CN113512525A - 一种间充质干细胞制剂及其应用 - Google Patents
一种间充质干细胞制剂及其应用 Download PDFInfo
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Abstract
本发明属于医药生物技术领域，具体涉及一种间充质干细胞制剂的制备方法与应用。本发明对人源间充质干细胞(MSCs)，经过由特定的多种生物活性分子和化合物组成的活化剂处理得到活化的间充质干细胞(Active MSCs)。所述Active MSCs特异性大量分泌白介素6，(IL6)，吲哚胺2，3加双氧酶(IDO)，骨形成蛋白6(BMP6)，中性粒细胞弹性蛋白酶，组织蛋白酶G，超氧化物歧化酶，组织金属蛋白酶抑制因子3(TIMP3)，蛋白质赖氨酸6氧化酶(LOX)，IGFL3，TNFAIP6，CYR61，TGFβ等多种生物活性物质，能够治疗自身免疫性疾病、提高机体的抗肿瘤能力、预防衰老，增加机体免疫力以及促进机体组织再生能力。本发明采用间充质干细胞活化剂对间充质干细胞进行诱导活化得到的Active MSCs无药物残留，并能高表达具有免疫调节和组织再生能力的生物活性物质。相较于化疗，Active MSCs移植的毒副作用更低，且具备低免疫原性，在肿瘤和免疫性疾病的治疗以及预防衰老方面具有重大的意义。
Description
技术领域
本发明属于干细胞治疗领域，具体涉及一种间充质干细胞制剂的制备方法与应用。
背景技术
间充质干细胞(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，如脂肪，骨髓等。间充质干细胞不仅具有同其他干细胞一样的强大的自我更新和多向分化的潜能，还具有如下三种特性：(1)来源广泛、易于分离、培养和纯化，多次传代后依然具有干细胞的特性；(2)MSCs通过直接接触或旁分泌的方式与天然和获得性免疫系统中的免疫细胞相互作用来发挥其免疫调节的功能；(3)MSCs的免疫原性低，因此异体移植时配型要求不严格，排异反应较轻；(4)MSCs具有向炎症和肿瘤部位“归巢”的特性。由于间充质干细胞所具备的这些生物学特性，使其在自身免疫病、炎症和癌症等治疗方面具有广阔的临床应用前景。并且也可作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。
临床上最常用的是骨髓来源的间充质干细胞(Bone marrow derived MSCs，BM-MSCs)，一般以自体移植为主，可用于治疗多种慢性疾病。但是在自体间充质干细胞的应用过程中逐渐发现许多不足之处。(1)扩增能力存在很大的个体差异；(2)培养时间长，无法及时满足病情的需要；(3)有研究报道癌症病人来源的BM-MSCs还能够保护肿瘤细胞免遭化疗药物的杀伤作用。因此，自体移植BM-MSCs无法达到缓解癌症病程的作用，甚至可能加剧肿瘤的生长。这些问题严重制约了自体间充质干细胞在临床上的应用。
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。基于以上，我们提出一种活化间充质干细胞的方法，使其能够特异性表达生物活性分子，增强其抗肿瘤和免疫调节能力。
脂肪干细胞(Adipose-derived stem cells，ADSCs)是一种从脂肪组织中分离得到的具有多向分化潜能的干细胞。脂肪干细胞能够分泌多种细胞因子和生长因子因子，通过旁分泌作用于调节损伤组织周围的微环境，干预细胞的增殖与凋亡，参与组织细胞生长、再生与重建。例如：ADSC分泌的成纤维生长因子促进长纤维细胞分泌I和III型胶原和纤连蛋白，促进胶原合成，发挥抗炎、抗氧化和损伤修复等作用。此外，ADSCs具有免疫调节作用，通过与免疫细胞的直接作用或旁分泌的作用，影响免疫细胞的分化和活化，重建机体的免疫平衡。基于以上，我们提出一种活化间充质干细胞的方法，使其能够特异性表达生物活性分子，增强其组织修复和免疫调节能力。
发明内容
本发明的第一目的在于提供一种间充质干细胞的活化剂的配伍方法。
本发明的第二目的在于提供一种间充质干细胞制剂的制备方法。
本发明的第三目的在于提供一种间充质干细胞制剂在免疫系统疾病中的应用。
本发明的第四目的在于提供一种间充质干细胞制剂在急性髓系白血病治疗中的应用。
本发明的第五目的在于提供一种间充质干细胞制剂在预防衰老，增加机体免疫力以及促进机体组织再生方面的应用。
所述的间充质干细胞的活化剂成分包括：10％胎牛血清的α-MEM培养基、0.01-10μM骨化三醇(1，25-Dihydroxyvitamin D3，1，25D3)，0.1-50μM巴多索隆(CDDO)，0.1-10μM积雪草苷，0.1-1μM罗格列酮(rosiglitazone)，0.2-10mM二甲双胍，0.1-2mM维生素C及0.5-1.5μM丙酮酸钠。
在一些实施方式中，活化剂的组分可替换成为其它同系或同类型效应化合物：
(1)1，25D3可替换为：阿法骨化醇(25(OH)2D3)、帕立骨化醇、度骨化醇、氟骨化醇以及马沙骨化醇等。
(2)罗格列酮可替换为：曲格列酮(troglitazone)、吡格列酮(pioglitazone)、环格列酮(ciglitazone)等噻唑烷二酮药物(TZDs)。
(3)二甲双胍可替换为：尿囊素和人参皂苷
(4)CDDO可替换为：齐墩果酸，CDDO-Me，CDDO-imidazolide，CDDO乙基酯
所述的间充质干细胞制剂的制备方法包含两个部分：间充质干细胞的提取、间充质干细胞的活化及制剂制备。
间充质干细胞的提取是分别从新生儿的脐带、成人的皮下脂肪或成人骨髓中通过酶消化法分离提取获得的。
间充质干细胞的活化是使用[0014]的间充质干细胞活化剂对提取获得的间充质干细胞进行孵育(12-72小时)，制备诱导型活化间充质干细胞(Active MSCs)
制剂制备可分为两种：
(1)使用[0015]中制备获得的Active MSCs与药用辅料进行混合，制备干细胞制剂。
(2)使用[0015]中制备获得的Active MSCs与[0011]的间充质干细胞活化剂以及药用辅料进行混合，制备干细胞制剂。
(3)使用[0014]中制备获得的间充质干细胞与[0011]的间充质干细胞活化剂以及药用辅料进行混合，制备干细胞制剂。
在一些实施方式中，药用辅料可包括：磷酸氢二钠，磷酸二氢钠，氯化钠，白蛋白，甘油。
本发明所述的Active MSCs发挥预防或治疗疾病的活性成分包括：白介素6，(IL6)，吲哚胺2，3加双氧酶(IDO)，骨形成蛋白6(BMP6)，中性粒细胞弹性蛋白酶，组织蛋白酶G，超氧化物歧化酶，组织金属蛋白酶抑制因子3(TIMP3)，蛋白质赖氨酸6氧化酶(LOX)，IGFL3，TNFAIP6，CYR61，TGFβ。
在一些实施方式中，可以使用活化剂成分中的任意一种，制备Active MSCs。在一些实施方式中，可以使用活化剂成分中的任意一种，并制备Active MSCs，并与药用辅料进行混合，制备间充质干细胞制剂。
所述的制备获得的Active MSCs应具有以下特征：
(1)表面标志物：CD14，CD34，CD45阴性表达；CD73，CD90，CD105阳性表达；
(2)能够分别诱导分化成脂肪细胞、成骨细胞和软骨细胞；
(3)高表达活性成分中的一种或几种活性成分组合。
所述的[0018]中的分泌因子应认定为该间充质干细胞制剂发挥疾病治疗与预防功能的功能活性因子。在一些实施方式中，通过在间充干细胞中使用基因编辑或药物诱导的方式促进本发明中所述的分泌因子的上调表达或释放的方式应认定为本发明的所述范围。
所述的间充质干细胞制剂在免疫系统疾病中的应用。免疫系统疾病包括：自身免疫性肝炎，特发性肺纤维化，糖尿病性皮肤病。制备获得的Active MSCs可以在体外抑制巨噬细胞的M1型极化，降低巨噬细胞TNF-α的表达，促进内皮细胞和成纤维细胞的增殖和迁移。制备获得含有Active MSCs的间充质干细胞制剂可在刀豆蛋白诱导的自身免疫性肝病(Autoimmune Liver，AIH)模型中有效减缓炎性反应和肝脏损伤；在肺纤维化模型中有效缓解肺部的纤维化水平。同时，该间充质干细胞制剂不会对主要脏器造成显著的毒副作用。
所述的间充质干细胞制剂在急性髓系白血病(AML)治疗中的应用。制备获得的Active MSCs可在体外显著促进AML细胞的凋亡，促进白血病细胞细胞周期阻滞并抑制增殖，促进白血病细胞向成熟单核细胞分化。制备获得含有Active MSCs的间充质干细胞制剂可在AML小鼠模型中有效延缓白血病进程，提高AML小鼠生存率。
所述的一种间充质干细胞制剂在预防衰老，增加机体免疫力以及促进机体组织再生方面的应用。制备获得的Active MSCs可在体外显著提高内皮细胞和成纤维细胞的增殖能力和迁移能力。制备获得含有Active MSCs的间充质干细胞制剂可在皮肤损伤模型中显著促进皮肤的伤口愈合和毛发再生。
Active MSCs的质量控制方法：
Active MSCs的培养上清中诱导分子残留含量的质量控制。
Active MSCs的细胞活力的检测。
Active MSCs表型和多向分化能力的评价。
Active MSCs的生物学效力评价。
附图说明
图1为高效液相色谱(HPLC)检测与同批次未处理MSCs(A)相比，Active MSCs(B)中制剂成分残留。
图2为CCK8检测诱导活化培养液对MSCs增殖的影响。
图3为Active MSCs流式鉴定，流式检测标记物CD73、CD90、CD105、CD14、CD34、CD45的表达。
图4为Active MSCs成骨(A)、成脂(B)、成软骨(C)诱导分化鉴定
图5为Active MSCs分泌组蛋白GO聚类分析
图6为Active MSCs IL-6和IDO的表达量。
图7为Active MSCs改善巨噬细胞炎性功能。流式细胞术检测(A)Q-pcr检测巨噬细胞相关炎性因子的表达，(B)含有Active MSCs的间充质干细胞制剂植入AIH小鼠体内，1周后分析肝脏形态与肝脏病理切片，(C)含有Active MSCs的间充质干细胞制剂治疗肝损伤小鼠的血清中ALT/AST的酶活性分析。
图8为Active MSCs缓解肺部的纤维化。Masson染色检测肺纤维化小鼠胶原沉积的水平。
图9为Active MSCs对U937(A)和THP-1(B)细胞的体外诱导凋亡。
图10为Active MSCs诱导急性单核白血病细胞分化的能力检测。Active MSCs处理白血病病人原代样本流式细胞术检测细胞表面CD14和CD11b的表达。
图11为Active MSCs延缓衰老能力检测。12月龄小鼠(A)肝脏脂质积累，(B)脂肪炎症，(C)外周血中白细胞数量。
图12为Active MSCs促进皮肤损伤修复的能力检测。(A)内皮细胞迁移能力，(B)含有Active MSCs的间充质干细胞制剂在小鼠皮肤损伤。
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
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；
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；
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；
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、80％饱和湿度培养箱中静置贴壁培养；
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；
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。
Active MSCs的质量控制方法及标准：
Active MSCs的培养上清中诱导活化培养液残留的质量控制：PBS清洗ActiveMSCs两遍，使用含有10％FBS的α-MEM完全培养基，37℃、5％CO2培养箱内培养2天，收集培养上清，高效液相色谱(HPLC)检测该上清中诱导分子的含量，上清中含量应在0.1nM以下。
Active MSCs的细胞活力的质量控制：以未经处理的同批次的MSCs对照，CFSE检测Active MSCs的细胞增殖能力。与处理前相比，增殖能力应不变化。
Active MSCs表型的质量控制：以未经处理的同批次的MSCs对照，流式细胞术鉴定Active MSCs细胞表面标志物CD14、CD34、CD45、CD73、CD90、CD105的表达。与处理前相比，表明标志物表达应不发生明显变化。
Active MSCs多潜能性评价：以未经处理的同批次的MSCs对照，同时将ActiveMSCs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，判断分化能力的强弱变化。与诱导前相比，三种谱系分化能力应不发生明显变化。
Active MSCs的效能评价方法及标准：以未经处理的同批次的MSCs对照，实时定量PCR(RT-qPCR)检测Active MSCs中IL-6、IDO，BMP6，中性粒细胞弹性蛋白酶，组织蛋白酶G，超氧化物歧化酶，组织金属蛋白酶抑制因子3，类胰岛素生长因子结合蛋白3的表达升高3倍及以上。
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 (10)
1.一种间充质干细胞活化剂的成分与配伍：
10％胎牛血清的α-MEM培养基、0.5-10μM骨化三醇(1，25-Dihydroxyvitamin D3，1，25D3)，1-10μM积雪草苷，0.1-1μM罗格列酮(rosiglitazone)，2-10mM二甲双胍，0.5-2mM维生素C及0.5-1.5μM丙酮酸钠。在一些实施方式中，药用辅料可包括：磷酸缓冲盐溶液(phosphate buffer saline，PBS)，0.9％的氯化钠水溶液，吐温-20(Tween-20)，二甲基亚砜(DMSO)。
2.一种间充质干细胞制剂：使用间充质干细胞活化剂对提取获得的间充质干细胞进行孵育，制备诱导型活化间充质干细胞(Active MSCs)3.一种间充质干细胞制剂在免疫系统疾病中的应用。
3.一种间充质干细胞制剂在自身免疫性疾病治疗、急性髓系白血病治疗、预防衰老，增加机体免疫力以及促进机体组织再生方面中的应用。
4.根据权利要求1中所述的间充质干细胞活化剂，可以使用活化剂成分中的任意一种或几种，制备Active MSCs。间充质干细胞活化剂的成分与配伍，其中，任一成分可替换成其它同系或同类型效应化合物，具体包括：
(1)1，25D3可替换为：阿法骨化醇(25(OH)2D3)、帕立骨化醇、度骨化醇、氟骨化醇以及马沙骨化醇等。
(2)罗格列酮可替换为：曲格列酮(troglitazone)、吡格列酮(pioglitazone)、环格列酮(ciglitazone)等噻唑烷二酮药物(TZDs)。
(3)二甲双胍可替换为：尿囊素和人参皂苷
(4)CDDO可替换为：齐墩果酸，CDDO-Me，CDDO-imidazolide，CDDO乙基酯。
5.根据权利要求2中所述的间充质干细胞制剂，其中所述的间充质干细胞(MSCs)是人脐带来源间充质干细胞(UC-MSCs)，或人骨髓来源间充质干细胞(BM-MSCs)，或人脂肪组织来源间充质干细胞(ADSCs)中的任意一种或几种。
6.根据权利要求2中所述的间充质干细胞制剂，其中的非细胞组分包括活化剂成分与药用辅料成分；活化剂成分为权利要求1中的活化剂组分的所有成分，或任意一种，或任意几种的配伍。制剂制备可包含以下三种：
(1)使用权利要求1中活化剂处理MSCs，制备获得的Active MSCs，并与药用辅料进行混合，制备干细胞制剂。
(2)使用权利要求1中活化剂处理MSCs，制备获得的Active MSCs；将获得的ActiveMSCs与活化剂以及药用辅料进行混合，制备干细胞制剂。
(3)MSCs与权利要求1中的活化剂直接混合，并于药用辅料混合，制备干细胞制剂。
7.根据权利要求3中的应用，Active MSCs需符合以下标准：
(1)细胞表面标志物：CD14，CD34，CD45阴性表达；CD73，CD90，CD105阳性表达。
(2)三谱系(成骨、成软骨、成脂)分化特征
(3)分泌因子：白介素6，(IL6)，吲哚胺2，3加双氧酶(IDO)，骨形成蛋白6(BMP6)，中性粒细胞弹性蛋白酶，组织蛋白酶G，超氧化物歧化酶，组织金属蛋白酶抑制因子3(TIMP3)，蛋白质赖氨酸6氧化酶(LOX)，IGFL3，TNFAIP6，CYR61，TGFβ。
8.根据权利要求7中的标准，分泌因子应认定为该间充质干细胞制剂发挥疾病治疗与预防功能的功能活性因子。功能活性因子应为权利要求11中的任意一种或多种的组合。在一些实施方式中，通过在间充干细胞中使用基因编辑或药物诱导的方式促进本发明中所述的分泌因子的上调表达或释放的方式应认定为本发明的所述范围。
9.根据权利要求3中的应用，免疫系统疾病包括：自身免疫性肝炎，肺纤维化，肝纤维化；急性髓系白血病包括：急性单核细胞白血病、急性粒-单核细胞白血病；预防衰老，增加机体免疫力以及促进机体组织再生方面应用包括：皮肤损伤修复(烧伤，摩擦损伤)，糖尿病病足，过敏性皮炎。
10.根据权利要求13-15中的疾病的应用，干细胞制剂的给药方式为：外周静脉输注，有效剂量为5×106/kg-10×107/kg。
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