瘤细胞株,来源于人淋巴瘤细胞裸鼠移植瘤模型中的淋巴结微环境,其细胞名称为小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF‑KLN,保藏编号为CCTCC NO:C2021107,保藏日期为2021年12月09日,保藏单位为中国典型培养物保藏中心。该细胞株为国内外首次建立的淋巴结来源淋巴瘤相关小鼠成纤维细胞肿瘤细胞株,可在构建小鼠成纤维细胞肿瘤动物模型、恶性淋巴结微环境模型、3D肿瘤培养体系和类器官体系模型、淋巴结微环境与淋巴瘤进展和耐药的关系模型中应用。
Description
技术领域
本发明属于生物医学技术领域,具体的说,是一种淋巴结来源淋巴瘤相关成纤维细胞肿瘤细胞株及其应用。
背景技术
淋巴瘤是一组起源于淋巴造血系统的恶性肿瘤,属于常见肿瘤之一,近年来发病率逐渐上升,主要表现为无痛性淋巴结肿大,肝脾肿大,全身各组织器官均可受累,伴发热、盗汗、消瘦、瘙痒等全身症状。根据瘤细胞分为霍奇金淋巴瘤(Hodgkin lymphoma,简称HL)和非霍奇金淋巴瘤(non Hodgkin lymphoma ,简称NHL)两类。
肿瘤的发生、生长及转移与肿瘤细胞所处的内外环境有着密切关系,称为肿瘤微环境,它不仅包括肿瘤所在组织的结构、功能和代谢,亦与肿瘤细胞自身的(核和胞质)内在环境有关。肿瘤微环境与肿瘤细胞相互作用,影响肿瘤的发生、发展和转归。
癌相关成纤维细胞(cancer-associated fibroblasts, CAFs)是构成实体肿瘤微环境的主要细胞成分,具有高度异质性、可塑性和多向分化潜能,通过重塑肿瘤微环境,在实体瘤的发生、发展、转移、化疗耐药及恶性转归中起着复杂而重要的作用。目前认为,CAFs的前体细胞主要有7种来源:骨髓间充质干细胞、组织中固有成纤维细胞、星形细胞、上皮细胞间质转化、内皮细胞间质转化、纤维细胞和其他细胞,如周细胞、平滑肌细胞、脂肪细胞和肿瘤干细胞等,因此,淋巴结微环境中有多种CAFs前体细胞。
由于不同来源的CAFs在肿瘤的进展中起不同的作用,因此构建不同来源的CAFs细胞有助于深入探索肿瘤病理。现有技术中,公开号为CN108795851A的发明专利公开了一种人唾液腺腺样囊性癌癌相关成纤维细胞系CAF-SA的建立方法及其应用,为唾液腺腺样囊性癌研究及药物研发提供了良好的实验模型。但目前针对淋巴瘤,尤其是淋巴结来源的癌相关成纤维细胞仍未见研究和报道。
发明内容
本发明的目的在于提供小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN,是一株淋巴结来源小鼠淋巴瘤相关成纤维细胞肿瘤细胞株,该细胞株为国内外首次建立的淋巴结来源淋巴瘤相关小鼠成纤维细胞肿瘤细胞株。为此,本发明还提供了该细胞株在构建小鼠成纤维细胞肿瘤动物模型、恶性淋巴结微环境模型、3D肿瘤培养体系和类器官体系模型、淋巴结微环境与淋巴瘤进展和耐药的关系模型中的应用。
本发明通过下述技术方案实现:一种淋巴结来源淋巴瘤相关成纤维细胞肿瘤细胞株,来源于人淋巴瘤细胞裸鼠移植瘤模型中的淋巴结微环境,其细胞名称为小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN,保藏编号为CCTCC NO:C2021107,保藏日期为2021年12月09日,保藏单位为中国典型培养物保藏中心,保存单位地址为中国湖北省武汉市武汉大学。
所述淋巴结来源小鼠成纤维细胞肿瘤细胞株来源于人间变性大细胞淋巴瘤细胞Karpas299裸鼠移植瘤模型,取自小鼠淋巴瘤细胞浸润淋巴结,是一株淋巴结来源淋巴瘤相关小鼠成纤维细胞肿瘤细胞株。该细胞株的原代细胞来源于人间变性大细胞淋巴瘤移植瘤模型小鼠;取肿大的淋巴结,剪碎成组织块,体外接种培养,去除悬浮细胞,获得贴壁生长细胞;细胞传代10代后,形态较前均一,以不规则多角形和纺锤形为主,核大,核仁清晰,胞浆有短粗突起,无接触抑制,可交叉重叠生长,细胞密集时成团聚集;在体外稳定无限传代,具高致瘤性;命名为小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN。
本发明涉及的小鼠成纤维细胞肿瘤细胞株HXLyAF-KLN具有以下生物学特性:
经转录组测序分析,为小鼠来源细胞。
经western bloting和流式细胞检测结果显示:该细胞高表达α-SMA,Vimentin,β-catenin,HSP47,PDGFR-α,PDGFR-β,为成纤维细胞来源肿瘤。
HXLyAF-KLN细胞在RPMI 1640完全培养基中贴壁生长,呈梭形或不规则多边形,体外培养生长良好,按1.5×105/55cm2密度接种,3~4天传代一次,已经体外连续培养超过10个月,传代超过60代,群体倍增超过170代,连续传代过程中细胞形态稳定、增殖动力学稳定、遗传学特征稳定,状态良好;经液氮或超低温冻存、复苏后性状不变,为一株永生化细胞株。
采用MTT和台盼蓝染色细胞计数法绘制HXLyAF-KLN细胞的生长曲线,计算细胞倍增时间为25~31小时,增殖速率稳定,可见分裂相,分裂后细胞仍贴壁;证实细胞株具有增殖动力学稳定性。
采用PI染色法和染色体核型分析证实,HXLyAF-KLN细胞为超6倍体细胞,具有细胞周期稳定性。
本发明还提供了小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN在构建小鼠成纤维细胞肿瘤动物模型中的应用,在构建恶性淋巴结微环境模型中的应用,在构建3D肿瘤培养体系和类器官体系模型中的应用,在构建淋巴结微环境与淋巴瘤进展和耐药的关系模型中的应用。可获取淋巴瘤细胞所导致的成纤维细胞肿瘤化生的相关机制,并为探索靶向CAFs的淋巴瘤治疗新方法提供更多新的思路和研究方向。
本发明与现有技术相比,具有以下优点及有益效果:
(1)本发明为目前首次建立的淋巴结来源小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN,该细胞株具有高致瘤性,可用于构建小鼠成纤维细胞肿瘤动物模型,恶性淋巴结微环境模型,3D肿瘤培养体系和类器官体系模型。
(2)本发明可将小鼠成纤维细胞肿瘤细胞株HXLyAF-KLN应用于淋巴瘤微环境的研究中,构建淋巴结微环境与淋巴瘤进展和耐药的关系模型,获取淋巴瘤细胞导致成纤维细胞肿瘤化生的相关数据,获取淋巴瘤相关成纤维细胞肿瘤与淋巴瘤微环境的关系的相关数据,为其在治疗淋巴瘤过程中获取淋巴瘤化疗耐药等相关数据。
附图说明
图1为倒置相差显微镜下HXLyAF-KLN细胞观察图(×200)。
图2 为透视电镜下HXLyAF-KLN细胞超微结构图(×6000)。
图3为HXLyAF-KLN细胞生长曲线图。
图4为HXLyAF-KLN细胞周期图。
图5为Western blotting检测HXLyAF-KLN细胞α-SMA,Vimentin,HSP47,β-catenin,PDGFR-α,PDGFR-β表达图。
图6 为HXLyAF-KLN细胞裸鼠皮下肿瘤形成实验图(一)。
图7为HXLyAF-KLN细胞裸鼠皮下肿瘤形成实验图(二)。
具体实施方式
下面结合实施例对本发明作进一步地详细说明,但本发明的实施方式不限于此。
实施例:HXLyAF-KLN的分离和建株
本实施例所述HXLyAF-KLN来源于人淋巴瘤细胞裸鼠移植瘤模型中的淋巴结微环境,其细胞名称为小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN,其构建方式如下:
(1)将处于对数生长期的人间变性大细胞淋巴瘤细胞Karpas299按8×106个/100µl/只,接种于雄性 Balb/c (nu/nu) 小鼠(8周龄,体重22~27g)裸区皮下;隔日观察接种部位肿瘤生成情况,小鼠未形成皮下瘤,于2周后,再次将人间变性大细胞淋巴瘤细胞Karpas299按3×106个/200µl/只,接种于小鼠前次接种部位对侧裸区皮下。第二次接种30余天后小鼠体重出现进行性下降,取小鼠双侧腹股沟淋巴结制成细胞悬液,悬在含10%胎牛血清(Thermo公司)的RPMI 1640培养基(Gibco公司)中,置37℃、饱和湿度、5% CO2培养箱培养。4天后可见贴壁细胞生长,去除上清和悬浮细胞,每2~3天换液,20天后培养皿中悬浮细胞基本去除,贴壁细胞多形性,呈簇状生长,胰酶消化传代,此后每10~14天传代一次,接种97天后细胞形态较之前均一,细胞增殖加快,130天后细胞增殖明显快,细胞以短梭形或不规则多角形为主。
(2)HXLyAF-KLN细胞传代时的初始密度为1.5×105个/55cm2,当细胞增殖到80~90%融合时1∶5~10传代。
(3)HXLyAF-KLN细胞冻存前48小时换液,使细胞处于对数生长期,计数细胞,无菌条件下,将细胞转入离心管,1200转/分离心5分钟,弃上清,加入冻存液,调整细胞浓度为3×106个/ml,充分混匀,移入无菌冻存管,4℃,30min; -20℃,60min;-80℃过夜,次日移入液氮。
(4)复苏时,从液氮中取出冻存管,迅速置于37℃温水中,待冻存物融化后,将细胞悬液移入加入5ml RPMI 1640培养基的离心管中,轻柔混匀,1200转/分,离心5分钟,弃上清,细胞沉淀中加入RPMI 1640完全培养基,混匀,转移入培养皿,置37℃、饱和湿度、5% CO2培养箱培养。
(5)HXLyAF-KLN细胞经反复如上述步骤(3)和步骤(4)冻存复苏,稳定生长,不影响细胞的生物学特征,显微镜下见细胞以不规则多角形和纺锤形为主,核大,核仁清晰,胞浆有短粗突起,贴壁生长,细胞倍增时间维持在25~31小时。
上述步骤中,RPMI 1640完全培养基的配方采用90% RPMI 1640培养基和10%胎牛血清。冻存液的配方采用45% RPMI 1640培养基,50%胎牛血清和5% DMSO(Sigma公司)。
实施例2:HXLyAF-KLN的生长和生物学特性鉴定
(1)细胞形态学观察:
取对数生长期的HXLyAF-KLN细胞于倒置显微镜下观察活细胞形态,如图1所示,细胞贴壁生长,以不规则多角形和纺锤形为主,核大,核仁清晰,胞浆有短粗突起,无接触抑制,可交叉重叠生长,细胞密集时成团聚集;3%戊二醛预固定,1%四氧化锇再固定,醋酸铀和枸橼酸铅染色JEM-1400FLASH透视电镜下观察细胞超微结构,如图2所示,可见细胞核呈不规则形,有切迹,核膜清晰,核仁明显,胞浆粗面内质网增多,游离核糖体丰富,可见内质网、线粒体等细胞器。
(2)生长曲线和倍增时间测定:
离心收集对数生长期的细胞,调整细胞浓度为2×104个/3.8cm2,接种于6孔板中,置37℃、5%CO2、21% O2培养箱中培养8天,每天用台盼蓝染色计数细胞。以时间为横坐标,细胞数为纵坐标,绘制细胞生长曲线,如图3所示,计算细胞倍增时间为25~31小时,可见细胞株经反复传代,群体倍增次数到170多代,仍然保持增殖动力学稳定性。
(3)PI染色法监测细胞周期:
收获1×106个细胞,4℃预冷PBS洗2次,4℃ 75% 乙醇固定过夜,4℃ PBS洗2次,PI室温避光染色10min,流式细胞仪检测细胞的G1/G0、S及G2/M期的比率,结果如图4所示,HXLyAF-KLN细胞为超六倍体细胞,经反复传代,能保持细胞周期稳定性。
(4)转录组测序证实HXLyAF-KLN细胞为小鼠来源细胞。
(5)western bloting检测结果显示:该细胞高表达α-SMA,Vimentin,HSP47,β-catenin,PDGFR-α,PDGFR-β,为成纤维细胞来源肿瘤,如图5所示。
以上结果显示,淋巴瘤细胞可以导致成纤维细胞肿瘤化生。
实施例3:小鼠成纤维细胞肿瘤动物模型的构建
具体涉及用小鼠成纤维细胞肿瘤细胞株HXLyAF-KLN构建小鼠成纤维细胞肿瘤动物模型,采用裸鼠成瘤实验,具体实验过程如下:
将处于对数生长期的HXLyAF-KLN细胞按3×106个/200µl/只,接种于雌性 Balb/c(nu/nu) 小鼠(4周龄,体重15~18g)裸区皮下;3~6天后即可见单或双侧腹股沟淋巴结长大(图6),30天后见接种部位肿瘤生成,50天后体积约605mm3,如图7所示。
以上所述,仅是本发明的较佳实施例,并非对本发明做任何形式上的限制,凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化,均落入本发明的保护范围之内。
Claims (5)
1.一种淋巴结来源淋巴瘤相关成纤维细胞肿瘤细胞株,其特征在于:来源于人淋巴瘤细胞裸鼠移植瘤模型中的淋巴结微环境,其细胞名称为小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN,保藏编号为CCTCC NO:C2021107,保藏日期为2021年12月09日,保藏单位为中国典型培养物保藏中心。
2.将权利要求1所述小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN在构建小鼠成纤维细胞肿瘤动物模型中的应用。
3.将权利要求1所述小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN在构建恶性淋巴结微环境模型中的应用。
4.将权利要求1所述小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN在构建3D肿瘤培养体系和类器官体系模型中的应用。
5.将权利要求1所述小鼠淋巴瘤相关成纤维细胞肿瘤细胞HXLyAF-KLN在构建淋巴结微环境与淋巴瘤进展和耐药的关系模型中的应用。
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