CN113549597A - 一种人原发性骨髓纤维化细胞株及其应用 - Google Patents
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Abstract
本发明公开了一种人原发性骨髓纤维化细胞株及其构建方法和应用。原发性骨髓纤维化细胞株命名为人原发性骨髓纤维化细胞株ZYXY‑M2，于2021年1月20日保藏于中国典型培养物保藏中心(武汉大学，中国武汉)，保藏编号为CCTCC NO:C202145。本发明通过从临床原发性骨髓纤维化患者外周血提取分离得到单个核细胞，体外培养连续自然传代而得到。该白血病细胞株JAK2、CALR、MPL突变阴性，ASXL1突变阳性,TP53突变阳性,IKZF1突变阳性，IDH1突变阳性，FLT3突变阳性，TET1突变阳性。具有良好的体外增殖能力，可作为研究人原发性骨髓纤维化发生发展机制研究及个体化治疗体外研究的细胞材料，同时可用于体内外研究人原发性骨髓纤维化药物筛选、评估，指导临床用药。
Description
技术领域
本发明涉及生物学和肿瘤学领域，具体涉及一种人骨髓纤维化细胞株及其构建方法和应用。
背景技术
血液系统源于髓系的恶性肿瘤依据发病时原始细胞分化水平、比例及细胞形态及分子特点分为急性髓系白血病及其相关肿瘤和慢性髓系肿瘤。慢性髓系肿瘤中最常见是BCR-ABL融合基因阳性的慢性髓系白血病和BCR-ABL阴性的骨髓增殖性肿瘤(MPN)。MPN中最为常见的是真性红细胞增多症(PV)、原发性血小板增多症(ET)和原发性骨髓纤维化(PMF)三种疾病。研究表明JAK2、CALR、MPL突变在PV、ET、PMF检测阳性的概率为：PV患者中分别为95％、0％、0％,ET患者中分别为60％、20％、3％,PMF患者中分别为60％、25％和7％。尽管在基因水平上三种疾病存在很多的相同，但是三种疾病在骨髓的病理、临床表现、疾病进展速度、临床转归及预后上具有明显的不同。
原发性骨髓纤维化(PMF)是MPN中以干细胞衍生的克隆性骨髓增殖为特征，通常但不总是伴有JAK2、CALR或MPL突变，约10％-15％的呈现JAK2、CALR或MPL突变阴性。临床特征包括骨髓网状蛋白/胶原纤维化、异常的炎性细胞因子表达、贫血、肝脾肿大、髓外造血(EMH)、全身症状(如疲劳、夜间出汗、发热)、恶病质、白血病进展和生存期缩短。相较于PV和ET，PMF的预后最差。研究表明三种疾病的总体中位生存时间PV是20年，ET是14年，PMF仅有6年。对于PMF目前为止国际上共先后提出5种预后分层体系，对于高危的PMF其中位生存时间不足2年。死亡原因包括大约20％的患者发生白血病进展，许多患者也死于合并症，包括心血管事件和血细胞减少症的后果，包括感染或出血。
当前PMF治疗手段极其有限，异基因造血干细胞移植(AHSCT)是当前唯一可以延长患者生存时间或使患者治愈的方法，但是接受AHSCT的患者至少存在50％的移植相关严重并发症或移植相关死亡。因此，对于个体患者，必须权衡AHSCT的风险与未进行AHSCT的预期生存率。研究表明，PMF患者匹配相关移植的5年无病生存率(DFS)和治疗相关死亡率(TRM)分别为33％和35％，无关移植分别为27％和50％，复发率高达29％。PMF传统药物治疗包括羟基脲和鲁索替尼，但是仅限于改善患者的临床症状，没有抗肿瘤活性，也没有已被证明可以逆转骨髓纤维化或诱导细胞遗传学或分子缓解。PMF患者对羟基脲和鲁索替尼有反映的仅在15％～25％,反应时间也仅持续1年左右。近年来新的JAK抑制剂，如费拉替尼、莫莫替尼和帕克替尼目前正在进行临床实验，也仅作为鲁索替尼耐药的替代治疗。由此可知当前PMF的药物治疗仅作为姑息性的治疗手段。JAK2突变在PMF中占65％左右，也有待进一步发掘JAK2阴性患者的治疗药物。
PMF的预后及转归与其分子特点密切相关。研究表明JAK2、CALR或MPL突变可能介导了PMF的骨髓纤维化，但是其向白血病的转化可能与其他基因突变相关，如TET2、ASXL1、IDH1/2、EZH2、DNMT3A、CBL、RAS、IKZF1、TP53、SF3B1、SRSF2、U2AF1基因均在PMF发现。ASXL1、SRSF2、EZH2、IDH1、IDH2、U2AF1Q157被纳入PMF的危险分层体系中。
综上所述，当前PMF是MPN中高转白血病率、预后最差，治疗手段有限。肿瘤的研究基础性工具是肿瘤细胞株，因为细胞株具有体外无限增殖能力和良好的体内成瘤能力，克服了原代细胞有限的局限性。肿瘤细胞也是进行抗肿瘤药物筛选和肿瘤治疗方案验证的有效工具。目前国内外均未建立起由PMF病人起源的细胞株。国外已建立起源于CML的细胞株K562和源于ET的细胞株SET-2，对这两种疾病的基础及临床研究具有巨大的推动作用。因此亟待建立起源于PMF的细胞株，以用于预后最差的PMF的发病机理，分子特点，新药筛选，化疗方案更新等的研究。
发明内容
本发明的目的在于针对现有技术的不足，提供一株人原发性骨髓纤维化永生化细胞株及其构建方法及应用。
本发明的目的是通过以下技术方案来实现的：一种人原发性骨髓纤维化细胞株，该细胞株命名为人骨髓纤维化细胞ZYXY-M2，于2021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:C202145。
本发明提供的人原发性骨髓纤维化细胞JAK2突变阴性、CALR突变阴性、MPL突变阴性，ASXL1突变阳性,TP53突变阳性,FLT3突变阳性，IKZF1突变阳性。细胞形态呈原始红细胞，细胞表面不表达CD34、CD11b、表达CD71。细胞株细胞呈悬浮生长或弱贴壁生长。
本发明还提供如上所述人原发性骨髓纤维化细胞株的用途，选自以下任一项或多项：
a.用于骨髓纤维化的分子特点及治病机理的研究；
b.制备肿瘤细胞模型或制备肿瘤动物模型；其中，肿瘤细胞模型包括通过本细胞株建立起来的子代细胞或在本细胞株建立起来的子代细胞通过转染带荧光标记的基因建立起来的细胞。动物肿瘤模型包括通过皮下荷瘤或尾静脉注射建模的方法，建立的PMF的动物模型。
c.筛选和/或评价肿瘤治疗药物；其中，筛选肿瘤治疗药物的方法可以为：通过向所述人原发性骨髓纤维化细胞株培养基中添加不同肿瘤治疗药物，观察细胞形态变化，获得初步有效的候选药物。然后，将候选药物施药于所述细胞，计算筛选出来的有效药物的半数抑制浓度(I50)，选择IC50最低的药物进一步作用于动物模型，与未施药组动物的存活期、肿瘤大小、转移情况等进行比较，筛选获得潜在的治疗PMF的药物。
d.开发肿瘤药物靶点；
e.制备肿瘤诊断产品，如特异性标志的流式抗体，基因检测试剂盒等；
f.筛选肿瘤生物治疗药物/试剂；所述的肿瘤生物治疗药物/试剂为肿瘤疫苗。
g.开发检测肿瘤相关生物工程产品。所述的肿瘤相关生物工程产品可以为PMF特异性的分子诊断PCR试剂盒，荧光原位杂交试剂盒等。
本发明还提供所述人原发性骨髓纤维化细胞株的构建方法，具体如下：
获得新鲜的初发诊断明确的原发性骨髓纤维化(PMF)患者外周血。取6ml外周血滴加入预先加6ml淋巴细胞分离液的15ml无菌离心管中，离心2000转，20分钟。离心结束后取单个核细胞层白色细胞沉淀至新的15ml无菌离心管中，加入5ml无菌1xPBS重悬细胞，离心2000转，5分钟。弃上清液后，加入无菌的红细胞裂解液常温下裂解细胞5分钟后离心，2000转，5分钟。弃上清，加入5mlIMDM完全培养基(IMDM 90％+胎牛血清10％)重悬细胞，离心，1500转，5分钟。弃上清，加入5mlIMDM完全培养基，重悬细胞。细胞计数板计数细胞，取1*108细胞加入25cm培养瓶后加IMDM培养基至6ml混匀细胞，放入37摄氏度，恒温恒湿培养箱培养细胞。1周后更换新的IMDM培养基，直至细胞开始增殖。
本发明的有益效果是：本发明的人原发性骨髓纤维化细胞株可无限传代，细胞体外形状稳定，并符合临床肿瘤生物学特性。所述的人原发性骨髓纤维化细胞株起源于PMF患者，JAK2突变阴性，CALR突变阴性，MPL突变阴性，ASXL1突变阳性,TP53突变阳性,FLT3突变阳性，IKZF1突变阳性。所述人原发性骨髓纤维化细胞株均能用于PMF的发生发展的机理研究。还可以利用所述细胞分析抗PMF新药及联合方案的疗效，进行PMF的药物筛选和评估，可用于指导临床用药。对于揭示PMF这一预后不良的MPN具有重要的意义。
附图说明
下面结合实例和附图对本发明进行进一步说明；
图1为所述人原发性骨髓纤维化细胞株瑞氏-吉姆萨染色后的结果图；
图2为所述人原发性骨髓纤维化细胞株细胞表面抗原表达图；
图3为所述人原发性骨髓纤维化细胞株不同细胞密度下的细胞生长曲线图；
图4为所述人原发性骨髓纤维化细胞株基因组变异Circos图。
具体实施方式
下面用实施例来进一步说明本发明，但本发明并不受其限制。下列实施例中未注明具体条件的实验方法，通常按照常规条件。
实例1ZYXY-M2细胞株制备
原代细胞培养：将浙江大学医学院附属第一医院获得的新鲜高白细胞分离标本(男性，62岁，PMF，白细胞178.1*109/L)立即分离白血病单个核细胞。在生物安全柜中，取6ml分离液滴加入预先加6ml淋巴细胞分离液的15ml无菌离心管中，离心2000转，20分钟。离心结束后取单个核细胞层至新的15ml无菌离心管中，加入5ml无菌1xPBS重悬细胞，离心2000转，5分钟。弃上清液后，加入无菌的红细胞裂解液常温下裂解细胞5分钟后离心，2000转，5分钟。弃上清，加入5mlIMDM完全培养基(IMDM 90％+胎牛血清10％)重悬细胞，离心，1500转，5分钟。弃上清，加入5mlIMDM完全培养基，重悬细胞。细胞计数板计数细胞，取1*108细胞加入25cm培养瓶后加IMDM培养基至6ml混匀细胞，放入37摄氏度，恒温恒湿培养箱培养细胞。1周后更换新的IMDM培养基去除细胞碎片，继续培养。以后每周更换培养基一次。
细胞传代培养：细胞在培养2周时出现细胞的凋亡。剩余未凋亡的细胞呈现极缓慢增殖状态，以后每周更换培养基。细胞培养2.5月时细胞开始增殖，悬浮生长。此时每隔72小时更换细胞培养基并开始传代，至目前细胞传代已超过50代，呈永生化细胞株。
本发明中，细胞呈悬浮状态生长或弱贴壁生长(无需胰酶消化)，抱团生长，细胞呈圆形或椭圆形，细胞生长速度稳定，将其命名为ZYXY-M2，于2021年1月20日保藏于中国典型培养物保藏中心(地址：中国.武汉.武汉大学，邮编430072)，保藏编号为CCTCC NO:C202145。
实例2人急性髓系白血病细胞系的生物学性状及应用
本发明采用含10％胎牛血清的IMDM培养基培养细胞株，使其可以体外稳定生长并稳定传代。显微镜下观察细胞呈悬浮或弱贴壁单个或抱团生长，圆形或椭圆形。瑞氏-吉姆萨染色，细胞呈原始红细胞为主，细胞体积大，胞浆深蓝色，部分有大量空泡，核周有淡染区，核染色质细致，呈颗粒状，有明显核仁。细胞株经流式检测不表达CD34和CD11b，高表达CD71。经对细胞株细胞进行全外显子测序发现细胞株细胞呈现JAK2、CALR、MPL突变阴性，PMF不良预后或向白血病转化相关基因的TP53、ASXL1、IKZF1突变阳性。除此之外与急性髓系白血病相关的基因FLT3、TET1突变阳性。该细胞株可以用于制备肿瘤细胞模型或制备肿瘤动物模型；筛选和/或评价/制备的肿瘤治疗药物；开发肿瘤药物靶点；制备肿瘤诊断产品；筛选肿瘤生物治疗药物/试剂；开发检测肿瘤相关生物工程产品。具体如下：
形态学观察
将培养的ZYXY-M2细胞株置于倒置的显微镜下观察细胞呈抱团的悬浮生长或弱贴壁生长，细胞呈圆形或椭圆形。将培养的细胞取1*106个于1.5mlEP管中，离心1500转，5分钟，弃上清后加10ul培养基重悬细胞后推片。待细胞涂片干燥后，加瑞氏-吉姆萨染液染细胞涂片5分钟后，冲洗，晾干。于倒置显微镜下观察细胞形态，如图1所示，细胞以原始红细胞为主，细胞体积大，胞浆深蓝色，部分有大量空泡，核周有淡染区，核染色质细致，呈颗粒状，有明显核仁。
细胞表面抗原检测
将培养的细胞取1*106，共3份，分装在3个干净无菌的EP管中，离心，1500转。5min，弃上清，用1xPBS洗细胞一遍。离心1500转，5min后弃上清，每个EP管加入100ul的1xPBS重悬细胞，第一管不加抗体，第二管加FITC标记的抗人CD34抗体和PE标记的抗人CD11b抗体，第三管加FITC标记的抗人CD71抗体，每种抗体加10ul，室温下孵育30min后加入1ml1xPBS混匀后，离心1500转，5min。弃上清，每管加入300ul的1xPBS重悬细胞后上流式分析仪，测CD34、CD11b、CD71的表达。结果如图2所示，该细胞株不表达CD34、CD11b抗原，高表达红系标志CD71(73.25％)，与形态学原红细胞相符合。
体外增殖能力观察
将培养的ZYXY-M2细胞株按1，2，4*105/ml的浓度铺板于96孔板，每孔铺100ul，在1，24，48，72，96小时，分别加20ul的MTS，4小时后酶标仪测96孔板的吸光值，绘制不同铺板浓度下细胞的增殖曲线如图3所示，细胞株细胞具有良好的体外增殖能力，呈恶性生长。
细胞全外显子测序
将培养的细胞计数后取含5*106的细胞培养液离心，1500转/5分钟，去上清留细胞团块，加1xPBS重悬细胞后离心，1500转/5分钟，去上清液留细胞团块。提取样本中的基因组DNA，电泳检测合格DNA样品先经过Covaris随机打断成150bp-220bp的片段，采用AgilentSureSelect Human All Exon V6试剂盒进行建库和捕获，DNA片段经过末端修复、加ployA尾、加测序接头、纯化、磁珠捕获、PCR扩增等步骤,最终完成文库构建。文库检验合格后利用测序仪进行双端测序。测序下机获得原始测序数据(Raw data)后，进入生物信息分析流程，分为两个阶段：1、测序数据质量评估：主要通过对测序错误率、数据量、比对率、覆盖度等进行统计，评估建库测序是否达到了标准，符合标准则进行后续分析。
2、变异信息分析：将高质量的测序序列比对到参考基因组上，检测样本中的变异信息，并对检测到的变异信息进行分析解读。基因组变异Circos如图4所示。ZYXY-M2细胞基因组SNP和InDel突变位点检测分析结果与血液系统恶性肿瘤密切相关的突变信息总结如表一。从表中可以知道所述细胞株细胞呈现JAK2、CALR、MPL突变阴性，PMF不良预后或向白血病转化相关基因的TP53、ASXL1、IKZF1突变阳性。除此之外与急性髓系白血病相关的基因FLT3、TET1突变阳性。这提示所述细胞在研究PMF转化上具有一定的价值，包括研究PMF转白血病的治病机制或发病机制以及针对上述突变的靶向药物筛选等。
表一：ZYXY-M2细胞SNP和InDel突变位点总结结果
| 基因名 | 突变位点 | 功能区 | 突变类型 |
| JAK2 | - | ||
| CALR | - | ||
| MPL | - | ||
| LNK | - | ||
| TET2 | - | ||
| ASXL1 | C20:31022959T<C | 外显子-非同义 | M |
| IDH1 | C2:209120640C<T | 上游 | M |
| IDH2 | - | ||
| EZH2 | - | ||
| DNMT3A | - | ||
| CBL | - | ||
| RAS | - | ||
| KRAS | - | ||
| HRAS | - | ||
| NRAS | - | ||
| IKZF1 | C7:50430033A<G | 外显子-非同义 | M |
| TP53 | C17:7579472G<C | 外显子-非同义 | WT/M |
| SF3B1 | - | ||
| SRSF2 | - | ||
| U2AF1 | - | ||
| FLT3 | C13:28611358C<T | 外显子-非同义 | WT/M |
| C13:28624294G<A | 外显子-非同义 | WT/M | |
| C13:28674628T<C | 外显子-非同义 | WT/M | |
| TET1 | C10:70332580A<G | 外显子-非同义 | WT/M |
| C10:70332672T<G | 外显子-非同义 | WT/M | |
| C10:70332862C<T | 外显子-非同义 | WT/M | |
| C10:70445539A<G | 外显子-非同义 | WT/M | |
| C10:70405855A<G | 外显子-非同义 | WT/M |
注：M代表所述细胞仅检测到突变型；WT/M代表所述细胞野生型、突变型均检测到。
细胞STR鉴定
将培养细胞送上海翼和生物进行细胞STR位点和Amelogenin位点的基因分型。结果提示所述细胞株与国际已有细胞株不匹配，是独一无二的。且与患者刚分离的细胞STR位点和Amelogenin位点的基因分型匹配度达97％，为同一来源细胞，即细胞来源正确培养过程中无交叉污染。细胞STR位点和Amelogenin位点的基因分型如表二所示。
表二：细胞的STR位点和Amelogenin位点的基因分型结果
以上实施例用来解释本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明做出的任何修改和改变，都落入本发明的保护范围。
Claims (6)
1.一种人原发性骨髓纤维化细胞株，其特征是，该细胞株命名为人骨髓纤维化细胞ZYXY-M2，于2021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：202145。
2.根据权利要求1所述的人原发性骨髓纤维化细胞株，其特征是，细胞形态呈原始红细胞，细胞表面不表达CD34、CD11b、表达CD71。细胞株细胞呈悬浮生长或弱贴壁生长。
3.根据权利要求1所述的人原发性骨髓纤维化细胞株，其特征是，细胞呈JAK2突变阴性,CALR突变阴性,MPL突变阴性,ASXL1突变阳性,TP53突变阳性,IKZF1突变阳性,IDH1突变阳性TET1突变阳性,FLT3突变阳性。
4.如权利要求1-3任一项所述的人原发性骨髓纤维化细胞株的子代细胞。
5.如权利要求1-3任一项所述的人原发性骨髓纤维化细胞株的用途，其特征是，选自以下任一项或多项：
a.用于骨髓纤维化的分子特点及治病机理的研究；
b.制备肿瘤细胞模型或制备肿瘤动物模型；
c.筛选和/或评价/制备的肿瘤治疗药物；
d.开发肿瘤药物靶点；
e.制备肿瘤诊断产品；
f.筛选肿瘤生物治疗药物/试剂；
g.开发检测肿瘤相关生物工程产品。
6.如权利要求5所述的应用，其特征是，所述制备肿瘤动物模型的动物为免疫缺陷小鼠或C57BL6小鼠。
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