CN113337470A - 一种人源皮肤白血病的pdx模型细胞和应用 - Google Patents
一种人源皮肤白血病的pdx模型细胞和应用 Download PDFInfo
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Abstract
本发明提供一种人源皮肤白血病的PDX模型细胞和应用，所述细胞的名称为LCutisSK‑PDX细胞，其保藏编号为：CCTCC NO:C202165。本发明建立了能稳定传代，细胞遗传学改变与病人一致，能够稳定定植于皮肤的皮肤白血病PDX模型细胞。采用静脉注射或皮下注射该细胞，均可使模型小鼠发生皮肤白血病，表现为多发皮肤肿块，同时伴有骨髓、脾脏等脏器白血病细胞的浸润。利用该PDX小鼠肿瘤组织，进行体外化合物筛选，可获得皮肤白血病的敏感化合物。本细胞模型对于研究人类皮肤白血病发病机制以及筛选新的靶点和小分子药物提供了重要的临床前研究模型，具有很好的推广应用价值。
Description
技术领域
本发明涉及细胞模型技术领域，尤其涉及一种人源皮肤白血病的PDX模型细胞和应用。
背景技术
皮肤白血病是指异常增生的白血病细胞浸润皮肤，出现特异性皮损或非特异性皮损的表现。皮肤白血病可与白血病患者全身器官包括脾脏、肝脏和淋巴结等受累同时发生，也可发生在诊断白血病之前或之后。单纯皮肤白血病也称为原发性皮肤白血病，比较罕见，常常是全身白血病的早期表现。
皮肤白血病的特异性皮损的形状有多种表现，多呈丘疹、结节或肿块。皮损颜色可呈棕褐色、红色、紫色或蓝色；皮损部位可遍布全身，并可见累及口唇、齿龈，部分皮损可有破溃。非特异性皮损为多形性皮损，常见的有荨麻疹、大疱性皮疹、多形性红斑，也可见到丘疹坏死性皮疹、湿疹样皮疹或带状疱疹，甚至弥漫性红皮症、出血性皮损与溃疡。
由于在生物学特性、分子特征和临床表现等方面的巨大异质性，目前皮肤白血病的治疗并无统一或特异的针对皮肤病灶的方案。原发白血病诊断明确的，可采用对应白血病的治疗方案。但相当一部分皮肤白血病发生时原发灶并不明确，缺乏针对性治疗手段。更重要的是，诸多研究已证实皮肤白血病往往是白血病预后不良、化疗复发或快速进展的重要“预警信号”。然而，由于对其机制认识的空白，尚无有效的干预措施，使大量病人因无法得到早期治疗而死亡。2019年一项针对1683名急性髓细胞白血病(AML)白血病患者的回顾性研究发现，出现皮肤白血病表现的患者5年生存率仅有8.6％,大大低于整体AML群体的28.3％。因此，建立有效的体内、外皮肤白血病模型，深入研究其发病机制并开发新型靶向治疗途径具有重要临床意义。
PDX(Patient-derived xenograft)模型是将患者的原位肿瘤接种到免疫缺陷小鼠体内的一种临床前研究模型。与传统的皮下接种肿瘤细胞株成瘤的小鼠模型相比，PDX模型能更好地反映原发病人的病理特性以及基因表达，更准确地预测临床疗效，因此已成为近年来研究疾病发病机制及新的抗肿瘤药物筛选中不可或缺的工具。大量研究表明以PDX模型开展临床II期试验，能够极大的提高新药的临床通过率，缩短研发周期，减少研发费用。因此，2016年美国FDA停止肿瘤细胞系作为抗肿瘤药物的药效评价，将PDX模型列为药物反应检测的必备环节，使得PDX模型在肿瘤及药物研究中的作用日益凸显。目前，小鼠PDX模型技术主要集中在肠癌、胰腺癌、乳腺癌等实体瘤，商品化的PDX小鼠模型大大推进了实体瘤研究靶向新药物的研发进程。然而，由于皮肤白血病的标本来源少、异质性高，细胞数少、植入率低等原因，尚无稳定的PDX模型供研究使用。
发明内容
本发明的第一个目的在于，提供一种能稳定传代，细胞遗传学改变与病人一致，并且能够稳定定植于皮肤，同时侵犯骨髓、脾脏等造血器官的人源皮肤白血病PDX模型细胞。
本发明的第二个目的在于，提供人源皮肤白血病的PDX模型细胞的应用。
为了实现上述目的，本发明提供了一种人源皮肤白血病的PDX模型细胞，所述细胞的名称为LCutisSK-PDX细胞，其保藏编号为：CCTCC NO:C202165。
为了实现上述第二个目的，本发明提供了一种人源皮肤白血病的PDX模型细胞在体外筛药中的应用，将所述PDX模型细胞体外培养，筛选化合物库，获得对皮肤白血病敏感的化合物。
本发明提供了人源皮肤白血病的PDX模型细胞在制备治疗皮肤白血病药物中的应用。
本发明提供了人源皮肤白血病的PDX模型细胞在建立人源皮肤白血病PDX动物模型中的应用。
本发明人源皮肤白血病的PDX动物模型制备方法包括如下步骤：
(a)筛选合适的出现明显皮肤浸润包块的白血病患者，穿刺采集皮下肿瘤组织标本；
(b)肿瘤组织与Matrigel基质胶混合，制成悬液；
(c)NSG小鼠用X-ray放射线辐照，然后将步骤(b)中的细胞注射至小鼠皮下，注射后4、8、12、16周采血通过流式细胞术初步判断模型是否构建成功；
(d)通过皮下肿块、脾脏和肝脏病理切片、基因检测等手段进一步明确发病情况(P1代PDX小鼠)，P1代细胞命名为LCutisSK-PDX细胞；
(e)P1代PDX小鼠的皮下肿块或者骨髓细胞移植入新的NSG小鼠体内，通过流式细胞术、病理HE染色和基因检测等手段，明确发病情况(P2代PDX小鼠)；
(f)取P2代小鼠皮下肿块或者骨髓细胞继续进行传代，至PDX模型到P3代小鼠，P2-P3代小鼠及其细胞均可作为皮肤白血病研究模型。
本发明所要保护的细胞命名为LCutisSK-PDX细胞，保藏在中国典型培养物保藏中心(保藏地址：湖北省武汉市武昌区珞珈山路16号武汉大学中国典型培养物保藏中心邮编430072)，保藏日期是2021年2月23日，保藏编号：CCTCC NO:C202165。
本发明的优点在于，本发明建立了能稳定传代，细胞遗传学改变与病人一致，并且能够稳定定植于皮肤的皮肤白血病PDX模型细胞。进一步，利用该PDX小鼠肿瘤组织，进行了体外化合物筛选，获得了皮肤白血病敏感化合物。本细胞模型对于研究皮肤白血病发病机制以及筛选新的靶点和小分子药物提供了重要的临床前研究模型，具有较好的推广应用价值。
附图说明
图1皮肤白血病LCutisSK-PDX细胞来源及模型构建。A来源患者皮肤肿块穿刺细胞瑞氏染色结果。B,C,D皮肤白血病LCutisSK-PDX P1代小鼠的皮肤肿块和明显增大的肿瘤浸润的器官脾脏、肝脏照片。E,F,G用皮肤白血病LCutisSK-PDX小鼠造P2代模型小鼠发病时皮肤肿块、脾脏和肝脏照片。
图2流式细胞术明确皮肤白血病LCutisSK-PDX细胞所造P2代小鼠发病情况(皮肤肿块与外周血)。皮肤白血病的PDX发病小鼠的皮肤肿瘤灶(Tumor)、外周血(PB)制成细胞悬液，流式细胞观察humanCD45+(hCD45)、hCD33细胞比例大于3％，表明造模成功。
图3流式细胞术明确皮肤白血病LCutisSK-PDX细胞所造P2代小鼠发病情况(骨髓和脾脏)。浸润脏器骨髓(Bone marrow)和脾脏(Spleen)的流式细胞术检测，表明造模成功。
图4利用皮肤白血病LCutisSK-PDX细胞体外化合物筛选。取皮肤白血病XXX细胞用化合物库中的化合物处理，检测细胞活率，筛选出能有效杀伤该细胞的化合物compound 1、compound 2和compound 3。
具体实施方式
下面结合具体实施例，进一步阐述本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。应理解，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。
一、皮肤白血病患者样本处理：
1.采集皮肤白血病患者皮下穿刺肿瘤组织样本，用生理盐水洗涤两遍，以0.5ml体积生理盐水重悬，细胞计数。
2.以体积比1：1加入基质胶matrigel(购自美国BD公司)，冰上孵育20分钟后，经皮下注射于小鼠体内，接种细胞数为1×107/只。
二、NSG小鼠饲养及接种：
1.小鼠饲养条件：8周龄雌性NSG小鼠，购自北京百奥赛图基因生物技术有限公司，SPF级条件下饲养。室温18-25℃,相对湿度40％-60％，饲料及饮水经高压灭菌。每周至少更换2次垫料。
2.NSG小鼠接种前辐照：取5只小鼠进行称重，记录，用半致死剂量(1.25Gy)X光辐照。
3.NSG小鼠接种：辐照后6-12h皮下注射上述细胞，数量为1×107个。注射细胞前细胞先用0.2μm滤膜过滤，重悬于PBS中，注射体积为200μl。
三、造模成功评判指标：
1.一般指标：接种后，观察小鼠有无体表肿块、活动减少、消瘦、弓背、腹泻、脱毛、偏瘫等表现。每周给小鼠称重并记录。
2.尾静脉采血：注射后8-32周每隔一周取小鼠尾静脉血300-500ul，用红细胞裂解液裂解20分钟，PBS洗一遍，加入500μl PBS重悬制成有核细胞悬液。标记hCD45、hCD33抗体组合，流式检测人细胞比率，以判断是否移植接种成功。
3.发病小鼠各组织器官检查：小鼠于皮肤肿块达到1立方厘米、活动度明显下降立即处死或死亡后立即解剖，采集小鼠的皮肤肿块、骨髓、脾脏、肝脏，拍照记录脏器尺寸(参见图1)。皮肤肿瘤、脾脏和骨髓细胞悬液标记hCD45、hCD3抗体组合，用流式细胞术分析免疫表型，明确植入细胞比例(参见图2)。脾脏和肝脏组织采用常规固定、石蜡包埋、切片，然后用苏木素-伊红染色法(HE染色)进行染色，观察肿瘤细胞浸润情况(参见图3)。植入成功的判定为P1代小鼠，细胞命名为LCutisSK-PDX细胞。
四、皮肤白血病细胞连续接种传代：
1.P1代皮肤白血病细胞的保种冻存：将P1代NSG小鼠的皮肤肿块制成单细胞悬液，用胎牛血清+10％DMSO长期冻存保存于液氮中。
2.皮肤白血病细胞连续接种传代：传代前将P1代NSG小鼠细胞迅速于42℃水浴中解冻，PBS洗涤，经皮下或尾静脉注射于用X-ray放射线辐照过的NSG小鼠背部或体内，细胞数为2×106/只。
3.P2代发病的评判：观察指标同P1代，包括接种后动态尾静脉取血和发病后的体表肿物、各脏器的病理和流式指标。
4.P2代PDX的传代：将P2代小鼠皮下肿物或骨髓细胞液氮冻存，接种步骤同2，将P2代细胞传至P3代。从P2代开始，造模所需细胞数量明显减少，均为2×106/只以下。同批次小鼠脾脏和肝脏浸润情况高度一致。
五、皮肤白血病小鼠PDX模型体外化合物筛选的技术应用。
取造模成功的皮肤白血病小鼠PDX模型的皮肤肿块或骨髓组织，制成单细胞悬液。以3×105密度接种至96孔板中，分别加入化合物库中的小分子，48小时后检测细胞活率。细胞活率低于50％的被判定为有效化合物。根据细胞活率进行排序，筛选出有效浓度最低的FDA批准的化合物作为对皮肤白血病有效的候选化合物(参见图4)。
综上，本发明提供了皮肤白血病的PDX模型的建立方法，可用于皮肤白血病发病机制研究和大规模药物筛选实验，具有良好的应用前景。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种人源皮肤白血病的PDX模型细胞，其特征在于，所述细胞的名称为LCutisSK-PDX细胞，其保藏编号为：CCTCC NO:C202165。
2.权利要求1所述的一种人源皮肤白血病的PDX模型细胞在体外筛药中的应用，其特征在于，将所述PDX模型细胞体外培养，筛选化合物库，获得对皮肤白血病敏感的化合物。
3.权利要求1所述的一种人源皮肤白血病的PDX模型细胞在制备治疗皮肤白血病药物中的应用。
4.权利要求1所述的一种人源皮肤白血病的PDX模型细胞在建立人源皮肤白血病PDX动物模型中的应用。
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