CN116410921B - 一种人源脐带间充质干细胞诱导培养基、诱导方法及应用 - Google Patents
一种人源脐带间充质干细胞诱导培养基、诱导方法及应用 Download PDFInfo
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Abstract
本发明实施例公开了一种人源脐带间充质干细胞诱导培养基、诱导方法及应用。该人源脐带间充质干细胞诱导培养基由基础培养基和添加剂组成，以最终浓度计，所述添加剂由0.05‑1mg/ml酪氨酸、1‑1000mg/ml神经节苷脂、1‑100ng/ml人血小板衍生生长因子、1‑200ng/ml巨噬细胞集落刺激因子、100‑10000μM二丁酰环磷酸腺苷组成。本发明提供的诱导培养基中能够明显增加脐带间充质干细胞的以表皮细胞生长因子(EGF)、成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)为主的营养因子的分泌量，分泌的因子种类较为丰富而且能够长期稳定分泌，同时在妇科黏膜损伤修复治疗中更有优势。
Description
技术领域
本发明实施例涉及生物医药技术领域，具体涉及一种人源脐带间充质干细胞诱导培养基、诱导方法及应用。
背景技术
随着干细胞研究的不断深入，给修复妇科粘膜损伤和增强局部血液供应带来了新的曙光。目前研究发现间充质干细胞(Mesenchymal stem cells，MSCs)通过多种机制参与粘膜损伤的修复，主要包括细胞迁移、新生血管重建、抑制细胞凋亡、分泌神经营养因子和免疫调节等等。
发明内容
为此，本发明实施例提供一种人源脐带间充质干细胞诱导培养基、诱导方法及应用。
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，本发明实施例提供一种人源脐带间充质干细胞诱导培养基，由基础培养基和添加剂组成，以最终浓度计，所述添加剂由0.05-1mg/ml酪氨酸、1-1000mg/ml神经节苷脂、1-100ng/ml人血小板衍生生长因子、1-200ng/ml巨噬细胞集落刺激因子、100-10000μM二丁酰环磷酸腺苷组成。
进一步地，所述添加剂由0.19mg/ml酪氨酸、5mg/ml神经节苷脂、5ng/ml人血小板衍生生长因子、10ng/ml巨噬细胞集落刺激因子、1000μM二丁酰环磷酸腺苷组成。
进一步地，所述基础培养基为含10％胎牛血清的α-MEM培养基。
根据本发明实施例的第二方面，本发明实施例提供一种人源脐带间充质干细胞诱导方法，采用如上任一项所述的诱导培养基进行诱导培养。
进一步地，所述方法包括：将第五代人源脐带间充质干细胞铺于六孔板内进行培养，待人源脐带间充质干细胞生长密度达到80％时，弃去培养基，用PBS缓冲液洗涤2-3次，弃去上清，沿六孔板壁加入所述诱导培养基，于37℃、5％CO2的培养箱中培养48小时。
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诱导方法在妇科黏膜损伤修复中的应用。
本发明实施例具有如下优点：
(1)本发明的人源脐带间充质干细胞诱导培养基中，酪氨酸是一种稳定的细胞添加剂；神经节苷脂主要参与细胞的分化；人血小板衍生生长因子是一种重要的促有丝分裂因子；巨噬细胞集落刺激因子是一种调节因子；二丁酰环磷酸腺苷是用于参与调节细胞功能的第二信使物质。研究发现，上述诱导培养基能够明显增加脐带间充质干细胞的以表皮细胞生长因子(EGF)、成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)为主的营养因子的分泌量，分泌的因子种类较为丰富而且能够长期稳定分泌。
(2)本发明使用人源脐带间充质干细胞诱导培养基及诱导方法使用的成分较为简单，程序简易，从而在很大程度上增强了细胞的安全性且易产业化。
(3)本发明的人源脐带间充质干细胞诱导方法采用的脐带间充质干细胞，相比于其他来源的间充质干细胞(骨髓、脂肪等)，免疫原性更低。
(4)本发明诱导后的人源脐带间充质干细胞，在妇科黏膜损伤修复治疗中更有优势。
(5)本发明的人源脐带间充质干细胞诱导方法通过细胞因子诱导的方法避免了其它方法如基因修饰等带来的风险和不确定性，更适用于产业化和临床应用。
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：第五代人源脐带间充质干细胞来源于足月产的健康胎儿。采集前，应对供者(新生儿)的生物学父母亲两个人都进行人类免疫缺陷病毒传染病检测，全部合格后取该新生儿新鲜的脐带20cm，用加双抗的PBS反复冲洗3遍，祛除血管内的血液，用剪刀将脐带剪成3cm左右的小段，剥出里面的华通氏胶组织，将所得组织剪至1-2立方毫米大小的小块，均匀铺至150平方厘米的培养瓶中，加入α-MEM培养基后翻转培养瓶置于37℃、5％的CO2培养箱中，1h后将培养瓶翻转，使培养液覆盖所有脐带组织块，培养液中含10％血清。培养5-7天后，可见有部分细胞从组织块周围爬出，形态呈梭形，9-10天后，细胞开始迅速增殖，待细胞融合达到培养瓶的80％左右时，用0.05％胰蛋白酶消化细胞并接种。当细胞传至第4代时，使用流式细胞仪对细胞进行鉴定。合格的脐带间充质干细胞表面标志物表达CD105,CD73,CD90,不表达CD45,CD34,CD19,HLA-DR。再传至第五代，得到第五代人源脐带间充质干细胞。
实施例1
本实施例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由0.19mg/ml酪氨酸、5mg/ml神经节苷脂、5ng/ml人血小板衍生生长因子、10ng/ml巨噬细胞集落刺激因子、1000μM二丁酰环磷酸腺苷组成。
实施例2
本实施例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由0.5mg/ml酪氨酸、10mg/ml神经节苷脂、10ng/ml人血小板衍生生长因子、20ng/ml巨噬细胞集落刺激因子、2000μM二丁酰环磷酸腺苷组成。
实施例3
本实施例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由0.05mg/ml酪氨酸、2.5mg/ml神经节苷脂、2.5ng/ml人血小板衍生生长因子、5ng/ml巨噬细胞集落刺激因子、500μM二丁酰环磷酸腺苷组成。
实施例4
本实施例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由1mg/ml酪氨酸、500mg/ml神经节苷脂、50ng/ml人血小板衍生生长因子、100ng/ml巨噬细胞集落刺激因子、5000μM二丁酰环磷酸腺苷组成。
对比例1
本对比例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由5mg/ml神经节苷脂、5ng/ml人血小板衍生生长因子、1000μM二丁酰环磷酸腺苷组成。
对比例2
本对比例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由2mg/ml酪氨酸、0.5mg/ml神经节苷脂、200ng/ml人血小板衍生生长因子、500ng/ml巨噬细胞集落刺激因子、10μM二丁酰环磷酸腺苷组成。
对比例3
本对比例提供一种人源脐带间充质干细胞诱导培养基，由含10％胎牛血清的α-MEM培养基和添加剂组成，以最终浓度计，添加剂由0.19mg/ml酪氨酸、5mg/ml神经节苷脂组成。
实施例5
本实施例提供一种人源脐带间充质干细胞诱导方法
将第五代人源脐带间充质干细胞铺于六孔板上使用α-MEM培养基进行培养，当人源脐带间充质干细胞生长密度达到80％时，将六孔板中的培养基上清吸出，用PBS缓冲液洗涤3次，然后将PBS缓冲液吸尽，沿六孔板壁分别加入实施例1-4、对比例1-3的诱导培养基，于5％CO2的37℃培养箱中培养48小时。
取细胞上清，采用Elisa试剂盒(武汉博士德生物工程有限公司)检测EGF(表皮细胞生长因子)、FGF(成纤维细胞生长因子)、VEGF(血管内皮生长因子)的分泌量。检测结果见表1。
表1
| 诱导培养基 | EGF(pg/106细胞) | FGF(pg/106细胞) | VEGF(pg/106细胞) |
| 实施例1 | 780.3 | 134.6 | 922.3 |
| 实施例2 | 760.3 | 134.5 | 900.2 |
| 实施例3 | 753.2 | 124.6 | 886.4 |
| 实施例4 | 800.8 | 100.2 | 845.2 |
| 对比例1 | 360.8 | 88.3 | 378.9 |
| 对比例2 | 321.5 | 70.9 | 444.2 |
| 对比例3 | 357.6 | 34.5 | 430.2 |
结果显示，与对比例1-3提供的诱导培养基相比，实施例1-4提供的诱导培养基能够明显增加脐带间充质干细胞营养因子的分泌量，其中，实施例1最佳。
实施例6
首先建立并验证了小鼠体内阴道损伤模型。使用异氟醚以0.5L/min的速率麻醉小鼠，并在阴道口远端500微米处的阴道管背外侧使用1毫米的活检冲头，用光滑的平底钳损伤阴道粘膜。小鼠在手术后，24小时后使用实施例1的诱导培养基按照实施例5的诱导方法获得的无菌培养液进行试验性治疗，在治疗3天后，与对照组(使用非诱导培养液)相比，黏膜损伤修复面积达到50％以上。
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 (6)
1.一种人源脐带间充质干细胞诱导培养基，其特征在于，由基础培养基和添加剂组成，以最终浓度计，所述添加剂由0.05-1mg/ml酪氨酸、2.5-500mg/ml神经节苷脂、2.5-50ng/ml人血小板衍生生长因子、5-100ng/ml巨噬细胞集落刺激因子、500-5000μM二丁酰环磷酸腺苷组成。
2.根据权利要求1所述的人源脐带间充质干细胞诱导培养基，其特征在于，所述添加剂由0.19mg/ml酪氨酸、5mg/ml神经节苷脂、5ng/ml人血小板衍生生长因子、10ng/ml巨噬细胞集落刺激因子、1000μM二丁酰环磷酸腺苷组成。
3.根据权利要求1所述的人源脐带间充质干细胞诱导培养基，其特征在于，所述基础培养基为含10%胎牛血清的α-MEM培养基。
4.一种人源脐带间充质干细胞诱导方法，其特征在于，采用权利要求1-3中任一项所述的诱导培养基进行诱导培养。
5.根据权利要求4所述的人源脐带间充质干细胞诱导方法，其特征在于，所述方法包括：
将第五代人源脐带间充质干细胞铺于六孔板内进行培养，待人源脐带间充质干细胞生长密度达到80%时，弃去培养基，用PBS缓冲液洗涤2-3次，弃去上清，沿六孔板壁加入所述诱导培养基，于37℃、5%CO2的培养箱中培养48小时。
6.权利要求1所述的诱导培养基在制备妇科黏膜损伤修复产品中的应用。
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| GM1体外诱导人脐带血间充质干细胞分化为神经样细胞研究;马延;郭金耀;刘雷;范春;;标记免疫分析与临床(12);第1464页摘要 * |
| 兔骨髓间充质干细胞体外诱导分化为神经样细胞的实验研究;李强, 张建生, 丁永忠, 任军, 张新定, 任海军, 潘亚文, 程得钧;中华神经外科疾病研究杂志(05);431-433 * |
| 单唾液酸四己糖神经节苷脂最佳质量浓度诱导人脐带间充质干细胞向神经元样细胞的分化;张鹏;赵宗茂;李建华;刘辉;刘永军;李敏捷;陈明伟;申仑;何磊;;中国组织工程研究(13);2039-2044 * |
| 骨髓间充质干细胞向神经元样细胞分化的研究进展;秦安清;刘黎青;于娜;周盛年;;陕西中医(03);378-380 * |
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