CN112852732A - Dc细胞培养方法、培养基和基于dc治疗策略的药物及酪氨酸激酶抑制剂在制备其的应用 - Google Patents
Dc细胞培养方法、培养基和基于dc治疗策略的药物及酪氨酸激酶抑制剂在制备其的应用 Download PDFInfo
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Abstract
本发明公开了DC细胞培养方法、培养基和基于DC治疗策略的药物及酪氨酸激酶抑制剂在制备其的应用；其中，DC细胞培养方法在诱导分化未成熟DC细胞的培养过程中，于培养基中加入酪氨酸激酶抑制剂，经诱导分化培养获得成熟DC细胞；培养基为含有酪氨酸激酶抑制剂的细胞培养基，并可用于未成熟DC细胞诱导分化为成熟DC细胞的培养；基于DC治疗策略的药物中含有酪氨酸激酶抑制剂；酪氨酸激酶抑制剂可用于制备基于DC治疗策略的药物。上述方案能解决目前DC细胞培养方法不能满足于临床疗效所需高度成熟或高活性的DC细胞的问题，达到提高DC细胞成熟度或活性的目的。
Description
技术领域
本发明涉及DC细胞相关技术领域，尤其涉及DC细胞培养方法、培养基和基于DC治疗策略的药物及酪氨酸激酶抑制剂在制备其的应用。
背景技术
DC细胞(树突状细胞)作为最重要的抗原呈递细胞，可以通过引发幼稚T细胞并促进其向Th细胞和细胞毒性T淋巴细胞(CTL)的增殖和分化，在抗肿瘤免疫中发挥重要作用。
在过去的几十年中，基于DC细胞的抗肿瘤治疗策略迅速发展；例如，培养自体树突状细胞以增强其活性、之后再回输患者体内的方式为当前研究及应用的前沿技术，其不仅可有效激发出肿瘤患者体内的抗肿瘤免疫应答、在研究与应用中显示出良好的疗效，而且对于健康人群或免疫力降低人群可以起到提高机体免疫力、防治疾病、预防疾病发生发展的保健作用。
但是，由于血液肿瘤慢性粒细胞白血病(chronic myeloid leukemia，CML)患者具有免疫力显著低下的特征，所以免疫活性低下或免疫缺陷的树突状细胞是导致体外培养、活化患者自体树突状细胞效果不理想，以及自体树突状细胞回输患者体内后临床疗效不理想的主要原因。
而现有的DC细胞培养方法中，通过外周血单个核细胞于基础培养基中先经GM-CSF和IL-4诱导产生未成熟DC细胞，再经TNF-α、PGE2和IL-6刺激诱导分化为成熟DC细胞；其虽然可以使得患者自体DC细胞一定程度上具有成熟度或活性，但是其与临床疗效所需的高度成熟或高活性DC细胞仍存在差距。
发明内容
本发明公开一种DC细胞培养方法、培养基和基于DC治疗策略的药物及酪氨酸激酶抑制剂在制备其的应用，以解决目前DC细胞培养方法不能满足于临床疗效所需高度成熟或高活性的DC细胞的问题，达到提高DC细胞成熟度或活性的目的。
为了解决上述问题，本发明采用下述技术方案：
第一方面，本发明提供了一种DC细胞培养方法，其在诱导分化未成熟DC细胞的培养过程中，于培养基中加入酪氨酸激酶抑制剂，经诱导分化培养获得成熟DC细胞。
可选地，所述酪氨酸激酶抑制剂为伊马替尼、达沙替尼和尼洛替尼中的至少一种。
可选地，DC细胞培养方法包括以下步骤：将外周血单个核细胞进行贴壁粘附2小时，之后留下其中贴壁细胞于DCⅠ培养基中培养，所述培养基为添加有血清、GM-CSF和IL-4的基础培养基，经诱导培养产生所述未成熟DC细胞；再于所述DCⅠ培养基中加入诱导分化刺激物和所述酪氨酸激酶抑制剂，经继续培养后获得所述成熟DC细胞。
可选地，所述诱导分化刺激物包括细胞因子TNF-α、IL-6和PGE2。
可选地，所述诱导分化刺激物还包括抗原肽MART-1aa26-35*A27L。
可选地，所述基础培养基为由RPMI 1640和L-谷氨酰胺组成的基础培养基。
第二方面，本发明提供了一种培养基，所述培养基为含有酪氨酸激酶抑制剂的细胞培养基，并可用于未成熟DC细胞诱导分化为成熟DC细胞的培养。
第三方面，本发明提供了一种基于DC治疗策略的药物，所述基于DC治疗策略的药物中含有酪氨酸激酶抑制剂。
可选地，所述基于DC治疗策略的药物为DC疫苗或用于DC细胞疗法的药物。
第四方面，本发明还提供了酪氨酸激酶抑制剂在制备基于DC治疗策略的药物中的应用。
本发明采用的技术方案能够达到以下有益效果：
本发明公开的DC细胞培养方法、培养基和基于DC治疗策略的药物及酪氨酸激酶抑制剂在制备其的应用，通过酪氨酸激酶抑制剂可以改善DC细胞成熟或活性分子的表达，既显示出作为DC佐剂的潜力，又表明对于血液肿瘤慢性粒细胞白血病患者可以实现与健康供体相似的高比例成熟DC细胞的体外生成；因此，可以显著地提高DC细胞的成熟度或活性，进而很好地满足于临床疗效所需的高度成熟或高活性的要求。
附图说明
此处所说明的附图用来提供对本发明的进一步理解，构成本发明的一部分，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为HD组和CML患者组之间CD80+、CD86+细胞群体在总细胞中所占比例的比较示意图；
图2为健康者的达沙替尼组和对照组(HD，n＝10)之间的moDCs(单核细胞来源的树突状细胞)中CD83+细胞比例的比较示意图；
图3为健康者的达沙替尼组和对照组(HD，n＝10)之间的moDCs中CD40+细胞比例的比较示意图；
图4为健康者的达沙替尼组和对照组(HD，n＝10)之间的moDCs中HLA-DR+细胞比例的比较示意图；
图5为患者的达沙替尼组和对照组(CML，n＝10)之间的moDCs中CD40+细胞比例的比较示意图；
图6为患者的达沙替尼组和对照组(CML，n＝10)之间的moDCs中HLA-DR+细胞比例的比较示意图；
图7为患者的达沙替尼组和对照组(CML，n＝10)之间的moDCs中CD83+细胞比例的比较示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明具体实施例及相应的附图对本发明技术方案进行清楚、完整地描述。显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下结合附图，详细说明本发明各个实施例公开的技术方案。
实施例1
本发明实施例提供了一种DC细胞培养方法，其包括以下具体步骤：
首先，将外周血单个核细胞进行贴壁粘附2小时，之后留下其中贴壁细胞于DCⅠ培养基中培养，该培养基为添加有5％人血清，800U/mL的GM-CSF(粒细胞-巨噬细胞集落刺激因子)和500U/mL的IL-4(白细胞介素-4)的基础培养基，经诱导培养7天，获得imDC细胞(未成熟DC细胞)。
然后，再于上述DCⅠ培养基中补充加入10ng/mL的TNF-α(tumor necrosis factor-α，肿瘤坏死因子-α)、1mg/mL的PGE2(***素E2)和1000U/mL的IL-6(白细胞介素-6)以及25nM达沙替尼，继续培养3天，获得mDC细胞(成熟DC细胞)。
其中，GM-CSF可以促进髓系细胞发育，是维持、诱导DC发育和分化的最根本的细胞因子；IL-4可以抑制粒细胞和巨噬细胞生长，从而使CD34+前体细胞或CD14+单核细胞向DC方向发展；该细胞因子组合培养的DC细胞纯度可达95％以上，但诱导生成的DC多为不成熟的DC，只有加入TNF-α、PGE2和IL-6等刺激物方可使非成熟的DC转化为成熟DC。
同时，本发明通过添加的达沙替尼可以提高DC细胞成熟或活性分子的表达，既显示出达沙替尼作为DC佐剂的潜力，又表明达沙替尼对于血液肿瘤慢性粒细胞白血病患者可以实现与健康供体相似的高比例成熟DC细胞的体外生成。
因此，相较于现有DC细胞的培养方法，本发明可以显著地提高DC细胞的成熟度或活性，进而很好地满足于临床疗效所需DC细胞高度成熟或高活性的要求。
优选地，在未成熟DC细胞诱导分化为成熟DC细胞的培养过程中，补充加入于基础培养基中的刺激物还可以包括抗原肽MART-1aa26-35*A27L，可以有效地加速DC细胞的成熟。
需要说明的是，上述各组分的用量及培养时间可以根据DC细胞培养的实际情况进行适应性地调整，本发明不限制其用量和培养时间；基础培养基为培养DC细胞的现有基础培养基，本发明不再对其进行赘述；达沙替尼还可以选用伊马替尼或尼洛替尼等其他的酪氨酸激酶抑制剂替代。
同时，抗原肽MART-1aa26-35*A27L为一种现有的抗原肽，具体可参见“Schmitt,M.,etal.,Frequency of expression and generation of T-cell responses againstantigens on multiple myeloma cells in patients included in the GMMG-MM5trial.Oncotarget,2017.8(49):p.84847-84862.”或.“Harada,T.and S.Ozaki,Targetedtherapy for HM1.24(CD317)on multiple myeloma cells.Biomed Res Int,2014.2014:p.965384.”或“Hundemer,M.,et al.,Identification of a new HLA-A2-restricted T-cell epitope within HM1.24 as immunotherapy target for multiple myeloma.ExpHematol,2006.34(4):p.486-96.”或“Christensen,O.,et al.,Melan-A/MART1 analogpeptide triggers anti-myeloma T-cells through crossreactivity with HM1.24.JImmunother,2009.32(6):p.613-21”等文献。
实施例2
本发明实施例进行了对比试验，具体如下：
1、获取人外周血单个核细胞
从健康供体(HD，n＝10)和接受伊马替尼治疗的MR4.5缓解的慢性粒细胞白血病患者(CML，n＝10)中各抽取20ml抗凝的外周血，并分别与PBS(磷酸缓冲盐溶液)按1：1稀释，再相应地缓慢加入400g淋巴细胞分离液，于4℃的条件下离心30min，收集白色细胞层；然后，用PBS洗涤血沉棕黄层获得PBMC(外周血单个核细胞)。
2、DC细胞的产生和成熟
将上述获得的各例PBMC以2×106个/ml的细胞浓度在由RPMI 1640(RPMI是Roswell Park Memorial Institute的缩写，代指洛斯维·帕克纪念研究所；RPMI 1640是指该研究所研发的一种细胞培养基，1640是该培养基代号)和2mM的L-谷氨酰胺组成的基础培养基中粘附2小时，并补充1％的青霉素/链霉素，并在孵育两小时后收集贴壁细胞。
然后，将上述各例收集的贴壁细胞于DC培养基I中培养7天，收集未成熟的树突状细胞(imDC)，并分为两组；其中，一组加入25nM达沙替尼，另一组加入等量的PBS，并分别于等量的DC培养基II中继续培养三天，收获成熟的树突状细胞(mDC)。
上述的DC培养基I为补充了5％人血清，800U/mL GM-CSF和500U/mL IL-4的基础培养基；上述的DC培养基II为由补充了10ng/mL的TNF-α、1000U/mL的IL-6、1μg/mL的PGE2和DC培养基I组成，并可加入了浓度为4μg/ml的MART-1aa26-35*A27L，用于刺激加速imDC的成熟。
3、细胞活性(免疫荧光分析)
本发明实施例选择了MHC分子HLA-DR(激活T细胞第一信号必需；MHC为majorhistocompatibility complex的缩写，表示主要组织相容性复合体，是一组编码动物主要组织相容性抗原的基因群的统称；HLA-DR是MHC-II类分子)、共刺激分子CD86和CD80、CD83(DC细胞成熟相关分子)以及CD40(T细胞活化的第二信号)这五个表面标志物作为DC细胞成熟的指标。
洗涤收集的mDC细胞，重悬于预冷的PBS中，然后用串联染料APC-Cy7标记的CD83单克隆抗体(mAb)，APC(别藻蓝蛋白)标记的CD40 mAb，PE(藻红蛋白)标记的CD86 mAb，FITC(异硫氰酸荧光素)标记的CD80 mAb和pacific blue(PB染料，是一种明亮的蓝色荧光染料)标记的HLA-DR mAb或相同浓度同型匹配的阴性对照mAb在4℃染色30分钟。
随后，将细胞洗涤两次并重悬于预冷的PBS中，以通过流式细胞术进行免疫荧光分析。使用GraphPad Prism 5.0软件进行统计分析；配对T检验(两组数据符合正态分布时)或秩和检验(两组数据不符合正态分布时)比较达沙替尼组与对照组CD40，CD83和HLA-DR的表达；而独立样本T检验用于比较HD组和CML组之间CD80+、CD86+细胞群体的比例所有p值均为正反两面，且p值<0.05被认为具有统计学意义；同时，FACS分析***中将总细胞中的CD80+、CD86+细胞群体选为DC，然后分别分析该群体中CD83，CD40或HLA-DR的表达。
(1)HD-moDCs与CML-moDCs的比较(未用达沙替尼处理)
通过比较HD组和CML患者组之间CD80+、CD86+细胞群体的差异、各10例(n＝10)，发现HD组与CML组之间的收获细胞中CD80+、CD86+细胞的比例无统计学差异(89.46％±9.70％vs 87.39％±9.34％，P＝0.690)，其结果如下表1和图1所示。
表1 HD组和CML组总细胞中CD80+、CD86+细胞的比例
(2)达沙替尼(Dasatinib)对HD-moDCs的影响
通过分析了10例HD-moDCs上CD83，CD40和HLA-DR的表达，并与对照组相比较发现，达沙替尼组moDCs上CD83的表达与对照组没有显著差异(P＝0.233)，但是相较于对照组显著增强了表面标志物CD40(P＝0.008)和HLA-DR(P＝0.044)的表达，如下表2-表4和图2-图4所示。
表2健康者的达沙替尼组和对照组中moDCs中的CD83+细胞比例
表3健康者的达沙替尼组和对照组moDCs中的CD40+细胞比例
表4健康者的达沙替尼组和对照组moDCs中的HLA-DR+细胞比例
其中，图2中的a图显示了每例HD在达沙替尼组和对照组之间的moDCs中CD83+细胞比例的比较；b图显示了moDCs中CD83+细胞的比例在达沙替尼组和对照组之间没有差异(95.76％±5.01％对96.78％±3.48％，P＝0.233)。
图3中的a图显示了达沙替尼组和对照组中每例HD在moDCs中CD40+细胞比例的比较；b图显示了达沙替尼组moDCs中CD40+细胞的比例显着高于对照组(95.49％±4.39％vs93.46％±5.06％，P＝0.008)。
图4中的a图显示了每例HD在达沙替尼组和对照组之间的moDCs中HLA-DR+细胞比例的比较；b图显示了达沙替尼组的moDCs中HLA-DR+细胞比例显着高于对照组(96.83％±5.87％对94.83％±7.21％，P＝0.044)。
(3)达沙替尼对CML-moDCs的影响
通过分析了10例CML-moDCs上CD83，CD40和HLA-DR的表达并与对照组相比较发现，达沙替尼组moDCs上表面标志物CD40(P＝0.023)，CD83(P＝0.038)和HLA-DR(P＝0.001)的表达均相较于对照组显著增强，如下表5-表7和图5-图7所示。
表5患者的达沙替尼组和对照组moDCs中的CD40+细胞比例
表6患者的达沙替尼组和对照组moDCs中的HLA-DR+细胞比例
表7患者的达沙替尼组和对照组moDCs中的CD83+细胞比例
其中，图5中的a图显示了每例CML患者在达沙替尼组和对照组之间的moDCs中CD40+细胞比例的比较；b图显示了达沙替尼组的moDCs中CD40+细胞比例显着高于对照组(95.49％±4.40％vs 93.46％±5.07％，P＝0.023)。
图6中的a图显示了每例CML患者在达沙替尼组和对照组之间的moDCs中HLA-DR+细胞比例的比较；b图显示了达沙替尼组的moDCs中HLA-DR+细胞比例显着高于对照组(97.84％±2.32％vs 95.75％±2.56％，P＝0.001)。
图7中的a图显示了每例CML患者在达沙替尼组和对照组之间的moDCs中CD83+细胞比例的比较；b图显示了达沙替尼组的moDCs中CD83+细胞比例显着高于对照组(95.42％±3.54％对93.70％±3.91％，P＝0.038)。
可见，经本发明对比实施例研究发现达沙替尼在DC细胞培养中可以表现出显著提升DC细胞成熟/活性相关分子表达水平的作用，尤其是在25nM的浓度下表现尤为明显；因此，达沙替尼显示出了作为DC佐剂的潜力，且结果表明对于CML患者可以实现与HD相似的高比例moDCs的体外生成，进而使得达沙替尼可用于基于DC的治疗策略，如用于DC疫苗或用于DC细胞疗法的药物的制备。
同时，达沙替尼还可以选用伊马替尼或尼洛替尼等其他的酪氨酸激酶抑制剂替代，其相较于现有技术也可以达到提高DC细胞成熟度或活性的效果。
实施例3
本发明实施例提供了一种基于DC治疗策略的药物，如DC疫苗和用于DC细胞疗法的药物，其中可添加达沙替尼组分作为佐剂，用于增强DC疫苗和用于DC细胞疗法的药物的药效。
当然，上述基于DC治疗策略的药物中也可以选用伊马替尼或尼洛替尼等其他的酪氨酸激酶抑制剂替代达沙替尼作为用于增强药效的佐剂组分。
实施例4
本发明实施例还提供了一种用于DC细胞的培养基，其中添加有达沙替尼，用于未成熟DC细胞诱导分化为成熟DC细胞的培养过程，用于提高所培养的成熟DC细胞的成熟度或活性。
当然，上述用于DC细胞的培养基中也可以选用伊马替尼或尼洛替尼等其他的酪氨酸激酶抑制剂替代达沙替尼；相较于现有DC细胞培养基，可以提高所培养的成熟DC细胞的成熟度或活性。
以上所述仅为本发明的实施例而已，并不用于限制本发明。对于本领域技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原理之内所作的任何修改、等同替换、改进等，均应包含在本发明的权利要求范围之内。
Claims (10)
1.一种DC细胞培养方法，其特征在于，在诱导分化未成熟DC细胞的培养过程中，于培养基中加入酪氨酸激酶抑制剂，经诱导分化培养获得成熟DC细胞。
2.根据权利要求1所述的DC细胞养方法，其特征在于，所述酪氨酸激酶抑制剂为伊马替尼、达沙替尼和尼洛替尼中的至少一种。
3.根据权利要求1或2所述的DC细胞培养方法，其特征在于，包括以下步骤：将外周血单个核细胞于基础培养基中培养，且所述基础培养基中添加有血清、GM-CSF和IL-4，经诱导培养产生所述未成熟DC细胞；再于所述基础培养基中加入诱导分化刺激物和所述酪氨酸激酶抑制剂，经继续培养后获得所述成熟DC细胞。
4.根据权利要求3所述的DC细胞培养方法，其特征在于，所述诱导分化刺激物包括细胞因子TNF-α、IL-6和PGE2。
5.根据权利要求4所述的DC细胞培养方法，其特征在于，所述诱导分化刺激物还包括抗原肽MART-1aa26-35*A27L。
6.根据权利要求5所述的DC细胞培养方法，其特征在于，所述基础培养基为由RPMI1640和L-谷氨酰胺组成的基础培养基。
7.一种培养基，其特征在于，所述培养基为含有酪氨酸激酶抑制剂的细胞培养基，并可用于未成熟DC细胞诱导分化为成熟DC细胞的培养。
8.一种基于DC治疗策略的药物，其特征在于，所述基于DC治疗策略的药物中含有酪氨酸激酶抑制剂。
9.根据权利要求8所述的基于DC治疗策略的药物，其特征在于，所述基于DC治疗策略的药物为DC疫苗或用于DC细胞疗法的药物。
10.酪氨酸激酶抑制剂在制备基于DC治疗策略的药物中的应用。
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