CN110734893B - 含维生素c的促进脐带间充质干细胞增殖的培养基 - Google Patents
含维生素c的促进脐带间充质干细胞增殖的培养基 Download PDFInfo
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Abstract
本发明公开了维生素C在促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化中的应用，在制备促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化的培养基中的应用。本发明发现，在使用现有干细胞培养基使干细胞贴壁后，加入AA(无需增加其它的复杂成分)，即可以促进干细胞的增殖，通过数据也证明即使AA能显著在短时间内提高干细胞的数量，但干细胞的活性，活率以及细胞周期均与不使用添加的对照培养基无差异，说明AA的补充仅会提高细胞增殖，而不会降低细胞活性，降低细胞活率，促进干细胞老化。
Description
技术领域
本发明涉及维生素C在促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化中的应用。以及含维生素C的促进脐带间充质干细胞增殖的培养基，属于细胞培养技术领域。
背景技术
目前，干细胞作为一种新的治疗手段正在广泛地应用于多种疾病的临床试验研究当中，绝大多数的临床试验均证明干细胞能够较好地改善病症。然而，干细胞在未来临床应用中则要解决如何在更短时间内增殖到符合使用需求的细胞数量，同时保证大规模扩增后间充质干细胞仍然保持良好的细胞活性，并且增殖的细胞保持多潜能性和归巢能力，从而保证干细胞的医用价值不打折扣。
间充质干细胞是成体干细胞的一种，即在成体中一类具有可分化可增殖的细胞。目前中国有51项利用干细胞进行的临床试验，其中脐带间充质干细胞项目占总项目的比例超过51％。因此，对于如何有效快速增殖脐带间充质干细胞，同时保证细胞的活性和稳定性是目前亟待解决的问题。
细胞周期是指通过细胞分裂产生的新细胞的生长开始到下一次细胞分裂形成子细胞结束为止所经历的过程。细胞周期可以分为四个时期，细胞按照G1-S-G2-M路线正常运转。G0/G1期被认为是细胞静默和生长期，S期是DNA合成期，G2期是有丝分裂准备期，M期为使染色体等分到两子细胞的过程。干细胞的相对静止(即大部分处于G0/G1期)可以防止细胞未成熟耗竭(Premature exhaustion)。目前有研究发现脐带间充质干细胞在传代培养到第33代，较第3代细胞的G0/G1减少17.9％、S+G2/M增加103.4％、细胞凋亡率增加316.7％，差异均有显著性意义(P<0.01)(Chao X,Ji-dong L,Jing L,Wen-zhe M,Yang-qiu L.Biologicalactivity and restriction of human umbilical cord mesenchymal stem cellsduring long-termculture in vitro.J Clin Rehabil Tissue Eng Reserach 2011；15:1750–4.doi:10.3969/j.issn.1673-8225.2011.10.009.)。另外，第12代脐带间充质干细胞的增殖能力和向成脂肪细胞分化诱导率显著下降，而向成骨细胞分化诱导率及细胞衰老率显著上升(程梅,王洪岩,于大海,李华伟,杜鹏程,魏负禾,张颢,邱林,马军.脐带间充质干细胞长期体外培养的生物学特性及遗传稳定性[J].中国生物制品学杂志,2014,27(3):356-360.)。因此，虽然细胞传代能增加细胞的数量，但细胞的老化和分化也随着细胞增殖而加快。因此，本发明期望通过筛选发现一种/多种能够促进脐带间充质干细胞增殖且不影响细胞活性和分化的配方，该配方将有助于突破干细胞应用的瓶颈。
维生素是人为维持正常的生理功能而必须从食物中获得的一类微量有机物质，在人体生长、代谢、发育过程中发挥着重要的作用。维生素C(Ascrobic acid,AA)是生命必须但人体无法合成的必要水溶性维生素。AA通过水解最后形成代谢产物(CaOx)(Linster CL,Van Schaftingen E.Vitamin C:Biosynthesis,recycling and degradation inmammals.FEBS J 2007；274:1–22.doi:10.1111/j.1742-4658.2006.05607.x.)。AA缺乏会导致坏血症(scurvy)。已知AA能促进成骨、肌细胞增殖、成软骨、细胞外基质的构成和胶原的形成(Franceschi T.The Role of Ascorbic Acid in MesenchymalDifferentiation.Nutrition1992；50:65–70.)(D’Aniello C,Cermola F,Patriarca EJ,Minchiotti G.Vitamin C in Stem Cell Biology:Impact on Extracellular MatrixHomeostasis and Epigenetics.Stem Cells Int 2017；2017:1–16.doi:10.1155/2017/8936156.)。近年来，成体细胞重编程研究中发现，AA通过调节组氨酸去甲基双加氧酶(histone demethylating dioxygenases)的活性促进诱导多能干细胞的产生(Chen J,GuoL,Zhang L,Wu H,Yang J,Liu H,et al.Vitamin C modulates TET1 function duringsomatic cell reprogramming.Nat Genet 2013:1–7.doi:10.1038/ng.2807.)。在已公开的干细胞培养基配方中大部分含有AA，例如公开号为CN103243071A的发明专利中陈述培养基中含有176.13mg/L的AA。在康宁的α-MEM培养基中含有50mg/L的AA。在公开号为CN104805054A的发明专利中表述含有20-100μg/mL的AA。在CN 108611320A的发明专利申请中表述培养基配方含有10mg/L的AA。在公开号为CN 108676773A的发明专利申请中表述培养基配方含有10-100mg/L的AA。在公开号为CN 108721606A的发明专利申请中表述产品含有10-50μg/mL的AA。在公开号为CN 108938618A的发明专利申请中表述AA的施用浓度是10μM-1000μM，优选地100μM-500μM；这说明不同浓度的AA对于干细胞的增殖发挥着重要作用(但不能说明细胞增殖是仅由AA带来的，部分培养基虽能延缓细胞衰老、分化等，但不能说明该作用是仅由AA带来的)。另一方面，在BI Biological Industries的间质干细胞成骨诱导分化培养基和成软骨诱导分化培养基中分别含有70.4μg(20mmol/L,400μL)和52.8μg(20mmol/L,300μL)的AA，这说明不同分化方向需要浓度不同的AA。但是，以上所涉及的培养基中除了AA外还含有其它非常复杂且各异的物质，例如：在公开号为108611320A的发明专利申请中描述“该抗衰老的干细胞培养基包括：低糖α-MEM培养基、胎牛血清、miR-705antagomir、维生素C、谷氨酰胺、青霉素和链霉素。该抗衰老的干细胞培养基能够延缓长期体外传代扩增所致的间充质干细胞衰老和功能下降”。又如在公开号为CN 108676773A的发明专利申请中描述“基础培养液和外源添加物，所述外源添加物为：葡聚糖为5-15g/L，脂类浓缩剂为0.1-0.5mL/L,氢化可的松为0.05-5μg/L，地塞米松为0.1-10μg/L，亚硒酸钠为1-5μg/L，β-巯基乙醇为10-50μM，维生素E为10-50μg/L，转铁蛋白为1-20mg/L,胰岛素5-50μg/L，生长激素(GH)为10-50μg/L，谷胱甘肽10-50μg/L，非必需氨基酸为1％”。再如在公开号为109790516A的发明专利申请中描述“包含5-10％的FBS和NAC(N-乙酰半胱氨酸)的DMEM或K-SFM”，还包括钙、rEGF、胰岛素和氢化可的松，以及0.1mM至1mM的阿司匹林。又如在公开号为CN 108938618A的发明专利申请中描述所述“本发明公开了选自槲皮素，大黄根酸，毛地黄黄酮，芒柄花黄素，和厚朴酚，黄连素，山奈酚，矮茶素，脱氧肾上腺素，甘草查尔酮A的化合物及其衍生产品在制备用于促进干细胞增殖、延缓干细胞衰老、促进干细胞向成骨、软骨、脂肪细胞分化、提高干细胞的血管形成能力和/或加强小鼠运动耐力的方法中使用的药物中的用途”。这些文献中均未揭示单独的AA在其中起到了何种具体的作用。因此，虽然不同浓度的AA对于间充质干细胞的增殖和分化都发挥着重要的作用，但如何保证干细胞快速增殖且又不发生分化是目前干细胞医学遇到的重要瓶颈问题之一。
发明内容
针对上述现有技术的不足之处，本发明通过筛选试验，发现了能够促进干细胞增殖且保持干细胞活性和不分化的培养基。本发明可以广泛地应用于干细胞扩增培养中。
本发明是通过以下技术方案实现的：
维生素C在促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化中的应用。维生素C在制备促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化的培养基中的应用。
进一步地，维生素C作为唯一有效成分在促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化中的应用。维生素C作为唯一有效成分在制备促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化的培养基中的应用。
进一步地，具体应用时，维生素C的添加浓度为0.01～10mg/mL，优选0.1mg/mL。
含维生素C的促进脐带间充质干细胞增殖的培养基，由基础培养基、维生素C和血小板分裂物组成，其中，维生素C的浓度为0.01～10mg/mL，优选0.1mg/mL；血小板分裂物占基础培养基质量的5％。
所述血小板分裂物，为现有技术中已有的产品，可商业购买得到，比如购自HeliosUltraGRO,货号：HPCPLCRL05。
含维生素C的促进脐带间充质干细胞增殖的培养基，由基础培养基，维生素C，血小板分裂物，以及维生素E或维生素K或萝卜硫素组成，其中，维生素C的浓度为0.01～10mg/mL，优选0.1mg/mL；血小板分裂物占基础培养基质量的5％。
进一步地，维生素E或维生素K或萝卜硫素的浓度为0.1mg/mL。
所述基础培养基，选自DMEM培养基、RPMI培养基、MEM培养基、BME培养基等(DMEMGibco货号：11885084；Gibco品牌DMEM high glucose货号：11995065；DMEM,Low Glucose货号：11885084；DMEM/F12货号：11330032；RPMI 1640货号：22400089；F-12K，货号：22400089；MEM货号：11095080；Opti-MEM货号：11058021；BME，货号：41010109)。这些基础培养基均为现有技术中已有的常规培养基，其成分组成均已公开，比如：DMEM培养基有DMEM(A)细胞培养基、DMEM(H)细胞培养基、DMEM(L)细胞培养基三种。本发明实施例所用的为DMEM(L)，Gibco货号：11885084，其具体组成成分如下所示：甘氨酸30mg/L；L-精氨酸盐酸盐84mg/L；L-胱氨酸二盐酸盐63mg/L；L-谷氨酰胺：584mg/L；L-组氨酸盐酸盐(一水)42mg/L；L-异亮氨酸105mg/L；L-亮氨酸105mg/L；L-赖氨酸盐酸盐146mg/L；L-蛋氨酸30mg/L；L-苯丙氨酸66mg/L；L-丝氨酸42mg/L；L-苏氨酸95mg/L；L-色氨酸16mg/L；L-酪氨酸二钠盐二水合物104mg/L；L-缬氨酸94mg/L；氯化胆碱4mg/L；D-泛酸钙4mg/L；叶酸4mg/L；烟酰胺4mg/L；维生素B6盐酸盐4mg/L；核黄素0.4mg/L；盐酸硫胺素4mg/L；无旋光肌醇7.2mg/L；无水氯化钙200mg/L；九水硝酸铁0.1mg/L；无水硫酸镁97.67mg/L；氯化钾400mg/L；碳酸氢钠3700mg/L；氯化钠6400mg/L；二水合磷酸二氢钠125mg/L；D-葡萄糖1000mg/L；酚红15mg/L；丙酮酸钠110mg/L。
本发明通过实验发现，人源脐带间充质干细胞(UC-MSCs)在DMEM+血小板分裂物培养基(以下简称为DMEM)培养24小时后，更换为含有AA 0.1mg/mL的DMEM培养基培养48小时，能够促进UC-MSCs增殖，相比于一直在DMEM中培养增殖速度高2.5倍，统计具有显著性差异。另外，本发明也检测了含有AA 0.1mg/mL和Vitamin E 0.1mg/mL(简称VE)，含有AA 0.1mg/mL和Vitamin K1 0.1mg/mL(简称VK)和含有AA 0.1mg/mL和萝卜硫素0.1mg/mL(简称Sulforaphane)。结果发现，相比于DMEM，所有添加剂配方(VE,VK和Sulforaphane)均能促进UC-MSCs的增殖，但相比于AA，其他添加剂对UC-MSCs的增殖并未显著性超过AA的作用，说明AA 0.1mg/mL对于UC-MSCs的增殖非常重要。另外，也同时对比了另外一种进口培养基(BI公司生产的alpha-MEM，简称BI)，该培养基中含有0.05mg/mL的AA，结果显示，相比于AA，BI的培养基不能显著的促进干细胞的增殖(如图2所示)，说明AA的浓度非常的重要。
本发明也检测了上述五种不同培养基对细胞活率和活力(MTT检测)的影响，结果发现在48小时培养后，UC-MSCs在各种培养基中的活率没有显著性差异(DMEM 93％；BI89.75％；AA92.75％；VE 92％；VK 91.5％；Sulforaphane90.5％)，同时对于细胞的活力也没有明显的差异(DMEM 1；BI 0.84；AA 1.07；VE 1.14；VK 1.07；Sulforaphane 1.07)(如图3所示)，可见维生素C不会对UC-MSCs的存活率和活力造成影响。最后检测了AA；VE；VK；Sulforaphane添加剂对细胞周期的影响，结果发现相比于有康干细胞培养基，这些添加剂不影响细胞周期(如图4所示)，说明维生素C不会在促进细胞增殖的时候导致细胞过早的分化和老化。
本发明的技术方案，相比于现有技术，本发明是通过简单的AA或含有AA的其它维生素组合在一个更加便捷的方式下，即在使用现有干细胞培养基使干细胞贴壁后，加入AA(AA作为唯一有效成分，无需增加其它的复杂成分)，即可以促进干细胞的增殖，通过数据也证明即使AA能显著在短时间内提高干细胞的数量，但干细胞的活性，活率以及细胞周期均与不使用添加的对照培养基无差异，说明AA的补充仅会提高细胞增殖，而不会降低细胞活性，降低细胞活率，促进干细胞老化。
本发明所引述的所有文献，它们的全部内容通过引用并入本文，并且如果这些文献所表达的含义与本发明不一致时，以本发明的表述为准。此外，本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。提及的术语和短语如有与公知含义不一致的，以本发明所表述的含义为准。
附图说明
图1：人源脐带间充质干细胞的培养过程示意图。
图2A：不同培养基对UC-MSCs增殖促进作用示意图。
图2B：不同培养基对UC-MSCs增殖促进作用示意图(照片)。
图3：不同培养基对UC-MSCs活性和活力影响示意图，其中，A：细胞活率；B：细胞活性。
图4：不同培养基对UC-MSCs的细胞周期影响示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。
实验 人源脐带间充质干细胞的培养
操作步骤：如图1所示。
1.细胞接种
为了与常用的培养基进行比较，本试验将DMEM+血小板分裂物培养基(血小板分裂物占基础培养基质量的5％)(以下简称为DMEM)和BI alpha-MEM(以下简称为BI)做为对照组。另外，首先采用对照组培养基让MSCs贴壁生长。细胞接种于24孔板中，接种密度为2.5×105，每孔加入0.1ml(24孔板加液量为1mL)培养基，1-10孔为DMEM+血小板分裂物培养基，11-12孔为BI alpha-MEM，置于37℃，5％CO2细胞培养箱。
2.准备配方
配方1：称取2mgAA溶于20mlDMEM中，用0.22μm滤器过滤。
配方2：称取2mgAA+2mgVE溶于20mlDMEM中，用0.22μm滤器过滤。
配方3：称取2mgAA+2mgVK溶于20mlDMEM中，用0.22μm滤器过滤。
配方4：称取2mgAA+2mg萝卜硫素溶于20mlDMEM中，用0.22μm滤器过滤。
3.换液
细胞在37℃，5％CO2培养箱中培养24h后，DMEM、BI培养基共4个孔做阴性对照不换液，剩下其余8个孔，弃掉原培养基，分别加入配方1、配方2、配方3、配方4，每孔加1ml。
4.细胞消化
细胞培养48h后，在镜下观察细胞生长情况，每个孔加胰酶消化，离心后计数，血球计数板计数，获得干细胞的增殖倍数。细胞悬液加入1％的台盼蓝进行染色，然后镜下计数染色的细胞(已死亡的细胞)并计算占总细胞数量的比例(活率)。依照MTT染色操作对每组的细胞进行MTT染色然后用酶标仪读取每组细胞的活性。用PI作为染料染色固定后的干细胞并通过流式细胞仪分析细胞周期。
结果：如图2、图3、图4所示。
人源脐带间充质干细胞(UC-MSCs)在DMEM+血小板分裂物培养基培养24小时后，更换为含有AA 0.1mg/mL的DMEM培养基培养48小时，能够促进UC-MSCs增殖，相比于一直在DMEM中培养增殖速度高2.5倍，统计具有显著性差异。
相比于DMEM，所有添加剂配方(VE,VK和Sulforaphane)均能促进UC-MSCs的增殖，但相比于AA，其他添加剂对UC-MSCs的增殖并未显著性超过AA的作用，说明AA 0.1mg/mL对于UC-MSCs的增殖非常重要。另外，相比于AA，BI的培养基不能显著的促进干细胞的增殖，说明AA的浓度非常的重要。
在48小时培养后，UC-MSCs在各种培养基中的活率没有显著性差异(DMEM 93％；BI89.75％；AA 92.75％；VE 92％；VK 91.5％；Sulforaphane90.5％)，同时对于细胞的活力也没有明显的差异(DMEM 1；BI 0.84；AA 1.07；VE 1.14；VK 1.07；Sulforaphane 1.07)(如图3所示)，可见维生素C不会对UC-MSCs的存活率和活力造成影响。
最后检测了AA；VE；VK；Sulforaphane添加剂对细胞周期的影响，结果发现相比于有康干细胞培养基，这些添加剂不影响细胞周期(如图4所示，如表1所示)，说明维生素C不会在促进细胞增殖的时候导致细胞过早的分化和老化。
表1
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。本说明书引述的所有出版物、专利和专利申请通过引用并入本文，如同这些出版物、专利和专利申请各自特别地和个别地表明通过引用并入本文。
Claims (2)
1.含维生素C的促进脐带间充质干细胞增殖的培养基，其特征在于：由基础培养基，维生素C，血小板分裂物，以及维生素E组成，其中，血小板分裂物占基础培养基质量的5％；
所述含维生素C的促进脐带间充质干细胞增殖的培养基能够促进脐带间充质干细胞增殖且不影响干细胞活性、活率和分化老化；
所述维生素C的浓度为0.1mg/mL；
所述基础培养基选自DMEM培养基、RPMI培养基、MEM培养基、BME培养基；
所述维生素E的浓度为0.1mg/mL。
2.根据权利要求1所述的含维生素C的促进脐带间充质干细胞增殖的培养基，其特征在于：所述DMEM培养基的组分组成为：甘氨酸30mg/L；L-精氨酸盐酸盐84mg/L；L-胱氨酸二盐酸盐63mg/L；L-谷氨酰胺：584mg/L；L-组氨酸盐酸盐一水42mg/L；L-异亮氨酸105mg/L；L-亮氨酸105mg/L；L-赖氨酸盐酸盐146mg/L；L-蛋氨酸30mg/L；L-苯丙氨酸66mg/L；L-丝氨酸42mg/L；L-苏氨酸95mg/L；L-色氨酸16mg/L；L-酪氨酸二钠盐二水合物104mg/L；L-缬氨酸94mg/L；氯化胆碱4mg/L；D-泛酸钙4mg/L；叶酸4mg/L；烟酰胺4mg/L；维生素B6盐酸盐4mg/L；核黄素0.4mg/L；盐酸硫胺素4mg/L；无旋光肌醇7.2mg/L；无水氯化钙200mg/L；九水硝酸铁0.1mg/L；无水硫酸镁97.67mg/L；氯化钾400mg/L；碳酸氢钠3700mg/L；氯化钠6400mg/L；二水合磷酸二氢钠125mg/L；D-葡萄糖1000mg/L；酚红15mg/L；丙酮酸钠110mg/L。
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