CN112481213A - 一种小分子活性肽在诱导hUC-MSCs分化方面的应用 - Google Patents

一种小分子活性肽在诱导hUC-MSCs分化方面的应用 Download PDF

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CN112481213A
CN112481213A CN202011369787.0A CN202011369787A CN112481213A CN 112481213 A CN112481213 A CN 112481213A CN 202011369787 A CN202011369787 A CN 202011369787A CN 112481213 A CN112481213 A CN 112481213A
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张川
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Abstract

本发明公开了一种小分子活性肽在诱导hUC‑MSCs分化方面的应用，所述小分子活性肽为(D‑ALA2)‑亮氨酸脑啡肽，所述分化指向神经元样细胞分化。本发明发现，小分子活性肽(D‑ALA2)‑亮氨酸脑啡肽具有诱导hUCMSCs向神经元样细胞分化的活性，且具有浓度效应。因此，(D‑ALA2)‑亮氨酸脑啡肽可以用于制备诱导hUCMSCs向神经元样细胞分化的培养基。

Description

一种小分子活性肽在诱导hUC-MSCs分化方面的应用
技术领域
本发明属于干细胞领域，涉及干细胞诱导分化，具体一种小分子活性肽在诱导hUC-MSCs分化方面的应用。
背景技术
间充质干细胞(MSCs)是一类来源于发育早期中胚层、具有多向分化及自我更新潜能的成体干细胞。MSCs的来源主要包括骨髓、脂肪、脐带、胎盘、羊膜等。其中，人脐带间充质干细胞(hUC-MSCs)因取材方便、来源广泛、免疫原性低且不受伦理学争议等众多优势，成为最具潜力的种子细胞。
研究显示，hUC-MSCs具有广阔的临床应用前景，可用于治疗神经系统疾病、肝肾损伤、自身免疫疾病、心脏疾病、骨科疾病、心血管疾病、糖尿病等。
诱导hUC-MSCs向神经元样细胞分化可以用于脊髓损伤等神经损伤相关疾病。
发明内容
本发明是为了提供一种小分子活性肽在诱导hUC-MSCs分化方面的应用。
技术方案：
一种小分子活性肽在体外诱导hUC-MSCs分化方面的应用，其中：所述小分子活性肽为(D-ALA2)-亮氨酸脑啡肽，所述分化指向神经元样细胞分化。
一种小分子活性肽在制备体外诱导hUC-MSCs向神经元样细胞分化的培养基方面方面的应用，所述小分子活性肽为(D-ALA2)-亮氨酸脑啡肽。
技术效果：
本发明发现，小分子活性肽(D-ALA2)-亮氨酸脑啡肽具有诱导hUCMSCs向神经元样细胞分化的活性，且具有浓度效应。因此，(D-ALA2)-亮氨酸脑啡肽可以用于制备诱导hUCMSCs向神经元样细胞分化的培养基。
附图说明
图1为人脐带间充质干细胞倒置显微镜观察结果；可见细胞呈梭形，呈漩涡状生长，符合人脐带间充质干细胞的生长特点。
图2为各组继续培养8d后倒置显微镜下观察结果；可见含有20μM或50μM小分子活性肽的加药诱导组可见明显的神经元样细胞，而正常对照组不明显。
图3为Westernblot检测结果；与正常对照组相比，含有20μM或50μM小分子活性肽的加药诱导组NSE蛋白显著升高，且小分子活性肽的浓度越高NSE蛋白含量越高。
具体实施方式
一、实验材料
人脐带间充质干细胞购自上海传秋生物科技有限公司。
胎牛血清、DMEM/F12(1:1)培养基购自购自美国Gibco公司。
小分子活性肽为(D-ALA2)-亮氨酸脑啡肽，序列为H-Tyr-D-Ala-Gly-Phe-Leu-OH(CAS号：64963-01-5)，购自源叶生物，纯度99％。
二、实验方法
1、人脐带间充质干细胞的培养、传代、形态观察和表型检测
将冻存的人脐带间充质干细胞按常规方法复苏后，用含10％胎牛血清的DMEM/F12培养基培养于T25培养瓶中，培养条件为37℃、5％CO2、饱和湿度。2～3d换一次液，待细胞长满瓶底面积80％～90％时传代。传代比例为1:5，步骤为：将0.25％胰酶-0.53mM EDTA消化液与DPBS置于37℃预热，倒掉培养瓶中的培养基，往培养瓶中加入3-5mLDPBS，轻晃洗涤后弃去。使用DPBS将胰酶稀释4倍，往瓶中加2mL稀释后的胰酶，置于室温孵育消化，消化好后加入2mL含血清的完全培养基终止消化。用移液枪轻轻吹打瓶壁上的细胞，使之完全脱落，然后收集细胞悬液并吹打成单个细胞悬液，1200rpm离心5min，弃上清，加入完全培养基重悬细胞，进行传代。
取传代后生长状态良好的人脐带间充质干细胞用于后续实验。
形态观察：在倒置显微镜下观察人脐带间充质干细胞的形态和生长情况，拍照。
表型检测：将人脐带间充质干细胞用0.25％胰酶消化，PBS重悬，制备1×106/mL的细胞悬液。然后取100μL细胞悬液与20μL标记一抗(CD73-PE、CD90-FITC、CD105-APC、CD34-PE、CD45-APC、HLA-DR-FITC)4℃避光孵育1h，上流式细胞仪检测细胞表型分子阳性率。
2、神经元样细胞分化检测
取生长状态良好的hUCMSCs，用含10％胎牛血清的DMEM/F12培养基制成细胞悬液，按每孔2×105个细胞接种于24孔培养板中，37℃、5％CO2、饱和湿度培养24h后，加药诱导组更换为含10％胎牛血清和20μM或50μM小分子活性肽的DMEM/F12培养基继续培养，正常对照组更换为含10％胎牛血清的DMEM/F12培养基继续培养，每2～3d更换一次对应的培养基。继续培养8d后，倒置显微镜下观察细胞形态。收集正常对照组和加药诱导组继续培养10d的细胞，PBS洗涤，裂解液中裂解，使用BCA法测量总蛋白。将蛋白样本通过SDS-PAGE凝胶电泳分离等量的总蛋白，转移到聚偏二氟乙烯膜上，用含5％脱脂牛奶的封闭溶液封闭，加入抗NSE(神经元特异性烯醇化酶)、β-Actin一抗，4℃摇床过夜，加入HRP标记的二抗室温孵育1h，化学发光法显色，凝胶成像仪采集图像。
3、统计学处理
数据采用GraphPad Prism 5.0软件进行统计处理，以x±s表示，两组间比较采用t检验，P＜0.05表示具有显著性差异。
三、实验结果
1、人脐带间充质干细胞形态观察和表型检测结果
倒置显微镜观察结果如图1所示，细胞呈梭形，呈漩涡状生长，符合人脐带间充质干细胞的生长特点。表型检测结果如表1所示，CD34、CD45、HLA-DR表达水平很低，CD73、CD90、CD105表达水平很高，符合人脐带间充质干细胞的表型特点。
表1细胞表型分子阳性率
细胞表型分子阳性率 阳性率(％)
CD73 98.5
CD90 98.8
CD105 98.2
CD34 2.73
CD45 1.69
HLA-DR 0.87
2、神经元样细胞分化检测
各组继续培养8d后，倒置显微镜下观察结果如图2所示，含有20μM或50μM小分子活性肽的加药诱导组可见明显的神经元样细胞，说明加药诱导组hUCMSCs向神经元样细胞分化，且小分子活性肽的浓度越高分化越明显，而正常对照组不明显。
Western blot检测结果如图3所示，与正常对照组相比，含有20μM或50μM小分子活性肽的加药诱导组NSE蛋白显著升高，且小分子活性肽的浓度越高NSE蛋白含量越高。NSE为神经元标志蛋白，常作为反应神经元样细胞分化水平的标志。
综合细胞形态观察和Western blot检测结果可知，小分子活性肽(D-ALA2)-亮氨酸脑啡肽具有诱导hUCMSCs向神经元样细胞分化的活性，且具有浓度效应。因此，(D-ALA2)-亮氨酸脑啡肽可以用于制备诱导hUCMSCs向神经元样细胞分化的培养基。

Claims (2)

1.一种小分子活性肽在体外诱导hUC-MSCs分化方面的应用，其中：所述小分子活性肽为(D-ALA2)-亮氨酸脑啡肽，所述分化指向神经元样细胞分化。
2.一种小分子活性肽在制备体外诱导hUC-MSCs向神经元样细胞分化的培养基方面方面的应用，所述小分子活性肽为(D-ALA2)-亮氨酸脑啡肽。
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