CN114438025B - 一种制备脂肪间充质干细胞因子的诱导培养基及培养方法 - Google Patents
一种制备脂肪间充质干细胞因子的诱导培养基及培养方法 Download PDFInfo
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Abstract
本发明公开了一种制备脂肪间充质干细胞因子的诱导培养基，包括基础培养基和添加在基础培养基中的诱导剂，诱导剂包括：维生素C、积雪草酸、弗斯可林、海藻酸钠。本发明提供的诱导培养基各组分协同作用，有效提高了培养液中细胞因子的含量和活性，保证了细胞因子产品在临床的应用效果。本发明还提供了一种制备脂肪间充质干细胞因子的方法，采用本发明提供的诱导培养基，在低氧条件下对脂肪间充质干细胞进行诱导培养，操作简便。
Description
技术领域
本发明涉及一种诱导培养基，尤其涉及一种制备脂肪间充质干细胞因子的诱导培养基及培养方法。
背景技术
间充质干细胞(mesenchymal stem cells，MSC)广泛存在于全身多种组织中，如骨髓、脂肪组织、牙髓、脐带、胎盘等。MSC除了具有多向组织细胞分化能力之外，还在造血、免疫炎症反应、血管新生等人体重要功能中起调节作用。
脂肪的间充质干细胞作为间充质干细胞的一种，具有取材方便，在体外培养条件下增殖速度的特点，并且可以多向分化，可以向脂肪细胞、成骨细胞、软骨细胞、心肌细胞，甚至神经细胞分化。目前研究证明，脂肪间充质干细胞在器官损伤修复中的应用是安全、有效的，而且已有研究证实脂肪的间充质干细胞具有促进皮肤再生修复的作用。
脂肪间充质干细胞可以分泌各种生长因子，包括血管内皮生长因子(VEGF)、表皮生长因子（EGF）、成纤维细胞生长因子（FGF）、角质细胞生长因子(KGF)、转化生长因子β(TGF-β)、胰岛素样生长因子(IGF)和血小板源性生长因子(PDGF)等。这些因子能够有效调控机体细胞信号传导，活化人体细胞进而生理性修复或替代机体损伤、衰老细胞，改善细胞生长微环境、促进伤口愈合、延缓衰老。传统方法培养脂肪间充质干细胞的细胞液中细胞因子存在浓度低活性差的问题，不能较好的发挥作用，影响产品的应用效果。因此，有必要提供一种诱导培养基，提高脂肪间充质干细胞因子的含量及活性。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种制备脂肪间充质干细胞因子的诱导培养基，提高脂肪间充质干细胞因子的制备效率。
本发明的目的之二在于提供一种制备脂肪间充质干细胞因子的培养方法。
本发明的目的之一采用如下技术方案实现：
一种制备脂肪间充质干细胞因子的诱导培养基，包括基础培养基和添加在基础培养基中的诱导剂，所述诱导剂包括：维生素C、积雪草酸、弗斯可林、海藻酸钠。
优选地，所述基础培养基为DMEM/F12，所述诱导剂中各成分在培养基中的浓度为：维生素C7.2-9.6μg/mL、积雪草酸12.5-15.5ng/mL、弗斯可林4.6-7.9ng/mL、海藻酸钠5.5-7.5μg/mL。
优选地，所述诱导剂中各成分在培养基中的浓度为：维生素C 8.4μg/mL、积雪草酸14.0ng/mL、弗斯可林6.2ng/mL、海藻酸钠6.5μg/mL。
本发明的目的之二采用如下技术方案实现：
一种制备脂肪间充质干细胞因子的培养方法，采用上述的诱导培养基进行诱导培养。
优选地，所述制备脂肪间充质干细胞因子的培养方法包括以下步骤：将脂肪间充质干细胞常规培养至融合度60-70%，弃去原培养基，加入诱导培养基，连续培养65-72h后收集培养上清液，经过滤浓缩后即得。
优选地，加入诱导培养基后在37℃，5%CO2，5-10%O2的条件下进行诱导培养。
优选地，所述脂肪间充质干细胞为传代培养得到的P1-P5代细胞。
优选地，所述诱导培养基中脂肪间充质干细胞的密度为3-5×104个/mL。
相比现有技术，本发明的有益效果在于：本发明提供了一种制备脂肪间充质干细胞因子的诱导培养基，在培养基中添加由维生素C、积雪草酸、弗斯可林、海藻酸钠组成的诱导剂，各组分协同作用，有效提高了培养液中细胞因子的含量和活性，保证了细胞因子产品在临床的应用效果。
本发明还提供了一种制备脂肪间充质干细胞因子的方法，采用本发明提供的诱导培养基，在低氧条件下对脂肪间充质干细胞进行诱导培养，操作简便。
具体实施方式
下面，结合具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
一种制备脂肪间充质干细胞因子的诱导培养基，包括DMEM/F12培养基和添加在培养基中的诱导剂，诱导剂由以下成分组成：维生素C、积雪草酸、弗斯可林、海藻酸钠；诱导剂中各成分在培养基中的浓度为：维生素C 8.4μg/mL、积雪草酸14.0ng/mL、弗斯可林6.2ng/mL、海藻酸钠6.5μg/mL。
一种制备脂肪间充质干细胞因子的培养方法，包括以下过程：取P1代脂肪间充质干细胞接种在添加LONZA培养基的T75培养瓶中，细胞的密度为1×104个/mL，在37℃，5%CO2的培养箱中培养至融合度60%，弃去培养基，加入诱导培养基，调整细胞的密度为3×104个/mL，在37℃，5%CO2，5%O2的条件下进行诱导培养，连续培养65h后离心，收集培养上清液，将上清液经0.22μm的滤膜过滤，然后用40KD超滤膜进行透析，取透析液再通过90D的超滤膜，收集截留液即为细胞因子浓缩液，可将截留液加入冻干保护剂壳聚糖（每100mL截留液中加入10g壳聚糖）后冷冻干燥保存。
实施例2
一种制备脂肪间充质干细胞因子的诱导培养基，包括DMEM/F12培养基和添加在培养基中的诱导剂，诱导剂由以下成分组成：维生素C、积雪草酸、弗斯可林、海藻酸钠；诱导剂中各成分在培养基中的浓度为：维生素C 7.2μg/mL、积雪草酸12.5ng/mL、弗斯可林4.6ng/mL、海藻酸钠5.5μg/mL。
一种制备脂肪间充质干细胞因子的培养方法，包括以下过程：取P3代脂肪间充质干细胞接种在添加LONZA培养基的T75培养瓶中，细胞的密度为1×104个/mL，在37℃，5%CO2的培养箱中培养至融合度65%，弃去培养基，加入诱导培养基，调整细胞的密度为4×104个/mL，在37℃，5%CO2，8%O2的条件下进行诱导培养，连续培养70h后离心，收集培养上清液，将上清液经0.22μm的滤膜过滤，然后用40KD超滤膜进行透析，取透析液再通过90D的超滤膜，收集截留液即为细胞因子浓缩液，可将截留液加入冻干保护剂壳聚糖（每100mL截留液中加入12g壳聚糖）后冷冻干燥保存。
实施例3
一种制备脂肪间充质干细胞因子的诱导培养基，包括DMEM/F12培养基和添加在培养基中的诱导剂，诱导剂由以下成分组成：维生素C、积雪草酸、弗斯可林、海藻酸钠；诱导剂中各成分在培养基中的浓度为：维生素C 9.6μg/mL、积雪草酸15.5ng/mL、弗斯可林7.9ng/mL、海藻酸钠7.5μg/mL。
一种制备脂肪间充质干细胞因子的培养方法，包括以下过程：取P5代脂肪间充质干细胞接种在添加LONZA培养基的T75培养瓶中，细胞的密度为1×104个/mL，在37℃，5%CO2的培养箱中培养至融合度70%，弃去培养基，加入诱导培养基，调整细胞的密度为5×104个/mL，在37℃，5%CO2，10%O2的条件下进行诱导培养，连续培养72h后离心，收集培养上清液，将上清液经0.22μm的滤膜过滤，然后用40KD超滤膜进行透析，取透析液再通过90D的超滤膜，收集截留液即为细胞因子浓缩液，可将截留液加入冻干保护剂壳聚糖（每100mL截留液中加入15g壳聚糖）后冷冻干燥保存。
对比例1
对比例1提供一种制备脂肪间充质干细胞因子的诱导培养基，和实施例1的区别为：省去积雪草酸，其余均和实施例1相同。
对比例2
对比例2提供一种制备脂肪间充质干细胞因子的诱导培养基，和实施例1的区别为：省去弗斯可林，其余均和实施例1相同。
对比例3
对比例3提供一种制备脂肪间充质干细胞因子的诱导培养基，和实施例1的区别为：省去积雪草酸和弗斯可林，其余均和实施例1相同。
对比例4
对比例4提供一种制备脂肪间充质干细胞因子的诱导培养基，和实施例1的区别为：省去积雪草酸，将弗斯可林的用量调整为20.2ng/mL，其余均和实施例1相同。
对比例5
对比例5提供一种制备脂肪间充质干细胞因子的诱导培养基，和实施例1的区别为：省去弗斯可林，将积雪草酸的用量调整为20.2ng/mL，其余均和实施例1相同。
以成纤维细胞生长因子（FGF）、表皮生长因子（EGF）、血管内皮细胞生长因子（VEGF）为代表，用ELISA试剂盒检测实施例1至3，对比例1至5中各细胞因子的含量，结果如表1所示。
表1
由表1可以看出，实施例1至3中成纤维细胞生长因子（FGF）、表皮生长因子（EGF）、血管内皮细胞生长因子（VEGF）的含量较高，对比例1至5中各细胞因子的含量不及实施例1。
对比例1中省去了积雪草酸、对比例2中省去了弗斯可林，对比例3中同时省去了积雪草酸和弗斯可林，成纤维细胞生长因子（FGF）、表皮生长因子（EGF）、血管内皮细胞生长因子（VEGF）的含量下降显著。对比例4中在省去积雪草酸后增加了弗斯可林的用量，对比例5中在省去弗斯可林后增加了积雪草酸的用量，各细胞因子的含量仍不及实施例1。由此可知，本发明通过添加弗斯可林和积雪草酸，和维生素C、海藻酸钠协同作用，有助于提高脂肪间充质干细胞分泌细胞因子的能力。
选取体重250-300左右的SD雄性大鼠42只，随机分为6组，每组7只，用3%戊巴比妥钠麻醉后去除背部毛发，用手术刀制作1cm×1cm的创面，用无菌纱布吸收创面血液，将实施例1和对比例1至5的冻干粉制成水溶液后涂抹于伤口，用纱布包扎，每日换药一次，统计各组的创面平均愈合时间，结果如表2所示。
表2
由表2可以看出实施例1中的大鼠创面愈合速度最快。对比例1至5中的创面愈合时间长于实施例1，说明对比例1至5的细胞因子冻干产品活性不及实施例1中的细胞因子活性，对比例1至5中调整了诱导培养基的组成，由此可知，本发明通过添加弗斯可林和积雪草酸，和维生素C、海藻酸钠协同作用，有助于提高脂肪间充质干细胞因子的。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (4)
1.一种制备脂肪间充质干细胞因子的方法，其特征在于，采用诱导培养基在37℃，5%CO2，以及5-10% O2的条件下对脂肪间充质干细胞进行诱导培养，所述诱导培养基由DMEM/F12培养基和添加在培养基中的诱导剂组成，所述诱导剂由以下成分组成：维生素C、积雪草酸、弗斯可林和海藻酸钠；所述诱导剂中各成分在培养基中的浓度为：维生素C 7.2-9.6 μg/mL、积雪草酸12.5-15.5 ng/mL、弗斯可林4.6-7.9 ng/mL、海藻酸钠5.5-7.5 μg/mL。
2.根据权利要求1所述的制备脂肪间充质干细胞因子的方法，其特征在于：所述诱导剂中各成分在培养基中的浓度为：维生素C 8.4 μg/mL、积雪草酸14.0 ng/mL、弗斯可林6.2ng/mL、海藻酸钠6.5 μg/mL。
3.根据权利要求1或2所述的制备脂肪间充质干细胞因子的方法，其特征在于，包括以下步骤：将传代培养得到的P1-P5代脂肪间充质干细胞常规培养至融合度60-70%，弃去原培养基，加入诱导培养基，连续培养65-72h后收集培养上清液，经过滤浓缩后即得。
4.根据权利要求1或2所述制备脂肪间充质干细胞因子的方法，其特征在于，所述诱导培养基中脂肪间充质干细胞的密度为3-5×104个/mL。
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