CN107083357A - 一种高效诱导培养骨髓间充质干细胞的方法 - Google Patents
一种高效诱导培养骨髓间充质干细胞的方法 Download PDFInfo
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Abstract
本发明提供了一种高效诱导培养骨髓间充质干细胞的方法，包括步骤：(1)分离骨髓间充质干细胞；(2)利用培养基A和培养基B依次进行培养；所述培养基A的成分为：DMEM/F12培养基、β‑磷酸甘油钠27mmol/ml、成纤维细胞因子4(EGF4)22ng/ml、10％胎牛血清、青霉素300U/ml、120mmol/ml谷氨酰胺；所述培养基B的成分位：DMEM/F12培养基、7mmol/ml的烟酰胺、10％胎牛血清、地塞米松1×10‑8mmol/ml、腺嘌呤30μg/ml、青霉素300U/ml、氢化可的松0.1μg/ml。本发明对骨髓间充质干细胞进行诱导培养时，心肌细胞的转化率较高，可达到48％。
Description
技术领域
本发明属于细胞技术领域，具体涉及一种高效诱导培养骨髓间充质干细胞的方法。
背景技术
细胞疗法作为一种重要的治疗方法，它的发展为许多疾病的治疗带来了新的希望。干细胞作为细胞疗法的一个重要组成部分，因其具有多向分化潜能，能被诱导分化成所需的细胞，而日益受到关注。其中，骨髓间充质干细胞以其来源丰富、易取材、分化能力强等优点受到越来越多的关注。骨髓间充质干细胞(Mesenchymai stem cells，MSCs)是在骨髓中的一种类似成纤维细胞的一类细胞，具有多方向分化潜能，可以向骨组织、软骨组织、皮肤、造血干细胞支持介质、骨骼肌细胞及心肌细胞转化。
目前，对于MSCs的诱导培养方面，主要的一些添加物为β-巯基乙醇、二甲基亚砜、丁基羟基茴香醚、5-氮胞苷等。目前对于获取具有高诱导效果的添加物的选择仍不是很清楚。在将MSCs向心肌细胞转化领域中，在仅利用培养基作为诱导条件方面，现有技术存在转化率过低的缺点。虽然一些技术通过特殊的设备实现了对干细胞转化的控制(如CN105087544 A)，但是对于一般的实验室或者医疗机构而言，如能仅利用特定的培养基便可以获得较高的MSCs向心肌细胞转化的转化率，那么对于后续的相关研究和临床治疗而言均是十分有利的。
发明内容
针对现有技术的缺点，本发明的目的在于提供一种高效诱导培养骨髓间充质干细胞的方法，所述方法包括如下步骤：
(1)分离骨髓间充质干细胞；(2)利用诱导培养基进行培养；
在步骤(2)中，利用培养基A进行培养之后再用培养基B进行培养，所述培养基A的成分如下：
DMEM/F12培养基、β-磷酸甘油钠27mmol/ml、成纤维细胞因子4(EGF4)22ng/ml、10％胎牛血清、青霉素300U/ml、120mmol/ml谷氨酰胺；
所述培养基B的成分如下：
DMEM/F12培养基、7mmol/ml的烟酰胺、10％胎牛血清、地塞米松1×10-8mmol/ml、腺嘌呤30μg/ml、青霉素300U/ml、氢化可的松0.1μg/ml。
利用培养基A进行培养时，培养时间为7～10天。优选的，培养时间为7天。
利用培养基B进行培养时，培养时间为17～19天。优选的，培养时间为18天。
利用培养基A进行培养时，培养至细胞密度为40～45％。
由培养基A替换为培养基B培养时，利用PBS对培养基A的残留液体进行清洗。
本发明在研究时发现，利用一般的诱导培养方法，仅用一种培养基一次性诱导时，很难获得高的转化率，通常而言，转化率不高于25％。在实验过程中，发明人发现，本发明的两种培养基并不能互换，如将培养基B作为第一次培养所用培养基，培养基A作为第二次培养所用培养基时，所得的转化率也较低，仅为21.1±2.3％。
有研究发现，利用烟酰胺可以作为未分化MSCs的培养基添加成分。本发明发现，利用烟酰胺、氢化可的松和地塞米松等添加物可以在对MSCs第一次培养后，实现对MSCs分化为心肌细胞转化率的有效提高。不过，本发明所述培养基A和培养基B中成分的含量对于转化率有着较大的影响，其中的机制还需要进一步的研究。
在一般培养基中，谷氨酰胺也常用作添加剂。不过，对于将其与其它哪些添加物共用可以提高干细胞向某种所需细胞进行转化方面，现有技术还没有明确的研究结论。
有研究者用β-磷酸甘油钠作为MSCs去分化诱导培养基的成分之一，并取得了较好的效果。不过，对于促进MSCs分化而言，如何利用β-磷酸甘油钠，在本发明之前，也同样还没有较为详细的研究结论。
如本发明的实验数据显示，本发明的培养方法对于MSCs转化为心肌细胞有着相对于现有技术而言十分优异的效果，所得的转化率有了很大的提高。可以理解的是，由于转化率的提高，在诱导MSCs转化为心肌细胞进行后续的研究或者临床需求而言，成本得到的大幅度的降低。
具体实施方式
下面通过实施例对本发明进行具体描述，有必要在此指出的是以下实施例只是用于对本发明进行进一步的说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员根据上述发明内容所做出的一些非本质的改进和调整，仍属于本发明的保护范围。
实施例1
本实施例所用的培养基共两种，分别为培养基A和培养基B。其中培养基A的成分如下：
DMEM/F12培养基、β-磷酸甘油钠27mmol/ml、成纤维细胞因子4(EGF4)22ng/ml、10％胎牛血清、青霉素300U/ml、120mmol/ml谷氨酰胺；
培养基B的成分如下：
DMEM/F12培养基、7mmol/ml的烟酰胺、10％胎牛血清、地塞米松1×10-8mmol/ml、腺嘌呤30μg/ml、青霉素300U/ml、氢化可的松0.1μg/ml。
参考文献(Leuk Lymphoma.2005；46(11)：1531-1544.)进行MSCs的分离获得MSCs。然后利用含100mg/L的胎牛血清、100ug/ml的青霉素和100ug/ml的链霉素的低糖DMEM培养基重新悬浮细胞，期间利用胰蛋白酶进行消化，待细胞呈圆形时，中止消化，制成细胞悬液，获得纯化的分离后的MSCs。
按照体积比1∶2，将上述所得的细胞悬液接种至上述培养基A中，隔夜换液，共培养7～10天；
将所得的细胞接种于培养基B中继续培养，每隔3天换液，共培养18天。
培养完毕后，分选出诱导后的心肌细胞(利用流式细胞仪)。
对照实施例1
将培养基A和培养基B互换，其它的方法和实施例1是相同的。
对照实施例2
将培养基A的β-磷酸甘油钠的浓度调整为30mmol/ml、浓度调整为成纤维细胞因子4(EGF4)18ng/ml，其它的与实施例1保持一致。
对照实施例3
将培养基B的腺嘌呤的浓度调整为35μg/ml、烟酰胺的浓度调整为10mmol/ml，其它的与实施例1保持一致。
实验项目和结果
取实施例1和对照组1～3培养第18天(培养基B)所收获的细胞，进行以下操作：用丙酮固定细胞15分钟；PBS溶液冲洗3次，每次5分钟；加入0.3％triton-100润湿剂，室温静置10分钟；PBS溶液冲洗3次，每次5分钟；滴加3％H2O2室温孵育20分钟，消除内源性过氧化物酶活性；PBS溶液冲洗3次，每次5分钟；加入10％山羊血清封闭液封闭20分钟；加入一抗4℃下静置12小时；PBS溶液冲洗3次，每次5分钟；加入二抗，室温静置一小时；PBS溶液冲洗3次，每次5分钟；DAB显色：加入稀释好的DAB显色液，室温环境显微镜下控制显色时间，显色适10度后，冲洗终止显色；苏木轻度复染30-60分钟；梯度酒精脱水；二甲苯透明；中性树胶封片；显微镜下观察染色结果；每组随机选4个非重复高倍镜视野(×400)，计数显黄色的阳性细胞数(N1)和总细胞数(N)，显黄色的阳性细胞即为心肌细胞，最后按公式(N1/N×100％)计算心肌样细胞转化率。
结果如表1所示：
表1
取实施例1和对照组中培养18天(培养基B)后的细胞，利用PBS冲洗后，再利用经过低温处理的丙酮固定13min。之后检测肌钙蛋白和间隙连接蛋白的表达。结果如表2和表3所示。
表2
表3
Claims (7)
1.一种高效诱导培养骨髓间充质干细胞的方法，所述方法包括如下步骤：
(1)分离骨髓间充质干细胞；(2)利用诱导培养基进行培养；
其特征在于，在步骤(2)中，利用培养基A进行培养之后再用培养基B进行培养，所述培养基A的成分如下：
DMEM培养基、β-磷酸甘油钠27mmol/ml、成纤维细胞因子4(EGF4)22ng/ml、10％胎牛血清、青霉素300U/ml、120mmol/ml谷氨酰胺；
所述培养基B的成分如下：
DMEM/F12培养基、7mmol/ml的烟酰胺、10％胎牛血清、地塞米松1×10-8mmol/ml、腺嘌呤30μg/ml、青霉素300U/ml、氢化可的松0.1μg/ml。
2.根据权利要求1所述的方法，其特征在于，利用培养基A进行培养时，培养时间为7～10天。
3.根据权利要求1所述的方法，其特征在于，利用培养基B进行培养时，培养时间为17～19天。
4.根据权利要求1或2所述的方法，其特征在于，利用培养基A进行培养时，培养至细胞密度为40～45％。
5.根据权利要求1所述的方法，其特征在于，由培养基A替换为培养基B培养时，利用PBS对培养基A的残留液体进行清洗。
6.根据权利要求3所述的方法，其特征在于，所述培养时间为19天。
7.根据权利要求2所述的方法，其特征在于，所述培养时间为7天。
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