CN117377754A - 外泌体产生促进剂及外泌体产生促进方法 - Google Patents
外泌体产生促进剂及外泌体产生促进方法 Download PDFInfo
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- CN117377754A CN117377754A CN202180097753.7A CN202180097753A CN117377754A CN 117377754 A CN117377754 A CN 117377754A CN 202180097753 A CN202180097753 A CN 202180097753A CN 117377754 A CN117377754 A CN 117377754A
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- exosome production
- stem cells
- exosome
- mesenchymal stem
- adipose
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Abstract
本发明提供一种促进脂肪源性间充质干细胞产生外泌体的外泌体产生促进剂。本发明的外泌体产生促进剂是促进在无血清培养基中培养的脂肪源性间充质干细胞产生外泌体的外泌体产生促进剂，其特征在于：具有表皮细胞生长因子(EGF)。优选还含有白细胞介素‑1β(IL‑1β)及海藻糖。优选不含有干细胞生长因子(SCGF)。优选以无纺布片作为支架进行培养。通过本发明所涉及的方法而大量产生的外泌体能用于例如美容整形等目的。
Description
技术领域
本发明涉及一种促进由间充质干细胞产生外泌体的外泌体产生促进剂及促进外泌体产生的方法。
背景技术
间充质干细胞(mesenchymal stem cell：MSC)是具有多能性的干细胞，其存在于间充质组织即来源于中胚层的***中。还明确已知间充质干细胞在未分化状态下分泌具有炎症抑制效果、细胞增殖促进效果、血管新生促进效果等的细胞因子·生长因子，并通过旁分泌来支持组织修复(非专利文献1)。未分化间充质干细胞所分泌的分子群所具有的治疗效果并不局限于有限的特定疾病，而是对各种疾病均具有治疗效果。通过使用幼稚间充质干细胞，无需将间充质干细胞诱导分化为欲修复的组织的细胞，而且无需对细胞进行任何转基因等人为操作，就能够进行组织再生。
外泌体能够通过使蛋白质、脂质以及RNA在细胞间迁移来介导细胞间的信息传递(非专利文献2、3)。已发现外泌体所含有的蛋白质、microRNA、mRNA等分子具有与其来源的细胞类似的功能。因此，期待作为针对广泛疾病的细胞疗法的来源而备受瞩目的间充质干细胞所分泌的外泌体具有与MSC相同的治疗效果(非专利文献4)。
专利文献1中记载了从已培养的神经干细胞系(NSCL)分离出的外泌体。该外泌体能够促进纤维细胞迁移，促进人脐带静脉内皮细胞分叉，还能够促进神经突起伸长。
与干细胞相比，外泌体含有相对较少的动物血清，从而也能够排除由动物血清感染而引起的症状的危险性。因此，利用了外泌体的细胞疗法能够克服现有干细胞疗法的局限性。
专利文献2中公开了以鞘氨醇碱(sphingoid base)为有效成分的外泌体产生促进剂。然而，专利文献2中记载的外泌体产生促进剂仅限于能够促进神经细胞中产生外泌体以治愈阿尔茨海默病的促进剂。
专利文献1：国际公开第2013/150303号
专利文献2：日本公开专利公报特开2019-156786号公报
非专利文献1：Chamberlain G,Fox J,Ashton B,Middleton J,Concise review:mesenchymal stem cells:their phenotype,differentiation capacity,immunologicalfeatures,and potential for homing.Stem Cells.25(2007)2739-2749
非专利文献2：Simons and Raposo、Curropin Cell Biology 2009；21:575-581
非专利文献3：Skog et al.,Nat Cell Biology 2008；10:1470～1476；Valadietal.,Nat Cell Biology 2007；9:654～659
非专利文献4：Katsuda T,Kosaka N,Takeshita F,Ochiya T.The therapeuticpotential of mesenchymal stem cell-derived extracellularvesicles.Proteomics.13(2013)1637-1653.
发明内容
－发明要解决的技术问题－
本发明正是为解决上述技术问题而完成的，其目的在于：提供一种促进脂肪源性间充质干细胞产生外泌体的外泌体产生促进剂及外泌体产生促进方法。
－用以解决技术问题的技术方案－
本发明所涉及的外泌体产生促进剂是促进在无血清培养基中培养的脂肪源性间充质干细胞产生外泌体的外泌体产生促进剂，其特征在于：所述外泌体产生促进剂具有表皮细胞生长因子。
本发明所涉及的外泌体产生促进方法是促进脂肪源性间充质干细胞产生外泌体的外泌体产生促进方法，其特征在于：所述外泌体产生促进方法为在含有表皮细胞生长因子的无血清培养基中培养脂肪源性间充质干细胞。
－发明的效果－
根据本发明，能够促进脂肪源性间充质干细胞产生外泌体。
附图说明
图1是示出添加了各种因子的情况下的脂肪源性间充质干细胞产生的外泌体的产生量的图。
具体实施方式
下面，参照附图对本发明的实施方式进行具体的说明，该实施方式只是为了易于理解本发明的原理，本发明的范围并不限于以下实施方式，本领域技术人员通过对以下实施方式的构成方式做适当的置换而得到的其他实施方式也包括在本发明的范围内。
本发明所涉及的外泌体产生促进剂促进在无血清培养基中培养的脂肪源性间充质干细胞产生外泌体。
脂肪源性间充质干细胞优选是人的脂肪源性间充质干细胞。与骨髓源性间充质干细胞相比，脂肪源性间充质干细胞具有细胞增殖快、分泌更多的再生促进因子、免疫抑制能力高的有利特征。此外，由于能够从腹部或臀部的脂肪组织得到脂肪源性间充质干细胞，因此，与需要采集骨髓的骨髓源性间充质干细胞相比，脂肪源性间充质干细胞还具有易于安全地确保足够的量的有利特征。将来源于患者自身脂肪组织的间充质干细胞用于患者的治疗，这在以下方面是优异的：没有伦理上的问题、没有免疫排斥反应、感染症等问题较少、通过静脉给药有治疗效果等。
脂肪源性间充质干细胞，例如CD73、CD90、CD105以及CD44表达为阳性，CD11b、CD19、CD34、CD45以及HLA-DR表达为阴性。来源于脂肪组织的间充质干细胞具有向成骨细胞、脂肪细胞以及软骨细胞分化的分化能力。
来源于脂肪组织的间充质干细胞是通过从脂肪组织中分离出间充质干细胞，在含有血清的培养基中原代培养间充质干细胞，并在无血清培养基中增殖培养从原代培养传代的间充质干细胞而得到的。脂肪组织可以通过外科从例如人类等哺乳动物身上切除而得到。在进行外科切除时，可以进行局部麻醉。或者，也可以通过将导管***腹部、大腿部或臀部的皮下脂肪组织，通过抽吸得到脂肪组织。能够得到的脂肪组织的量为例如为1g至100g、2g至50g、2g至40g、或者2g至20g，但不限于此。
将已得到的脂肪组织用例如生理盐水洗涤，在37℃的条件下浸泡在含有胶原酶的溶液中70分钟，使脂肪组织分散，其中，胶原酶是用汉克斯(Hanks)平衡盐溶液稀释成浓度为0.1％的胶原酶。将从脂肪组织中分离出的来源于脂肪组织的细胞在1800rpm下离心分离10分钟。此外，将通过离心分离得到的基质血管细胞群用溶液稀释并用100μm尼龙网进行过滤。用MEMα培养基洗涤过滤后的细胞。
无血清培养基是间充质干细胞用无血清培养基，能够列举出：例如，DMEM、MEMα、DMEM/F12、MEM等。
本发明所涉及的外泌体产生促进剂具有表皮细胞生长因子(EGF：EpidermalGrowth Factor)。如后述的实施例所示，含有多种因子的情况下的由脂肪源性间充质干细胞产生的外泌体产生量，比含有从这些多种因子中仅除去了表皮细胞生长因子后的因子的情况下的由脂肪源性间充质干细胞产生的外泌体产生量多。本发明人发现了新知识，即表皮细胞生长因子是促进外泌体产生的因子，并在此基础上完成了本发明。
表皮细胞生长因子是分子量为6045Da的蛋白质，由53个氨基酸残基及三个分子内二硫键组成。表皮细胞生长因子作为配体与存在于细胞表面的表皮生长因子受体(EGFR)结合，在调节细胞生长和增殖中起着重要的作用。培养基中的表皮细胞生长因子的含量没有特别限定，优选终浓度为0.05～300ng/ml，更优选终浓度为1～100ng/ml。
由于白细胞介素-1β(IL-1β：Interleukin-1β)也促进由脂肪源性间充质干细胞产生的外泌体产生量，因此本发明所涉及的外泌体产生促进剂优选还含有白细胞介素-1β。培养基中的白细胞介素-1β的含量没有特别限定，优选终浓度为0.001～10ng/ml，更优选终浓度为0.01～1ng/ml。
由于海藻糖也促进由脂肪源性间充质干细胞产生的外泌体产生量，因此本发明所涉及的外泌体产生促进剂还含有海藻糖。海藻糖是由两个α-葡萄糖通过1,1-糖苷键连接而成的二糖类。培养基中的海藻糖的含量没有特别限定，优选终浓度为1～50mg/ml，更优选终浓度为10～30mg/ml。
本发明所涉及的外泌体产生促进剂优选不含有干细胞生长因子(SCGF：Stem CellGrowth Factors)。如后述的实施例所示，含有多种因子的情况下的由脂肪源性间充质干细胞产生的外泌体产生量，比含有从这些多种因子中仅除去了干细胞生长因子后的因子的情况下的由脂肪源性间充质干细胞产生的外泌体产生量少。本发明人发现了新知识，即干细胞生长因子是阻碍外泌体产生的因子。因此，本发明所涉及的外泌体产生促进剂中优选不含有干细胞生长因子。干细胞生长因子是不具有N型糖链且分子量为29kDa的蛋白质，属于C型凝集素家族。
由于肿瘤坏死因子-α(TNF-α：tumor necrosis factor-α)也阻碍由脂肪源性间充质干细胞产生外泌体，因此本发明所涉及的外泌体产生促进剂中优选不含有肿瘤坏死因子-α。
在本发明中，脂肪源性间充质干细胞优选以无纺布作为支架进行培养。在以无纺布作为支架来培养脂肪源性间充质干细胞的情况下，能进一步促进外泌体的产生。
无纺布的单位面积重量为1～500g/m2即可，例如优选为50～200g/m2。无纺布也可以是经过了亲水化处理的无纺布。无纺布的亲水化是能够通过氟气处理、常压等离子体处理、真空等离子体处理、电晕处理、亲水性单体的接枝聚合处理、磺化处理或表面活性剂赋予处理来进行的，优选氟气处理、常压等离子体处理。
构成无纺布的纤维优选为聚烯烃类聚合物，作为聚烯烃类聚合物能够列举出：例如，乙烯、丙烯、1-丁烯、1-戊烯、3-甲基-1-丁烯、1-己烯、1-辛烯、1-十二烯、1-十八烯等。
构成无纺布的纤维优选为纤维直径小的纤维，平均纤维直径优选为200μm以下，更优选为10～100μm，特别优选为15～50μm。纤维中也可以混合存在纤维直径相对较大的纤维和纤维直径相对较小的纤维。
无纺布优选具有多孔性。多孔性具有以下重要意义：向使组织再生时所需的细胞补充充分的氧及营养、迅速排出二氧化碳、废物。通过具有多孔性，比表面积变大，细胞粘附性提高。这种情况下的平均孔径例如为5μm～200μm、20μm～100μm、25μm～100μm、30μm～100μm、35μm～100μm、40μm～100μm、50μm～100μm或60μm～100μm，优选为50μm～300μm。
无纺布的形态没有特别限定，但优选为无纺布片。优选地，无纺布片包括沿厚度方向贯穿无纺布片的多个通孔。无纺布片的总膜厚例如可以为5μm以上、10μm以上、20μm以上或25μm以上，也可以为500μm以下、300μm以下、100μm以下、75μm以下或50μm以下。优选为30～2000μm，更优选为500～1000μm。
使用本发明所涉及的外泌体产生促进剂而产生的外泌体能够用作含有外泌体的制剂。含有外泌体的制剂的形态没有特别限定，例如可以是注射剂、悬浮剂、溶液剂、喷雾剂之类的液剂，也可以是片状制剂、凝胶状制剂。
含有外泌体的制剂的使用方法没有限定，例如，能够用于直接对受试者给药，或者用作在生物体外进行的、用于组织、器官的重建的供给源。
含有外泌体的制剂能够用于治疗疾病或残疾。能够列举出可作为对象的疾病或残疾有：免疫性疾病、缺血性疾病(下肢缺血、缺血性心脏疾病(心肌梗塞等)、冠心病、脑血管缺血、肾缺血、肺缺血等)、神经疾病、克罗恩氏病、移植物抗宿主病(GVHD)、包括溃疡性大肠炎的炎症性肠病、包括全身性红斑狼疮的胶原病、肝硬化、脑梗死、脑内血肿、脑血管痉挛、放射性肠炎、特应性皮炎、多发性硬化、类风湿关节炎、牛皮癣、红斑狼疮、糖尿病、蕈样真菌病(Alibert-Bazin综合症)、硬皮症、由软骨等***的变性和/或炎症引起的疾病、眼疾、血管新生相关疾患、充血性心力衰竭、心肌病、创伤、上皮损伤、纤维化、肺病、癌等疾病或残疾。
含有外泌体的制剂也能够用于美容目的的治疗、处理或改善。美容目的不仅仅是单纯的以成为健康状态的美容为目的，还包括对手术后或外部创伤后的变形及先天性的变形的美容治疗。例如，能够用于乳腺的组织增大术(***、乳腺再造)、针对脸颊或上下眼睑的凹陷的组织增大术、以及对半侧面部萎缩症、面部或漏斗胸的组织增大术。
含有外泌体的制剂除了外泌体以外还可以含有药学上可接受的载体、添加物。作为这样的载体、添加物，能够列举出：例如，等渗剂、增粘剂、糖类、糖醇类、防腐剂(保存剂)、杀菌剂或抗菌剂、pH调节剂、稳定剂、螯合剂、油性基剂、凝胶基剂、表面活性剂、悬浮剂、结合剂、赋形剂、润滑剂、崩解剂、发泡剂、流动助剂、分散剂、乳化剂、缓冲剂、助溶剂、抗氧化剂、甜味剂、酸味剂、着色剂、呈味剂、香料或清凉助剂等，但并不限于这些。
根据本发明，能够稳定且大量地供给来自脂肪源性间充质干细胞的外泌体。利用基因重组技术使mRNA、miRNA、蛋白质等具有治疗效果的分子在间充质干细胞中过度表达(overexpression)，产生携带有这些分子的外泌体，从而也能够应用于各种治疗及美容。利用基因重组技术使针对靶组织特异性表达的蛋白质的受体表达于外泌体的表面等来修饰外泌体的表面，从而也能够将外泌体更特异性地递送至靶组织。
实施例
将4×104个脂肪源性间充质干细胞(ADSC)接种到了各孔中。细胞培养，使用了无纺布(3D、BioNOCII、CESCO公司、非粘附性的24孔板(PrimeSurface(注册商标)住友电木))。基础培养基为1.5ml的含10％FBS的DMEM F12培养基(Sigma)。培养天数为六天。从第二天开始，每天更换1.5ml培养基，测量了使用完毕的培养液的糖消耗量。
接着，在第七天及第八天即48小时的细胞培养中，为进行外泌体回收而用DMEMF12作了培养基。在该48小时的培养中，在试验#1中添加了10％的ExoFBS(Funakoshi)。ExoFBS是除去了牛源性的外泌体的培养基，从而抑制了其对外泌体研究的影响。在48小时的培养中，在试验#2中按下述表1所示的浓度添加了A到H共八种因子。
【表1】
| 因子 | 浓度 | |
| A | IGF-1 | 10ng/ml |
| B | bFGF | 10ng/ml |
| C | TNFα | 1ng/ml |
| D | SCGF | 1ng/ml |
| E | PDGF | 1ng/ml |
| F | EGF | 10ng/ml |
| G | IL-1β | 1ng/ml |
| H | 海藻糖 | 10mg/ml |
在48小时的培养中，在试验#3中，从表1所示的八种因子中仅除去因子A并添加了其它因子，在试验#4中，从表1所示的八种因子中仅除去因子B并添加了其它因子，在试验#5中，从表1所示的八种因子中仅除去因子C并添加了其它因子，在试验#6中，从表1所示的八种因子中仅除去因子D并添加了其它因子，在试验#7中，从表1所示的八种因子中仅除去因子E并添加了其它因子，在试验#8中，从表1所示的八种因子中仅除去因子F并添加了其它因子，在试验#9中，从表1所示的八种因子中仅除去因子G并添加了其它因子，在试验#10中，从表1所示的八种因子中仅除去因子H并添加了其它因子。
从回收到的1.5ml的培养上清液中每次提取50μl，用外泌体计数仪(Exo Counter)(Kenwood)对由脂肪源性间充质干细胞(ADSC)产生的外泌体的量进行了测量。外泌体计数仪(Exo Counter)是用光盘和连接抗体的纳米微球(nanobeads)以表面抗原特异性的方式通过夹心免疫检测法检测外泌体的。通过检测外泌体表面的CD81，对外泌体量进行了定量。
结果示于图1。如图1所示，在除去了表皮细胞生长因子的情况下，所产生的外泌体的量减少，从而可知表皮细胞生长因子是促进外泌体产生的因子。在除去了白细胞介素-1β的情况下，所产生的外泌体的量也减少，从而可知白细胞介素-1β也是促进外泌体产生的因子。在除去了海藻糖的情况下，所产生的外泌体的量也减少，从而可知海藻糖也是促进外泌体产生的因子。
相反，在除去了干细胞生长因子的情况下，所产生的外泌体的量增大，从而可知干细胞生长因子是阻碍外泌体产生的因子。在除去了肿瘤坏死因子-α的情况下，所产生的外泌体的量也增大，从而可知肿瘤坏死因子-α也是阻碍外泌体产生的因子。
－产业实用性－
能够用于产生外泌体。
Claims (12)
1.一种外泌体产生促进剂，其促进在无血清培养基中培养的脂肪源性间充质干细胞产生外泌体，其特征在于：
所述外泌体产生促进剂具有表皮细胞生长因子。
2.根据权利要求1所述的外泌体产生促进剂，其特征在于：
所述外泌体产生促进剂还含有白细胞介素-1β。
3.根据权利要求1或2所述的外泌体产生促进剂，其特征在于：
所述外泌体产生促进剂还含有海藻糖。
4.根据权利要求1到3中任一项权利要求所述的外泌体产生促进剂，其特征在于：
所述外泌体产生促进剂不含有干细胞生长因子。
5.根据权利要求1到4中任一项权利要求所述的外泌体产生促进剂，其特征在于：
所述外泌体产生促进剂不含有肿瘤坏死因子-α。
6.根据权利要求1到5中任一项权利要求所述的外泌体产生促进剂，其特征在于：
所述脂肪源性间充质干细胞是以无纺布作为支架进行培养的。
7.一种外泌体产生促进方法，其促进脂肪源性间充质干细胞产生外泌体，其特征在于：
在含有表皮细胞生长因子的无血清培养基中培养脂肪源性间充质干细胞。
8.根据权利要求7所述的外泌体产生促进方法，其特征在于：
所述无血清培养基中还含有白细胞介素-1β。
9.根据权利要求7或8所述的外泌体产生促进方法，其特征在于：
所述无血清培养基中还含有海藻糖。
10.根据权利要求7到9中任一项权利要求所述的外泌体产生促进方法，其特征在于：
所述无血清培养基中不含有干细胞生长因子。
11.根据权利要求7到10中任一项权利要求所述的外泌体产生促进方法，其特征在于：
所述无血清培养基中不含有肿瘤坏死因子-α。
12.根据权利要求7到11中任一项权利要求所述的外泌体产生促进方法，其特征在于：
所述脂肪源性间充质干细胞是以无纺布作为支架进行培养的。
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