CN116333980B - 提高胎盘间充质干细胞分泌活性因子水平的诱导培养基及方法与应用 - Google Patents
提高胎盘间充质干细胞分泌活性因子水平的诱导培养基及方法与应用 Download PDFInfo
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Abstract
本发明实施例公开了一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基及方法与应用。所述诱导培养基由基础培养基、转化生长因子‑α、环磷酸腺苷、银杏提取物和维甲酸组成，所述转化生长因子‑α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：8‑16 ng/ml、10‑20 ng/ml、500‑700 sg/ml、15‑25 ng/ml。本发明解决现有脐带间充质干细胞的分泌因子成分种类单一，且因子分泌水平较低持续稳定分泌性差等导致干细胞治疗缺血性脑中风效果不佳的问题。
Description
技术领域
本发明实施例涉及生物医药技术领域，具体涉及一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基及方法与应用。
背景技术
随着干细胞研究的发展，干细胞移植为有效治疗缺血性脑中风带来了新的曙光。在种类繁多的干细胞中，现有使用脐带间充质干细胞（UC-MSCs），通过静脉注射的方式，将细胞移植到缺血性脑中风大鼠模型体内，结果发现所移植的脐带间充质干细胞主要集中分布在脑中风病灶部位，使大鼠神经功能缺损症状得以改善，证实脐带间充质干细胞治疗的确减轻了疾病模型的症状。
经深入研究后发现，脐带间充质干细胞之所以能改善大鼠缺血性脑中风症状，主要是因为细胞所分泌的营养因子来发挥治疗作用，现有技术中使用不同药物组合物，对细胞进行诱导，促进神经营养因子（BDNF）的分泌，用于大鼠缺血性脑中风模型治疗，但是通过这种方式诱导的脐带间充质干细胞所分泌因子成分种类较为单一，且因子分泌水平较低持续稳定分泌性差，不一定能达到有效浓度，加上诱导所使用的细胞代次低一般为3-4代，导致每个单位原代细胞可用于诱导的细胞量有限，成本升高，不能够更高效的用于缺血性脑中风的临床治疗研究。
鉴于此，特提出本发明。
发明内容
为此，本发明实施例提供一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基及方法与应用，以解决现有脐带间充质干细胞的分泌因子成分种类单一，且因子分泌水平较低持续稳定分泌性差等导致干细胞治疗缺血性脑中风效果不佳的问题。
为了实现上述目的，本发明实施例提供如下技术方案：
根据本发明实施例的第一方面，本发明提供一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基，所述诱导培养基由基础培养基、转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸组成，所述转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为8-16 ng/ml、10-20 ng/ml、500-700 sg/ml、15-25 ng/ml。
转化生长因子-α（TGF-α）是一种在细胞增殖和分化中发挥作用的蛋白质。它可以促进细胞增殖、细胞分化、细胞迁移和细胞存活。TGF-α主要通过与表面上的受体结合，触发信号转导通路来发挥作用。这些信号通路包括RAS/MAPK、PI3K/AKT和JAK/STAT等，这些信号通路可以调节基因表达，从而影响细胞增殖和分化。TGF-α在胚胎发育、生长和再生过程中扮演着重要角色，也与多种疾病的发生和发展有关。
环磷酸腺苷（cAMP）是一种重要的细胞信使分子，它在多种生物过程中发挥作用，包括细胞增殖、分化、信号转导、代谢调控等。cAMP的作用包括：1. 激活蛋白激酶A（PKA）：cAMP可以结合PKA的调节亚基，使其释放出催化亚基，从而激活PKA。PKA在细胞中参与多种生物过程，如糖原的合成和分解、脂肪酸的合成和分解、蛋白质的磷酸化等。2. 调节离子通道：cAMP可以调节许多离子通道，如钾通道、钙通道等。通过调节这些离子通道的开放程度，cAMP可以影响细胞的兴奋性、肌肉收缩等生理功能。3. 促进基因转录：cAMP可以通过激活转录因子CREB，促进基因的转录和表达。这些基因涉及多种生物过程，如细胞增殖、分化、凋亡等。4. 调节蛋白质的磷酸化：cAMP可以影响多种蛋白质的磷酸化状态，从而影响它们的功能。例如，cAMP可以促进肌肉收缩的蛋白质肌动蛋白的磷酸化。
银杏提取物是以银杏叶为原料，采用适当溶剂提取有效成分的产品，即从银杏叶中提取的一种天然草药提取物，被广泛用于中医药和保健品中。其主要成分为银杏内酯、黄酮类化合物、酚酸类化合物、三萜类化合物等。银杏提取物的作用包括：1. 改善记忆力和认知功能：银杏提取物可以促进脑血流量的增加，增强脑部供氧能力，从而改善记忆力和认知功能。2. 抗氧化作用：银杏提取物中含有大量黄酮类化合物和酚酸类化合物，具有很强的抗氧化作用，可以清除体内自由基，减轻氧化应激对身体的损害。3. 促进血液循环：银杏提取物可以增加血管内皮细胞的活性，促进血管扩张，从而改善血液循环，降低血压，预防心血管疾病。4. 抗炎作用：银杏提取物中的黄酮类化合物和酚酸类化合物具有抗炎作用，可以减轻炎症反应，缓解疼痛和肿胀。5. 保护神经系统：银杏提取物可以促进神经细胞的生长和发育，保护神经系统的健康，预防神经退行性疾病。
维甲酸是维生素A的一种活性形式，也是一种重要的药物。它主要作用于皮肤和黏膜组织，具有多种作用。1. 促进细胞分化：维甲酸可以促进皮肤细胞的分化，促进表皮细胞向角质细胞的转化，从而使皮肤更加细腻光滑。2. 抑制角质过度增生：维甲酸可以抑制皮肤角质过度增生，减少角质堆积，使皮肤更加健康。3. 抗炎作用：维甲酸可以减轻炎症反应，抑制炎症细胞的活性，缓解疼痛和肿胀。4. 促进胶原蛋白合成：维甲酸可以促进胶原蛋白的合成，从而增加皮肤弹性，减少皱纹和松弛。5. 抗氧化作用：维甲酸具有抗氧化作用，可以清除体内自由基，减轻氧化应激对皮肤的损害
本发明通过大量试验发现，含有上述终浓度范围的转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的诱导培养基能显著提高胎盘间充质干细胞分泌活性因子水平。
进一步地，所述转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：12 ng/ml、17 ng/ml、600 sg/ml、20 ng/ml。
进一步地，所述基础培养基为α-MEM培养基。
根据本发明实施例的第二方面，本发明提供一种提高胎盘间充质干细胞分泌活性因子水平的方法，采用如上任一项所述的诱导培养基对胎盘间充质干细胞进行诱导培养。
进一步地，所述方法包括：将第7-8代胎盘间充质干细胞铺于六孔板上进行培养，待细胞生长密度达到80%时，且细胞生长状态良好，细胞活率>95%，形态符合胎盘间充质干细胞形态，将六孔板中的培养基上清吸出，用PBS缓冲液反复洗涤2-3次，将PBS缓冲液残余液吸干，贴着孔板壁缓慢加入所述诱导培养基，于37℃、5%CO2的培养箱中培养72小时。
进一步地，所述将第7-8代胎盘间充质干细胞铺于六孔板上进行培养的步骤中，所采用的培养基为α-MEM培养基。
根据本发明实施例的第三方面，本发明提供如上所述的诱导培养基或如上所述的方法在制备缺血性脑中风的药物中的应用。
本发明实施例具有如下优点：
1、本发明提供的诱导培养基成分较为简洁，利用多因子诱导的方法，明显促进了神经生长因子（NGF）、神经营养因子（BDNF）、血管内皮生长因子（VEGF）、表皮生长因子（EGF）、血小板衍生因子（PDGF）等多因子的分泌。
2、本发明提供的培养方式简单，使用普通α-MEM培养基即可，增强了细胞的安全性且易产业化，更适用于临床应用，避免了其它处理方法（如：基因修饰等带来的风险和不确定性）。
3、经本发明诱导培养基诱导后的胎盘间充质干细胞（PMSCs）所分泌的因子含量较高，且因子分泌量长期稳定。
4、本发明诱导用的细胞代次高，一般第7-8代的细胞，每单位原代细胞可用于诱导的量更多，成本降低，因此能够更高效的用于缺血性脑中风的临床治疗研究。
附图说明
为了更清楚地说明本发明的实施方式或现有技术中的技术方案，下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍。显而易见地，下面描述中的附图仅仅是示例性的，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图引伸获得其它的实施附图。
图1为本发明提供的诱导1组和对照组的细胞倒置显微镜观察对比图；
图2为本发明提供的不同组大鼠脑组织缺血性脑中风模型TTC染色对比图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例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。
以下内容中，银杏提取物购自山西子哲生物科技有限公司，型号为YXHT。
实施例1
本实施例提供一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基，将转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸加入α-MEM培养基中，轻柔吹打至混合均匀，其中，转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：8 ng/ml、10ng/m、500 sg/ml、15 ng/ml。
实施例2
本实施例提供一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基，通过将转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸加入α-MEM培养基中，轻柔吹打至混合均匀，其中，转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：16 ng/ml、20 ng/ml、700 sg/ml、25 ng/ml。
实施例3
本实施例提供一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基，通过将转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸加入α-MEM培养基中，轻柔吹打至混合均匀，其中，转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：12 ng/ml、17 ng/ml、600 sg/ml、20 ng/ml。
对比例1
本对比例提供一种诱导培养基，其与实施例3的区别仅在于，将转化生长因子-α替换成等终浓度的转化生长因子-β1。
对比例2
本对比例提供一种诱导培养基，其与实施例3的区别仅在于，转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度不同。本对比例中转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：12 ng/ml、30 ng/ml、600 sg/ml、10 ng/ml。
实施例4
本实施例提供一种提高胎盘间充质干细胞分泌活性因子水平的方法：
将第7代胎盘间充质干细胞铺于六孔板上使用α-MEM培养基进行培养，其生长密度达到80%，细胞生长状态良好，细胞活率>95%，形态符合胎盘间充质干细胞形态，细胞表面分子流式结果符合胎盘间充质干细胞相关分子指标：CD105、CD90、CD73呈强阳表达，造血标记物 CD34、CD45、CD14以及HLA-DR呈阴性表达，显微镜下观察细胞形态符合胎盘间充质干细胞形态。将六孔板中的培养基上清吸出，吸干净残液后用PBS缓冲液反复洗涤3次，洗好后将PBS缓冲液残余液吸干，吸干后贴着孔板壁分别缓慢加入2ml诱导培养基，操作完成后将孔板放入培养箱（5%CO2、37℃）内培养72小时。
对比例3
本对比例提供一种提高胎盘间充质干细胞分泌活性因子水平的方法，其与实施例4的区别仅在于，将诱导培养基替换为α-MEM培养基。
试验例1
实施例3、对比例1、对比例2的诱导培养基分别按照实施例4的方法对胎盘间充质干细胞进行培养（依次设为诱导1组、诱导2组和诱导3组）。对诱导1-3组和对比例3（设为对照组）收取细胞上清并进行细胞计数，后续进行NGF、BDNF、VEGF、EGF、PDGF等因子的Elisa检测。
图1为诱导1组和对照组的细胞倒置显微镜观察对比图。结果显示，对照组与诱导1组细胞形态对比没有显著差异，细胞都为贴壁生长，形态呈长梭形，少数为多角形，但是镜下观察诱导组的细胞量明显大于对照组。
诱导1-3组和对照组的每106的细胞分泌的NGF量、BDNF量、VEGF量、EGF量和PDGF量的检测结果见下表1。
表1
结果显示，本发明提供的诱导培养基能有效提高胎盘间充质干细胞分泌活性因子水平。
试验例2 动物实验
1、收取诱导组与对照组细胞上清液做elisa
将PMSCs分为 PMSCs-NTF(诱导组)和PMSCs(对照组)，分别按照实施例4（使用实施例3的诱导培养基）和对比例1的方法进行培养，收集两组细胞上清液，离心、-20℃保存，做elisa检测。
2、收集PMSCs-NTF和PMSCs细胞做脑立体注射
将收集完上清之后的细胞，2000r/5min离心两遍，进行细胞计数，然后用生理盐水对细胞进行重悬，重悬浓度为10微升/4*105，然后进行脑立体注射。
3、大脑中动脉(MCA)闭塞的短暂性缺血模型
采用成年雄性SD大鼠(230-250g)进行缺血性脑卒中实验，PMSCs-NTF 6-8只，PMSCs 6-8只，共造模16只，脑立体注射细胞时进行分组，术后24h进行脑立体注射实验。通过闭塞右侧大脑中动脉(MCA)90分钟来模拟短暂性脑缺血。通过吸入异氟醚来诱导并维持大鼠的麻醉。在颈部中线切口后，暴露、分离右侧颈总动脉、颈外动脉(ECA)和颈内动脉(ICA)。消毒硅胶涂层单丝从ECA切口插入，并向MCA推进，直到阻塞MCA的起源。采用激光多普勒血流测定法证实可成功诱导脑缺血（血流减少超过80%）。在MCAO作用90min后，轻轻地去除单丝。手术在温度控制（37℃）加热灯上进行，术后移至温控室。
4、收集两组诱导组与对照组细胞进行脑立体注射
将PMSCs-NTF和PMSCs的细胞移植到MCAO模型的脑缺血梗死部位内囊附近。位置位前囟后1.2mm，左侧旁开3.2 mm，垂直进针4.7mm处。
5、对注射细胞后的MCAO模型进行TTC染色
用TTC染色测试了两组细胞移植3周后，大鼠缺血部位的恢复情况。结果显示，两组实验都对中风大鼠脑梗死体积都有所改变，但PMSCs-NTF的治疗效果更为明显（图2）。
虽然，上文中已经用一般性说明及具体实施例对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (7)
1. 一种提高胎盘间充质干细胞分泌活性因子水平的诱导培养基，其特征在于，所述诱导培养基由基础培养基、转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸组成，所述转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：8-16 ng/ml、10-20 ng/ml、500-700 sg/ml、15-25 ng/ml。
2. 根据权利要求1所述的诱导培养基，其特征在于，所述转化生长因子-α、环磷酸腺苷、银杏提取物和维甲酸的终浓度分别为：12 ng/ml、17 ng/ml、600 sg/ml、20 ng/ml。
3.根据权利要求1所述的诱导培养基，其特征在于，所述基础培养基为α-MEM培养基。
4.一种提高胎盘间充质干细胞分泌活性因子水平的方法，其特征在于，采用如权利要求1-3中任一项所述的诱导培养基对胎盘间充质干细胞进行诱导培养。
5.根据权利要求4所述的提高胎盘间充质干细胞分泌活性因子水平的方法，其特征在于，所述方法包括：
将第7-8代胎盘间充质干细胞铺于六孔板上进行培养，待细胞生长密度达到80%时，且细胞生长状态良好，细胞活率>95%，形态符合胎盘间充质干细胞形态，将六孔板中的培养基上清吸出，用PBS缓冲液反复洗涤2-3次，将PBS缓冲液残余液吸干，贴着孔板壁缓慢加入所述诱导培养基，于37℃、5%CO2的培养箱中培养72小时。
6.根据权利要求5所述的提高胎盘间充质干细胞分泌活性因子水平的方法，其特征在于，所述将第7-8代胎盘间充质干细胞铺于六孔板上进行培养的步骤中，所采用的培养基为α-MEM培养基。
7.权利要求1所述的诱导培养基或权利要求4所述的方法在制备缺血性脑中风的药物中的应用。
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