CN114214281A - 人胶质母细胞瘤细胞系gwh04及其培养方法与应用 - Google Patents
人胶质母细胞瘤细胞系gwh04及其培养方法与应用 Download PDFInfo
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Abstract
本发明公开了一种人胶质母细胞瘤细胞系GWH04及其培养方法与应用,属于神经肿瘤细胞系技术领域。它包括从一位右侧颞叶占位胶质母细胞瘤患者的肿瘤组织中培养得到保藏于中国典型培养物保藏中心CCTCC,保藏号CCTCC NO.C202163的人胶质母细胞瘤细胞系GWH04。该新胶质母细胞瘤细胞系能够在体外稳定传代培养、裸鼠成瘤性好,可用于研究胶质瘤发生机理、耐药机理或筛选治疗药物等。
Description
技术领域
本发明涉及一种人胶质母细胞瘤细胞,属于神经细胞系技术领域,具体地涉及一种人胶质母细胞瘤细胞系GWH04及其培养方法与应用。
背景技术
胶质瘤是颅内最常见的恶性肿瘤,约占所有颅内肿瘤的26.6%,其中胶质母细胞瘤(WHO IV级)占所有胶质瘤的56.1%,占所有颅内恶性肿瘤的47.1%,中位生存时间约14~16.6个月,通过手术、放疗和化疗等多种治疗仅有约5.5%的患者存活达到5年。即使预后相对较好的低级别胶质瘤(WHO II级)也不可避免地会复发进展为高级别胶质瘤(WHO III级和IV级)并最终吞噬病人的生命。关于胶质母细胞瘤的治疗,以手术为主,即便术后辅助同步放化疗+替莫唑胺续贯化疗等,或辅助贝伐单抗等靶向治疗,预后近十年也未见明显改善,其病因和进展转化的机制仍不十分清楚。揭示其发病机制,寻找有效的治疗策略是亟待解决的问题。此例人胶质母细胞瘤细胞系的建立,为了解胶质母细胞瘤的生物学特性、研究其癌变、分子遗传以及耐药复发演变机制等提供了丰富的实验材料,可以为胶质瘤发生发展方面的研究和化疗耐药机制相关研究等做出贡献。
由于胶质母细胞瘤的原代培养及体外稳定传代培养在技术上存在一定的难度,相对于胶质母细胞瘤表型的多样性和肿瘤的异质性,目前已建立的胶质母细胞瘤细胞系仍然十分有限。作为重要研究工具的细胞系非常少见,目前常用细胞系主要有U87,U251,U373,HS683,A172等,这些细胞系的建立时间均较早。此外,ATCC保藏的U87已被证实与最初在Uppsala实验室建立的U87不是同一来源细胞;U251和U373被证实为相同来源;HS683和A172因成瘤性不佳,在基础研究中使用很少。2016年WHO更新的第四版中枢神经系统肿瘤分类中明确推荐胶质瘤相关分子诊断,新的基因诊断标志物不断被大家认可并应用,如判断替莫唑胺化疗敏感性的MGMT启动子甲基化水平,判断预后的IDH突变、染色体1p/19q共缺失、TERT启动子区域突变情况等。在胶质瘤工具细胞中这些突变背景也越来越被科研工作者重视,有助于进行深入研究。因此建立新的胶质母细胞瘤细胞系,同时丰富呈现相应的突变背景,以丰富完备现有的胶质母细胞瘤细胞库和细胞相关背景信息,为研究胶质瘤提供合适的体外模型,促进胶质母细胞瘤的基础和临床研究是很有必要的。
发明内容
为实现上述技术目的,本发明公开了一种人胶质母细胞瘤细胞系GWH04及其培养方法与应用。本发明探究得到的新胶质母细胞瘤细胞系能够在体外稳定传代培养、裸鼠成瘤性好,可用于研究胶质瘤发生机理、耐药机理或筛选治疗药物等。
为更好的实现本发明技术目的,本发明公开了一种人胶质母细胞瘤细胞系GWH04,它是保藏于中国典型培养物保藏中心CCTCC,保藏号CCTCC NO.C202163的人胶质母细胞瘤细胞系GWH04。
进一步地,它为来源于一位右侧颞叶占位胶质母细胞瘤患者的肿瘤组织进行体外培养的细胞和/或子代细胞。
进一步地,所述子代细胞包括保藏于中国典型培养物保藏中心CCTCC,保藏号CCTCC NO.C202164的单克隆细胞珠GWH317。
其中,所述细胞系GWH04具备具有TERT基因启动子区C228T突变、TP53基因突变、PTEN基因突变、19q有缺失及IDH野生、MGMT启动子区域甲基化阴性的特征。
与此同时,本发明还公开了一种人胶质母细胞瘤细胞系GWH04的培养方法,它包括如下步骤:
1)取材:术中留取胶质母细胞瘤患者的肿瘤组织0.5cm3于含DMEM的无菌取样管中,置于4℃冰盒中并快速送到实验室;
2)消化:在无菌超净台中用PBS清洗弃去步骤1)所得样本表面残余血细胞后,在平皿中用剪刀将组织反复剪碎,加入高糖DMEM培养基3mL,继续剪碎组织至1~2mm颗粒状,加入3mL含EDTA的胰酶继续吹打混匀,并移至15mL离心管内,在4℃摇床中,快速振摇20分钟;
3)过滤:用不小于200目网径的滤网过滤步骤2)所得悬液,并离心、沉淀,用5mL培养基重悬,所述培养基内包含高糖DMEM+10%FBS,然后转移至T25培养瓶培养,次晨换液;
4)常规传代培养:T25培养瓶细胞长满后,用1mL含EDTA的胰酶室温消化2~3min后,培养基中和,控制离心条件为1000r/min,5min离心处理后,继续1:5传代。
为更好的实现本发明技术方案,本发明其中一个技术目的是公开了一种人胶质母细胞瘤细胞系GWH04的用途,它包括人胶质母细胞瘤细胞和/或子代细胞在哺乳动物中产生胶质瘤瘤块。
进一步地,它包括胶质母细胞瘤细胞系和/或子代细胞系用于在哺乳动物中产生胶质瘤瘤块并用来研究胶质瘤发生机理及耐药机理。
进一步地,所述哺乳动物是免疫缺陷小鼠,本发明优选为裸鼠。
本发明其中一个技术目的是公开了一种人胶质母细胞瘤细胞系GWH04在筛选胶质瘤治疗药物中的应用。
进一步地,它包括建立胶质瘤动物模型并用来筛选胶质瘤治疗药物。
进一步地,它包括将测试化合物施用于体外细胞模型中,施用后抑制细胞增殖或导致细胞凋亡的测试化合物为治疗人胶质母细胞瘤的候选化合物。
有益效果:
1、本发明筛选得到的人胶质母细胞瘤细胞系GWH04经STR鉴定为新的单一细胞株,为人颅内原发胶质母细胞瘤研究提供了新的与临床肿瘤生物学接近的实验材料。
2、本发明筛选得到的人胶质母细胞瘤细胞系GWH04具有较好的贴壁性,且细胞性状稳定,可持续稳定传代,其生长速度与经典的胶质瘤细胞系U87相当,可良好耐受转染等操作,研究发现其具有非常强的裸鼠皮下成瘤和颅内成瘤能力,可以成功制备胶质母细胞瘤动物模型,实验发现颅内成瘤50天,裸鼠可出现明显症状;皮下成瘤30天,可见明显直径10mm肿瘤形成。
3、本发明筛选得到的人胶质母细胞瘤细胞系GWH04,基因检测提示TP53基因错义突变c.614A>G,蛋白水平变化为p.Tyr205Cys,IHC染色提示为突变无功能p53蛋白强阳性表达。此外,二代测序也显示PTEN的无义突变c.945T>A,氨基酸序列变化为p.Tyr315Ter。TP53和PTEN同为重要的抑癌基因,两者在该细胞系中的突变概率均为100%,故其可作为有效的靶点研究细胞系,在筛选治疗胶质瘤药物中具备较好应用前景。
附图说明
图1为本发明探究得到的GWH04细胞所来源的病人术前病灶的影像学图像,其中,图1A为术前CT,图1B为MR平扫T1像,图1C为MR平扫T2像,图1D为MR平扫T2压水像,图1E、1F为MR增强。
图2为图1病人切除的胶质瘤病灶组织石蜡切片HE及IHC染色情况。HE中红箭头标记核分裂象,蓝色箭头标记核异型,虚线框标记微血管增生带。IHC示GFAP(+),Nestin(+),EMA(+),Olig2(+),MGMT(+),Neun(-),Syn(-),p53(+),EGFR(+),c-myc(+),Ki67 10%。
图3为图1中肿瘤组织培养出来的胶质母细胞瘤细胞系GWH04明场细胞形态学及细胞免疫荧光染色情况。其中,图3A为P1、P2、P10、P50代细胞的光学形态图片,图3B为GFAP及Nestin染色情况。
图4为图1中GWH04细胞系的细胞增殖能力和锚定非依赖生长能力相对于U87和U251的对比图。图4A为细胞增殖曲线图,图4B为判断锚定非依赖生长能力的软琼脂克隆形成实验明场图片,图4C、图4D为克隆形成的细胞球数量多少和大小的统计分析图。
图5为皮下成瘤、颅内成瘤及颅内成瘤组织对比病人原生肿瘤染色结果。图5A为第60天裸鼠皮下成瘤结果;图5B为GWH04表达荧光素酶(luciferase)后颅内成瘤第30天活体成像结果及对应的HE染色情况;图5C为病人原发肿瘤和GWH04细胞系裸鼠颅内成瘤组织的HE及相关免疫组化指标GFAP,Nestin,EMA,Ki67在表达情况。
图6为第10代GWH04细胞核型分析情况,其中,图6A为计数40个细胞核型染色体数目分布情况,染色体数目分布范围为61~125条,上下四分位数是86和82,图6B为其中一核型染色体镜下拍照情况,图6C为该例染色体核型分析情况。
图7为对GWH04细胞系胶质瘤相关基因突变情况检测报告。其中,TERT启动子C228T突变阳性,MGMT启动子未甲基化,IDH野生型,1p19q无共缺失。
图8为GWH04的STR分析图谱。
图9为中国科学院生物化学与细胞生物学研究所苏州研究院的STR检测和污染鉴定结果,其中细胞曾用名为Fu。
图10为药物TMZ、CCNU、5-Fu、oxaliplatin、SN-38、VP-16在GWH04的IC50检测结果。
具体实施方式
下面结合具体实例,进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法,可采用本领域中的常规方法,例如参考《分子克隆实验指南》(第三版,纽约,冷泉港实验室出版社,New York:ColdSpringHarbor Laboratory Press,1989)或按照供应商所建议的条件。
本发明涉及生物材料保藏,其中,保藏单位为中国典型培养物保藏中心,地址为中国武汉,武汉大学,邮编:430072,保藏时间为2021年03月31日。
下列实施例中未特别说明的各种仪器、原料和试剂均为本领域熟知的市售产品,可通过商业途径获得。在下列实施例中列出的所使用的具体材料及其来源,仅仅是示例性的,并不意图限制本发明,与如下组织、细胞、试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。
实施例1
本实施例公开了人胶质母细胞瘤细胞系GWH04的建立,它包括如下步骤:
1.1标本的来源:
该肿瘤组织标本来源于一名72岁右侧颞叶占位的胶质瘤女性患者,2016年11月因“头痛10余天,加重4天”入院,病人MR平扫+增强+波谱提示:右侧颞叶占位,考虑为肿瘤性病变,伴右侧海马旁回疝,大脑镰下疝,后于我院行开颅占位显微切除术。术后病检示:(右颞叶)胶质母细胞瘤(WHO IV级)。免疫组化:GFAP(+),NEUN(-),Nestin(+),EMA(+),Syn(-),Olig2(+),MGMT(+),Ki67 30%,EGFR(+),p53(+),C-myc(灶+)。术前签署病人知情同意书。病人术前CT及MR影像学表现见图1。病人肿瘤组织切片染色情况见图2。
1.2标本处理:
取材:术中无菌留取约0.5cm3肿瘤活性较高部分标本,快速送至实验室。
消化:用PBS清洗去除表面残余血细胞后,在平皿中用剪刀将组织反复剪碎,加入高糖DMEM培养基3mL,继续剪碎组织至大小约1~2mm颗粒状后,加入3mL含EDTA胰酶吹打混匀,并移至15mL离心管,至4℃摇床内,快速振摇20分钟。
过滤:用不小于200目的粗网径滤网过滤悬液,并离心,沉淀用5mL培养基重悬,所述培养基为高糖DMEM+10%FBS,转移至T25培养瓶培养,次晨换液。
常规传代培养:T25培养瓶细胞长满后,用含EDTA的胰酶1mL室温消化2~3min后,培养基中和,1000r/min,5min离心后,1:5传代。5~7天可传代一次。
1.3单克隆筛选:
种单克隆板:用1×BME基础培养基配制0.5%的Agar溶液,所述Agar溶液包含10%FBS,2mM L-谷氨酰胺,25μg/mL庆大霉素,置于60℃水浴中。准备48孔板中,每孔加150μL该溶液,铺3张,常温放置1h待其凝固。取第10代生长状态良好的细胞,消化制备细胞悬液,90个/mL,取细胞悬液6mL混于12mL上述0.5%的Agar溶液中,制备成0.33%的细胞Agar悬液(约3个/100μL),向底层胶已凝固的3张培养板中添加细胞悬液100μL/孔。置于37℃细胞培养箱中培养两周后观察。
获得单克隆细胞:培养过程中观察并标记单细胞孔,在第14天将成团的单细胞挑出置于新的24孔板中,单孔加1mL含血清培养基培养,待其贴壁并逐渐爬开扩增,1周后使用胰酶消化并于原孔传代培养。第2周传至6孔板,第3周传至T25培养瓶,经过6周时间3次传代建立起GWH04细胞系来源的单克隆细胞多株,其中,曾用名Fu1.4,Fu1.5,Fu3.4,Fu3.17,其中对Fu3.17进行相关测序及STR鉴定等,命名为GWH317,其为GWH04的一株单克隆。目前细胞生长良好,形态均一,传代情况稳定。并于2021年03月31日,一并保藏于中国典型培养物保藏中心,保藏编号为C202164。
实施例2
本实施例公开了人胶质母细胞瘤细胞系GWH04的生物学特性。
2.1GWH04细胞系的形态学观察
取传代培养的GWH04细胞,在日本Olympus IMT-2倒置显微镜100×下观察活细胞生长情况。结果见图3,依次分别为GWH04细胞系P0、P1、P2、P10、P20、P40代明场细胞形态图片,由图3可见:本细胞系生长旺盛,背景清晰,杂质少见,贴壁生长细胞为梭形。
2.2GWH04细胞系的生长动力学研究
细胞增殖能力检测:取对数期生长的GWH04、U87、GL15细胞,制备2×104/mL细胞悬液,铺96孔板,100uL/孔,即2×103/孔,细胞生长5天,期间不更换培养基,每个细胞30个副孔,分6次检测用,每次检测5个孔,依次记为第0、1、2、3、4、5天检测。种细胞当天、细胞贴壁后及后续每天相同时间点,每孔加入10ul CCK8试剂,置培养箱培养1小时后,酶标仪检测波长450nm及参比波长620nm的吸光度值,即OD值,记OD450~OD620,每组去掉最高、最低的两个值,其余数据行统计学分析。第n天增殖率=(第n天OD值-第0天OD值)/第0天OD值。绘制GWH04、U87、GL15细胞系生长曲线如图4A所示。由图4A可知,GWH04细胞第1、2、3、4天进入倍增状态,具有较强增殖能力,生长速度最初比U87慢,后与U87相当。
2.3GWH04锚定非依赖生长
6孔板下层铺0.5%的Agar Mix 3mL,待室温凝固后,取对数期生长的GWH04、U87、GL15细胞,分别制备0.33%的Agar Mix细胞悬液3.6mL,8000个细胞/mL。每个细胞3个副孔,在已室温凝固的0.5%的Agar Mix上层铺上述0.33%的Agar Mix细胞悬液1mL/孔。置培养箱培养,14天后取出镜下拍照记录代表图片见图4B。图4C、4D为克隆形成数量和克隆形成大小分析图。
2.4GWH04细胞系的核型分析
待T25细胞培养瓶中细胞长满且状态良好时,加秋水仙素0.25μg/mL继续培养处理3h,消化离心,于37℃水浴中0.075mmol/L KCl低渗液处理15min后,加固定液固定(V甲醇/V乙酸=3:1),悬液滴片,进行Gemisa染色,油镜下观察分析。计数40个细胞核染色体数目,25%~75%区间为82~86条,如图6A所示。图6B、6C为同一个细胞染色体制片染色图片,及核型分析图片。
2.5GWH04细胞系的基因检测
进行胶质瘤相关基因检测,MGMT启动子区域甲基化水平检测,1p19q共缺失检测,IDH突变,TERT启动子区域C228T、C250T突变检测等,检测报告如图7所示。且全外显子测序相关基因点突变情况见表1。
表1 全外显子测序相关基因点突变情况列表
2.6GWH04细胞系的成瘤性检测及染色分析
将GWH04细胞种于裸鼠右腋窝皮下,200万细胞/裸鼠,60天后,裸鼠皮下成瘤情况见图5A。luciferace慢病毒感染GWH04细胞并稳定筛选后,种于裸鼠颅内,70万细胞/裸鼠,30天老鼠有明显症状,行走不稳,活体成像情况及脑组织切片HE染色见图5B。病人颅内肿瘤(GBM)及原代培养后裸鼠颅内成瘤(Xenograft)的HE及免疫组化染色情况见图5C。
2.7GWH04细胞系的遗传物质STR分析
短串联重复序列(short tandem repeat,STR)又称为微卫星DNA,是指染色体上,由数个碱基对作为核心单位,一般2~6个碱基对,串联重复形成的一类DNA序列,重复次数为10~60多次,基因片段在400碱基对以下;每个核心单位重复的次数会出现个体差异,从而形成片段长度不同的等位基因。因此,一组STR序列的重复次数在不同个体中几乎是唯一的,是个体的基因身份特征,也是细胞生物学对细胞身份和来源进行鉴定的主要方法。
将收集的第9代GWH04细胞(送检时曾用名Fu)及术中切除并速冻的病人肿瘤组织,寄送至中国科学院生物化学与细胞生物学研究所苏州研究院,进行STR位点和性别基因Amelogenin的检测。报告情况如图8、9,可知本发明筛选得到的GWH04细胞是真实存在的。
STR位点包括:Amelogenin、D3S1358、D1S1656、D6S1043、D13S317、PentaE、D16S539、D18S51、D2S1338、CSF1PO、PentaD、TH01、vWA、D21S11、D7S820、D5S818、TPOX、D8S1179、D12S391、D19S433、FGA。
在德国微生物菌种保藏中心DSMZ数据库(Deutsche SammlungvonMikroorganismen und Zellkulturen)、美国典型培养物保藏中心(ATCC)数据库,进行STR序列检索,未发现相同STR检测结果。即,未找到与该细胞STR位点完全匹配的细胞系,表明此为新细胞株;未发现多等位点,表明此细胞株为单一细胞,无其他细胞污染。
其中,图8为GWH04细胞系的STR位点和Amelogenin位点的基因分型结果,报告中Fu-C为GWH04细胞样品,Fu-T为病人的肿瘤组织样本。综合以上实验观察与验证,体外生长的GWH04细胞具有较好的贴壁性,且细胞性状稳定,可稳定多次传代,且呈恶性生长。遗传物质STR鉴定该细胞为新的单一细胞株。免疫组化分析和病理学鉴定表明其GFAP阳性表达,EGFR阳性表达,MGMT高表达,Nestin强阳性,TP53为突变蛋白无功能阳性表达,基因检测示TERT启动子区域C228T突变阳性,IDH野生型,这些都提示该细胞系为典型的颅内原发胶质母细胞瘤来源的细胞系。
实施例3
本实施例公开了具备上述生物特征的人胶质母细胞瘤细胞系GWH04在建立胶质瘤动物模型中的应用。具体如上述步骤2.6及图5可知,人胶质母细胞瘤细胞具备非常强的裸鼠皮下成瘤和颅内成瘤能力,可以成功制备胶质母细胞瘤动物模型,实验发现颅内成瘤30天,裸鼠可出现明显症状;皮下成瘤30天,可见明显直径10mm肿瘤形成。
实施例4
本实施例公开了具备上述生物特征的人胶质母细胞瘤细胞系GWH04在用于筛选治疗胶质瘤药物中的应用。
相关化疗药物IC50检测:
将处于对数生长期的GWH04细胞消化计数并种96孔板,3000个cell/孔,于72h后换梯度药物培养基(TMZ,CCNU,SN-38,VP-16,5-Fu,Oxaliplatin),3天换液一次,药物作用6天后通过CCK8检测细胞活性,计算对应药物IC50。如图10所示。GWH04细胞MGMT蛋白阳性表达,理论上对烷化化疗药物TMZ耐药,实际检测的IC50为980.3uM,结合既往相关文献研究知其为TMZ耐药细胞株。
综上所述,本发明筛选得到的人胶质母细胞瘤细胞系GWH04,可作为有效的靶点研究细胞系,在筛选治疗胶质瘤药物中具备较好应用前景。
Claims (9)
1.一种人胶质母细胞瘤细胞系GWH04,其特征在于,它是保藏于中国典型培养物保藏中心CCTCC,保藏号CCTCC NO.C202163的人胶质母细胞瘤细胞系GWH04。
2.根据权利要求1所述人胶质母细胞瘤细胞系GWH04,其特征在于,它为来源于一位右侧颞叶占位胶质母细胞瘤患者的肿瘤组织进行体外培养的细胞和/或子代细胞。
3.根据权利要求2所述人胶质母细胞瘤细胞系GWH04,其特征在于,所述子代细胞包括保藏于中国典型培养物保藏中心CCTCC,保藏号CCTCC NO.C202164的单克隆细胞珠GWH317。
4.一种人胶质母细胞瘤细胞系GWH04的培养方法,其特征在于,包括如下步骤:
1)取材:术中留取胶质母细胞瘤患者的肿瘤组织0.5cm3于含DMEM的无菌取样管中,置于4℃冰盒中并快速送到实验室;
2)消化:在无菌超净台中用PBS清洗弃去步骤1)所得样本表面残余血细胞后,在平皿中用剪刀将组织反复剪碎,加入高糖DMEM培养基3mL,继续剪碎组织至1~2mm颗粒状,加入3mL含EDTA的胰酶继续吹打混匀,并移至15mL离心管内,在4℃摇床中,快速振摇20分钟;
3)过滤:用不小于200目网径的滤网过滤步骤2)所得悬液,并离心、沉淀,用5mL培养基重悬,所述培养基内包含高糖DMEM+10%FBS,然后转移至T25培养瓶培养,次晨换液;
4)常规传代培养:T25培养瓶细胞长满后,用1mL含EDTA的胰酶室温消化2~3min后,培养基中和,控制离心条件为1000r/min,5min离心处理后,继续1:5传代。
5.一种权利要求2所述人胶质母细胞瘤细胞系GWH04的用途,其特征在于,它包括人胶质母细胞瘤细胞和/或子代细胞在哺乳动物中产生胶质瘤瘤块并用来研究胶质瘤发生机理及耐药机理。
6.根据权利要求5所述人胶质母细胞瘤细胞系GWH04的用途,其特征在于,所述哺乳动物是免疫缺陷小鼠。
7.一种人胶质母细胞瘤细胞系GWH04在制备用于筛选治疗胶质瘤药物中的应用。
8.根据权利要求7所述人胶质母细胞瘤细胞系GWH04在制备用于筛选治疗胶质瘤药物中的应用,其特征在于,它包括建立胶质瘤动物模型并用来筛选胶质瘤治疗药物。
9.根据权利要求7或8所述人胶质母细胞瘤细胞系GWH04在制备用于筛选治疗胶质瘤药物中的应用,其特征在于,将测试化合物施用于体外细胞模型中,施用后抑制细胞增殖或导致细胞凋亡的测试化合物为治疗人胶质母细胞瘤的候选化合物。
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