CN117660320A - 一种间充质干细胞cd317+亚群及其制备和其应用 - Google Patents
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Abstract
本发明涉及一种间充质干细胞CD317+亚群及其制备和其应用，涉及生物医药领域。本发明所述的间充质干细胞CD317+亚群的细胞表面高表达CD317，将CD317作为流式分选间充质干细胞CD317+亚群的表面标志分子，其可以在体外培养并增殖，具有较强的免疫抑制功能，并具有改善治疗的效果；利用得到的间充质干细胞CD317+亚群对小鼠急性炎症模型进行治疗，发现治疗后的模型小鼠临床症状有所改善，且治疗效果比间充质干细胞CD317‑亚群细胞明显。
Description
技术领域
本发明涉及生物医药领域，具体涉及一种间充质干细胞CD317+亚群及其制备和其应用。
背景技术
间充质干细胞(mesenchymal stem cell，MSC)是一种具有多能分化潜能的体细胞来源的干细胞，在适宜的体内外微环境下，传统MSC能诱导分化成各种中胚层来源的组织细胞，如骨细胞、脂肪细胞和软骨细胞等。由于其具有低免疫原性和免疫调控能力，在自身免疫或炎症疾病中，MSC可发挥免疫负调控功能，抑制自身免疫反应或炎症，被广泛应用于再生医学和疾病治疗的相关研究中。然而，具有较强免疫抑制功能的MSC亚群尚缺乏。鉴于此，本发明提供一种间充质干细胞CD317+亚群及其制备和其应用。
发明内容
本发明所要解决的技术问题是提供一种间充质干细胞CD317+亚群及其制备和其应用。目的是提供具有强的免疫抑制功能，并具有改善治疗效果的间充质干细胞亚群。
本发明解决上述技术问题的技术方案如下：
第一个方面是提供一种间充质干细胞CD317+亚群，其细胞表面具有高表达的CD317。
本发明的有益效果：
(1)本发明所述的间充质干细胞CD317+亚群的细胞表面高表达CD317，将CD317作为流式分选间充质干细胞CD317+亚群的表面标志分子。
(2)本发明的间充质干细胞CD317+亚群(CD317+MSC)，可以在体外培养并增殖，具有较强的免疫抑制功能，并具有改善治疗效果；
(3)本发明利用得到的间充质干细胞CD317+亚群对小鼠急性炎症模型进行治疗，发现治疗后的模型小鼠临床症状有所改善，且治疗效果比间充质干细胞CD317-亚群细胞明显。
第二个方面是提供一种间充质干细胞CD317+亚群的制备方法，包括如下的步骤：利用抗CD317-PE抗体或IgG-PE抗体对间充质干细胞进行分选得到间充质干细胞CD317+亚群。
进一步，一种间充质干细胞CD317+亚群的制备方法，所述间充质干细胞为脐带间充质干细胞、骨髓间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带血间充质干细胞、牙髓间充质干细胞中的任意一种。
进一步，所述分选具体为采用细胞分选仪进行分选。
第三个方面是提供所述一种间充质干细胞CD317+亚群在制备免疫负调控类药物中的应用。
第四个方面是提供所述一种间充质干细胞CD317+亚群在制备治疗自身免疫性疾病和/或炎症疾病的药物中的应用。
进一步，所述自身免疫性疾病包括SLE、RA、细胞移植或器官移植引起的免疫排斥中的至少一种，所述炎症疾病包括炎性肠炎或肺炎。
第五个方面是提供一种免疫调控药物，包括所述的间充质干细胞CD317+亚群。
第六个方面是提供一种治疗自身免疫性疾病和/或炎症疾病的药物，包括权利要求1所述的间充质干细胞CD317+亚群。
附图说明
图1为本发明MSCs的鉴别与表征结果图；其中，图A为脂肪细胞分化效率通过OilRed O染色分析并用qPCR对LPL和PPARγ基因进行分析；图B为脂肪细胞分化由Oil Red O进行染色；图C为骨细胞分化效率通过茜素红染色和OSTERIX与RUNX2(n＝3)基因的qPCR定量分析；图D为骨细胞分化由Oil Red O进行染色；图E为软骨细胞分化效率通过阿利新蓝染色和SOX9与BMP2(n＝3)基因的qPCR定量分析；图F为阿利新蓝染色分化的软骨细胞；
图2为本发明单细胞RNA-seq测序并分析结果图；其中，图A为细胞簇鉴定通过UMAP非线性降维分析的结果；图B为基于KEGG和GO的生物学功能聚类分析；
图3为本发明细胞增殖和小鼠急性炎症模型及细胞移植结果分析图；图A为在用或不用20ng/ml IFN-γ刺激48小时的条件下对CD317+或CD317-MSCs共培养后的PBMC增殖情况分析(n＝3)；图B为LPS脂多糖作用后7天肺组织的HE染色图；图C为LPS刺激7天后通过流式细胞术检测肺内CD45+细胞(n＝8)；图D为BAL中中性粒细胞数量通过LPS刺激24小时后流式细胞术分析的CD45+CD11b+Ly-6G+Ly-6Cmed情况评估得出(n＝8)；图E为LPS作用24小时后定量测定MPO活性(n＝8)；图F为血清中IL-6、TNF-α、IFN-γ和IL-1β水平由LPS刺激后24小时后通过ELISA测定。*表示P<0.05。
具体实施方式
以下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，并非用于限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购得的常规产品。
实施例1:人间充质干细胞的分离、扩增和鉴定及生物信息学分析
通过高通量单细胞测序，生物信息学分析，表明CD317是MSCs的功能亚群之一。
(1)人间充质干细胞的分离、扩增和鉴定及单细胞RNA-seq测序并分析
取人脐带组织并切碎，并用1mg/mL胶原酶B(STEMCELL Technologies)进行消化，用我们开发的全合成培养体系(NBVbe，在文献Xu J,Lian W,Chen J,Li W,Li L,HuangZ.Chemical-defined medium supporting the expansion of human mesenchymal stemcells.Stem Cell Res Ther.2020Mar 19；11(1):125.doi:10.1186/s13287-020-01641-7.PMID:32192530；PMCID:PMC7083066.中已经公开，具体成分如表1)进行培养使用TrypLE(Thermo Scientific)传代MSCs，并使用20ng/ml的IFN-γ进行刺激。通过Adipogenesis Differentiation Kit(Gibco)试剂盒、/>OsteogenesisDifferentiation Kit(Gibco)试剂盒、/>Chondrogenesis Differentiation Kit(Gibco)试剂盒进行MSCs的鉴别与表征。结果如图1A-F；图1为本发明MSCs的鉴别与表征结果图；其中，图A为脂肪细胞分化效率通过Oil Red O染色分析并用qPCR对LPL和PPARγ基因进行分析；图B为脂肪细胞分化由Oil Red O进行染色；图C为骨细胞分化效率通过茜素红染色和OSTERIX与RUNX2(n＝3)基因的qPCR定量分析；图D为骨细胞分化由Oil Red O进行染色；图E为软骨细胞分化效率通过阿利新蓝染色和SOX9与BMP2(n＝3)基因的qPCR定量分析；图F为阿利新蓝染色分化的软骨细胞；由图1A-F表明了CD317+MSC具有较强的分化能力和干细胞活性。
表1 NBVbe培养基的成分
(2)单细胞RNA-seq测序并分析
用于scRNA-seq(single-cell RNA sequencing)单细胞RNA测序的为人来源于脐带并通过NBVbe进行培养的MSCs。具体来说，用TrypLE分离MSCs，并用含有0.04％ BSA的HBSS(Hank's Balanced Salt Solution，即Hank's平衡盐溶液)进行重悬(1×106个细胞/mL)。通过10×Genomics Chromium平台完成建库，测序通过Illumina NovaSeq 6000系统进行测序(双端测序模式)。数据使用10xGenomics pipeline Cell Ranger(v2.1.0)进行处理，并通过Seurat package in R(v4.0.0)进行分析。
结果如图2；图2为本发明单细胞RNA-seq测序并分析结果图；其中，图A细胞簇鉴定通过UMAP非线性降维分析，共检测到0至6共7个不同的群集；图B为基于KEGG和GO的生物学功能聚类分析。数据分析显示，MSCs根据其生物学功能可分为3个不同的组，包括外泌体分泌功能增强的亚群、细胞外基质修饰以及对刺激的反应(再生和免疫反应)。
实施例2：CD317+MSC亚群，具有更强的干细胞活性、分化能力和更强的免疫抑制能力
1、CD317+/CD317-MSCs的筛选
来源于人脐带的MSCs，通过NBVbe进行培养。用TrypLE消化MSCs并使用CD317-PE抗体或IgG-PE(Thermo Fisher Scientific)进行标记，然后将CD317+和CD317-的MSCs用BDFACSAria SORP(BD Biosciences)细胞分选仪进行分选。所有的RNA测序均由华大基因完成。
2、ELISA和qPCR
将CD317+或CD317-MSCs分别培养在12孔板上(每孔20x104细胞)，3天后收集细胞培养的上清。通过Human MCP-1/CCL2 ELISA Kit(Sigma)试剂盒和Human TSG-6ELISA Kit(Thermo Fisher Scientific)试剂盒检测CCL2和TSG6蛋白水平。从小鼠的眼睛中采取外周血，血清中IL-6(BioLegend)、TNF-α(BioLegend)和IFN-γ(BioLegend)水平通过ELISA试剂盒测定。提取总RNA并进行反转录后完成qPCR。发现CD317+MSC分泌更高水平的CCL2和TSG6。
3、MSC-PBMC共培养
人外周血单核细胞(PBMCs)由EasySepTMDirect Human PBMC Isolation Kit(STEMCELL167 Technologies)试剂盒提取获得。用Human T-Activator CD3/CD28(Thermo Fisher Scientific)刺激PBMCs 24小时，然后与筛选出的CD317+或CD317-(20×104PBMCs vs 5×104MSCs)共培养72小时。细胞增殖情况通过Cell Proliferation Kit I(Roche)试剂盒评估，并通过全自动酶标仪(Bio-Rad)在波长570nm条件下进行量化评估。
3.1细胞增殖分析
用流式细胞术纯化CD317+和CD317-MSCs，以每孔10×104个细胞的浓度涂于6孔板上，当细胞融合度达到80-90％时，用TrypLE分离MSCs并用血细胞计数仪计数，然后死细胞通过Cytotoxicity Detection Kit(Sigma)试剂盒鉴定。结果如图3A，图A为在用或不用20ng/ml IFN-γ刺激48小时的条件下对CD317+或CD317-MSCs共培养后的PBMC增殖情况分析(n＝3)；结果表明CD317+MSC具有更强的免疫抑制功能(抑制淋巴细胞增殖)。
4、小鼠急性炎症模型及细胞移植
小鼠(C57BL/6J，雌性，8周龄)购自广东省医学实验动物中心并在无菌条件下饲养。本实验采用内毒素脂多糖(LPS)诱导小鼠急性炎症模型。LPS通过腹腔注射到小鼠体内(20mg/kg,Sigma)，等待十分钟后，PBS、CD317+MSCs(1x106细胞/小鼠)和CD317-MSCs(1x106细胞/小鼠)分别腹腔移植到小鼠模型中，n＝8。
4.1肺分析
免疫细胞浸润分和髓过氧化物酶(MPO)在支气管肺泡灌洗术中的CD45+淋巴细胞和中性粒细胞(CD45+CD11b+Ly-6G+Ly-6Cmed)由流式细胞术测定。髓过氧化物酶(MPO)的活性通过MPO Activity Assay Kit(Abcam)测定。HE(苏木精和伊红)染色对肺组织进行染色。
结果如图3B-F；图B为LPS脂多糖作用后7天肺组织的HE染色图；图C为LPS刺激7天后通过流式细胞术检测肺内CD45+细胞(n＝8)；图D为BAL中中性粒细胞数量通过LPS刺激24小时后流式细胞术分析的CD45+CD11b+Ly-6G+Ly-6Cmed情况评估得出(n＝8)；图E为LPS作用24小时后定量测定MPO活性(n＝8)；图F为血清中IL-6、TNF-α、IFN-γ和IL-1β水平由LPS刺激后24小时后通过ELISA测定。*表示P<0.05。结果表明CD317+MSC在体内具有更强的免疫抑制功能。
综上可知，本发明所述的间充质干细胞CD317+亚群的细胞表面高表达CD317，将CD317作为流式分选间充质干细胞CD317+亚群的表面标志分子，其可以在体外培养并增殖，具有较强的免疫抑制功能，并具有改善治疗效果；利用得到的间充质干细胞CD317+亚群对小鼠急性炎症模型进行治疗，发现治疗后的模型小鼠临床症状有所改善，且治疗效果比间充质干细胞CD317-亚群细胞明显。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (9)
1.一种间充质干细胞CD317+亚群，其特征在于，其细胞表面具有高表达的CD317。
2.基于权利要求1所述一种间充质干细胞CD317+亚群的制备方法，其特征在于，包括如下的步骤：利用抗CD317-PE抗体或IgG-PE抗体对间充质干细胞进行分选得到间充质干细胞CD317+亚群。
3.根据权利要求2所述一种间充质干细胞CD317+亚群的制备方法，其特征在于，所述间充质干细胞为脐带间充质干细胞、骨髓间充质干细胞、脂肪间充质干细胞、胎盘间充质干细胞、脐带血间充质干细胞、牙髓间充质干细胞中的任意一种。
4.根据权利要求2所述一种间充质干细胞CD317+亚群的制备方法，其特征在于，所述分选具体为采用细胞分选仪进行分选。
5.权利要求1所述一种间充质干细胞CD317+亚群在制备免疫负调控类药物中的应用。
6.权利要求1所述一种间充质干细胞CD317+亚群在制备治疗自身免疫性疾病和/或炎症疾病的药物中的应用。
7.根据权利要求6所述应用，其特征在于，所述自身免疫性疾病包括SLE、RA或细胞移植或器官移植引起的免疫排斥中的至少一种，所述炎症疾病包括炎性肠炎或肺炎。
8.一种免疫调控药物，其特征在于，包括权利要求1所述的间充质干细胞CD317+亚群。
9.一种治疗自身免疫性疾病和/或炎症疾病的药物，其特征在于，包括权利要求1所述的间充质干细胞CD317+亚群。
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