CN113750220A - 间充质干细胞联合tpo及其类似物在治疗慢性髓性白血病中的应用 - Google Patents
间充质干细胞联合tpo及其类似物在治疗慢性髓性白血病中的应用 Download PDFInfo
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Abstract
本发明属于医药生物技术领域，具体涉及一种脐带来源间充质干细胞联合TPO及其类似物在制备治疗慢性髓性白血病中的应用。本发明提供的间充质干细胞为人来源的脐带间充质干细胞(UC‑MSCs)，在联合TPO类似物Eltrombopag后可高效诱导慢性髓性白血病细胞分化，该种能力是通过MSCs分泌TPO并诱导白血病细胞表达TPO受体(MPL)来实现的；相较于化疗，间充质干细胞移植的毒副作用更低，且具有低免疫原性，满足临床需求，且Eltrombopag为临床批准药物。该种方法为髓性白血病的干细胞移植治疗提供了新的思路，可应用于髓性白血病的分化治疗。
Description
技术领域
本发明属于医药生物技术领域，具体涉及一种间充质干细胞与TPO及其类似物的联合方法及其在慢性髓性白血病治疗中的应用。
背景技术
慢性髓性白血病(chronic myeloid leukemia，CML)是一种在造血干细胞(hematopoietic stem cell，HSC)中由于染色体易位t(9；22)(q34；q11)导致干/祖细胞不受控制的增殖和停滞的分化引起的增殖性肿瘤，其由BCR-ABL具有组成型酪氨酸激酶活性的融合蛋白驱动白血病细胞的克隆扩增。酪氨酸激酶抑制剂(TKIs)，如甲磺酸伊马替尼(imatinib mesylate，IM)，能够针对该致病蛋白BCR-ABL，是目前CML的标准治疗方案。但发生IM耐药的CML患者病情进展迅速、预后不良，因此IM耐药成为CML治疗亟需解决的问题。找寻另一个靶点抑制CML细胞的增殖和促进其分化可能是有效治疗的重要途径。分化疗法已经被成功应用于白血病的临床治疗，以全反式维甲酸和砷类化合物为代表的促分化小分子药物，已被证明能够促进急性早幼粒白血病(Acute promyelocytic leukemia，APL)向成熟粒细胞分化，进而抑制白血病细胞的增殖并降低其恶性程度。因此找到更为适合CML的分化疗法有望解决目前临床治疗的瓶颈。
间充质干细包(Mesenchymal stem cells，MSCs)是一种多潜能成体干细胞，广泛分布于间充质组织中，如脐带、脂肪、骨髓等。因其广泛的分泌能力和体内迁移能力已被证明能发挥免疫抑制作用和抗肿瘤作用。脐带间充质干细胞(Umbilical cord MSCs，UC-MSCs)是一种从新生儿脐带组织中分离获得的间充质干细胞。相比于其他成体间充质干细胞，UC-MSCs具备一些独特的优势，例如生长迅速，细胞分泌能力旺盛，免疫原性更低等。有证据表明，来自于健康新生儿的异体UC-MSCs具备更强的免疫调节活力，因而被成功应用于治疗多种自体免疫性疾病。同时，也有一些报道指出UC-MSCs具备一定的抗肿瘤活性。临床上间充质干细胞的移植可用于治疗多种慢性疾病，加上其具有较强的抑制增殖和促进恶性增殖细胞分化的能力，为我们探索CML的新治疗策略提供了线索。
造血干/祖细胞的发育过程中依赖血小板生成素(thrombopoietin，TPO)信号进行维持和扩增。血小板生成素通过与其受体(MPL)结合，是造血干细胞稳态维持和进一步分化成熟的关键调节因子。在正常造血干细胞和巨核细胞中持续表达MPL，而研究发现随着CML的疾病进展该受体的表达逐渐降低，这可能成为CML未来治疗的突破口。在提高CML细胞中MPL表达的同时联合TPO或其类似物增强TPO/MPL信号，有望使CML细胞跨越分化障碍。艾曲波帕eltrombopag(CAS号：496775-62-3)是一种非肽类的TPO类似物可与MPL的跨膜结构域结合，已被临床批准成功用于血小板减少症。本发明利用MSCs联合TPO及其类似物，增强CML细胞的分化能力，从而达到控制CML病程的作用。
发明内容
本发明目的在于提供一种使用间充质干细胞(可简称为MSCs)与TPO及其类似物治疗慢性髓性白血病的方法和组合。其中包含MSCs与TPO及其类似物的联用技术方法，以及评价促进慢性髓性白血病分化效能的方法与标准。
本发明提供了一种用于治疗慢性髓性白血病的细胞治疗组合物。该组合物包括间充质干细胞(可以是脐带或其他来源)和TPO及其类似物(肽类或非肽类TPO类似物，例如艾曲波帕)。二者联合使用可以使肿瘤细胞的增殖受阻并向更成熟的谱系分化，从而建立一种全新的CML分化疗法。
本发明所述的脐带间充质干细胞的分离及原代培养的方法为：取新生婴儿脐带组织，无菌条件下清洗干净，剪碎，并采用酶消化法分离其中间充质干细胞，将得到的细胞用含有10％胎牛血清，100U/mL青霉素和0.1mg/mL链霉素的α-MEM培养基置于37℃、5％CO2、饱和湿度培养箱中贴壁培养。待细胞长至80％丰度时，用含0.25％胰酶和0.02％EDTA的细胞消化液消化细胞，重悬细胞，然后接种到新的培养皿中，传代细胞。待细胞长至90％融合后，进行下一次传代培养。
本发明所述脐带间充质干细胞的鉴定方法如下：
流式细胞术检测分离的MSCs细胞表面标志物，根据2006年“The InternationalSociety for Cellular Therapy”发布的间充质干细胞准则，要求该细胞在标记物检测中应符合：细胞表面标记物CD11b、CD34、CD45、CD19、HLA-DR表达阴性；标记物CD44、CD73、CD90、CD105表达阳性，经测定实施方式中的全部间充质干细胞均符合要求。
MSCs多潜能性评价：将脐带来源的间充质干细胞进行成脂、成骨和成软骨诱导分化及鉴定：采用广州赛业生物的MSCs成骨和成脂分化诱导培养基以及诱导方案进行诱导成脂肪和成骨分化；采用Stem Cell的MSCs成软骨分化诱导培养基以及诱导方案进行诱导成软骨分化。通过形态学染色和观察，判断分化能力。
MSCs联合TPO及其类似物促进CML细胞分化的效能评价方法：
将MSCs以1×105/孔的数目接种到12孔板中，放到培养箱中细胞贴壁生长12h，弃培养上清。将3×105/孔的慢性髓性白血病细胞(原代细胞或K562细胞株)接种在MSCs培养皿中并加入2000ng/ml的eltrombopag，共培养48h，以在相同培养条件下单独培养的K562细胞株、只加入eltrombopag培养和只与MSCs共培养的K562细胞为对照。
实时定量PCR测定白血病细胞中巨核生成标志物的表达。
蛋白质印迹法检测白血病细胞中TPO/MPL下游信号通路的激活情况。
有益效果：本发明中，MSCs显著抑制白血病细胞的增殖并促进其凋亡。与艾曲波帕的联合应用更能够显著诱导白血病细胞分化。此种联用方法规避了IM治疗时的耐药现象，可作为一种治疗慢性髓性白血病的新型细胞疗法。
附图说明
图1为MSCs成脂肪、成骨和成软骨细胞的诱导分化鉴定(a)；MSCs流式检测标记物CD14、CD34、CD44、CD45、CD90、CD105的表达(b)的结果。
图2为MSCs影响K562细胞增殖(a)、细胞周期(b)和凋亡(c)的结果。
图3为MSCs与K562细胞共培养，MSCs中TPO的表达和K562细胞中MPL的表达结果。
图4为MSCs与艾曲波帕体外诱导K562细胞巨核分化的结果。(图4a为K562细胞巨核分化标志物的表达情况，图4b为巨核分化相关转录因子的表达情况)。
图5为western blot检测MSCs与艾曲波帕联用引起慢性髓系白血病细胞TPO/MPL下游信号通路激活的结果。
具体实施方式
以下结合实例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并未用于限定本发明的范围。
脐带间充质干细胞的分离及传代培养
1.配置组织消化酶(II型胶原酶250U/ml，中性蛋白酶100U/ml，透明质酸酶10U/ml)，37℃溶解于α-MEM培养基，用0.22μm的滤器过滤除菌备用；
2.取新生婴儿的脐带组织，将脐带放入培养皿中用含0.1％青霉素-链霉素双抗的PBS清洗干净，放入α-MEM培养基中，剥离三根血管，将脐带剪碎成1-2mm3的组织块；
3.将剪碎的组织块与配置的组织消化酶溶液按1∶1的体积比例混合于50ml离心管中，37℃，200rpm，消化3h，待组织块基本消化完全即可；
4.将消化后的组织液4℃，300g离心5min，弃上清。PBS重悬于50mlα-MEM培养基中，4℃，300g离心5min，弃上清。PBS洗两次，将沉淀重悬于含有10％胎牛血清、1％双抗的α-MEM培养基中，接种于直径10cm细胞培养皿中，放置在37℃、5％CO2、饱和湿度培养箱中静置贴壁培养；
5. 3天后，半量换液。此后，每两天换液，MSCs沿着贴壁的组织块或者贴壁的细胞长出；
6.待细胞长至80％丰度时，用含0.25％胰酶和0.02％EDTA细胞消化液消化下细胞；重悬细胞，1000rpm离心5min，弃上清，PBS洗一遍，离心后弃上清，获得的细胞沉淀用新鲜的培养基重悬，然后接种到新的培养皿中，传代细胞。待细胞长至90％融合后，进行下一次传代培养。
流式细胞术鉴定MSCs细胞表面标志物：预处理UC-MSCs，用细胞消化液消化下细胞，重悬细胞，离心弃上清，用PBS洗两遍后，用PBS重悬成1-2×106/ml的单细胞悬液，分别加入荧光标记抗体CD14-FITC、CD34-FITC、CD45-FITC、CD73-FITC、CD90-FITC、CD105-FITC、IgG1，κ-FITC、IgG2α，κ-FITC，冰上避光染色；PBS洗去未结合的抗体，然后加入500ulPBS重悬，上机检测。
间充质干细胞与TPO及其类似物的联用治疗方案的优化
1.MSCs以1×105/孔的数目接种到12孔板中，使用含有10％FBS的α-MEM完全培养基，37℃，5％CO2培养箱内培养过夜，将人源白血病细胞系(K562细胞)或白血病病人来源的原代白血病细胞按照与MSCs数量比为3∶1的比例铺进含MSCs的培养皿中，更换为含有10％FBS的1640培养基，加入艾曲波帕，使其在培养液中的终浓度为10-2000ng/ml。在此条件下，共培养48小时。以单独培养的K562细胞株为对照。。
2.作为本发明MSCs联合eltrombopag的一种优选实施方案，所述eltrombopag在培养液中的终浓度为2000ng/ml。
以上实施例进一步说明本发明的内容，但不应理解为对本发明的限制。对于本领域的技术人员来说，在不背离本发明精神和实质的情况下对本发明方法、步骤或条件所做的修改和替换，均属于本发明的范围。
Claims (9)
1.一种间充质干细胞联合TPO及其类似物在治疗慢性髓性白血病中的应用，其特征在于，人源的间充质干细胞联合TPO及其类似物艾曲波帕(eltrombopag)联合使用，进而发挥诱导慢性髓性白血病细胞凋亡和分化的功能。
2.根据权利要求1所述的细胞治疗组合物，其特征在于：该组合物包括间充质干细胞和TPO及其类似物。
3.根据权利要求2的细胞治疗组合物，其特征在于：
所述TPO类似物eltrombopag的使用终浓度为10-2000ng/ml；
所述间充质干细胞的密度为2-5×107/ml。
4.根据权利要求1所述的用途，其特征在于，间充质干细胞联合TPO及其类似物在制备作为慢性髓性白血病细胞治疗组合物中的应用。
5.根据权利要求4所述的用途，其特征在于，所述间充质干细胞联合TPO及其类似物可抑制慢性髓性白血病细胞增殖，并诱导其凋亡。
6.根据权利要求5所述的用途，其特征在于：所述间充质干细胞与TPO及其类似物的联用恢复慢性髓性白血病细胞MPL的表达，促进白血病细胞的巨核分化。
7.根据权利要求6所述的用途，间充质干细胞与TPO及其类似物的联合使用，其特征在于间充质干细胞诱导慢性髓性细胞高表达TPO受体(MPL)联合使用TPO或其类似物(如eltrombopag)激活白血病细胞MPL下游信号通路，进而高效诱导白血病细胞向巨核细胞分化的功能。
8.根据权利要求1～7任一项所述的使用的间充质干细胞，其特征在于使用异体来源的间充质于细胞，如：新生儿的脐带组织来源的间充质干细胞。
9.根据权利要求8所述的一种间充质干细胞联合TPO及其类似物在治疗慢性髓性白血病中的应用，间充质干细胞与TPO及其类似物的联合方法，作为一种全新的慢性髓性白血病分化疗法。
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