CN114716511A - 一种多肽及其促进脐带MSCs增殖、制备脐带MSCs培养基的用途 - Google Patents
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Abstract
本发明公开了一种多肽及其促进脐带MSCs增殖、制备脐带MSCs培养基的用途。本发明提供了一种氨基酸序列为VFGWMTED的多肽，该多肽在体外可以有效促进脐带MSCs增殖，具有开发成脐带MSCs增殖培养基的应用前景。
Description
技术领域
本发明属于生物化学领域，涉及多肽及干细胞培养，具体涉及一种多肽及其促进脐带MSCs增殖、制备脐带MSCs培养基的用途。
背景技术
近年来，干细胞治疗逐渐成为一个前景十分广阔的先进科学课题，干细胞临床治疗方法的发展引起了人们的极大期待。由于多种机制的共同作用，间充质干细胞(MSCs)移植被认为是最有效的细胞治疗工具之一，影响受损组织再生的各个阶段。脐带MSCs是指存在于新生儿脐带组织中的一种多功能干细胞，因其来源丰富而具有广阔的临床应用前景。如何促进脐带MSCs增殖是脐带MSCs研究的重要方向。
多肽是氨基酸连结成的化合物，由肽键相连，通常指的是由三个或三个以上氨基酸组成的化合物。多肽可包括活性多肽和人工合成多肽，活性多肽作为蛋白质水解产物，具有一定生理作用。人工合成多肽可以根据需要自由控制氨基酸连接顺序，为构建多肽库提供了可能，成为活性筛选的重要来源。
多肽结构和活性的多样性为探索发现促进脐带MSCs增殖的化学物质提供了丰富的机会。
发明内容
本发明目的是克服现有技术的不足，提供一种多肽及其促进脐带MSCs增殖、制备脐带MSCs培养基的用途。
本发明目的通过下述技术方案得以实现：
一种氨基酸序列为VFGWMTED的多肽。
上述多肽在促进人脐带间充质干细胞体外增殖中的应用。
上述多肽在制备促进人脐带间充质干细胞体外增殖的培养基中的应用。
有益效果：
本发明提供了一种氨基酸序列为VFGWMTED的多肽，该多肽在体外可以有效促进脐带MSCs增殖，具有开发成脐带MSCs增殖培养基的应用前景。
附图说明
图1为多肽的液相检测图；保留时间16.537min，HPLC纯度97.516％。
图2中A为脐带MSCs的倒置显微镜观察形态，呈长梭形；B为流式检测结果，高表达CD73、CD90、CD105、HLA-DR，低表达CD34和CD45；结果均符合脐带MSCs特点。
图3为western blot检测结果，多肽组脐带MSCs的干细胞特性蛋白SOX2、OCT4的表达水平与对照组无明显差异。
具体实施方式
以下实施例仅用于具体介绍本发明的实质内容，但不以此限定本发明的保护范围。
一、实验材料
序列为VFGWMTED(Sequence NO.1)的多肽K由南京肽谷生物科技有限公司采用常规固相合成法合成。多肽K的分子式为C45H61N9O14S，分子量为984.08，质谱检测到分子离子峰[M+H]+985.1。HPLC纯度97.516％，见图1。
DMEM/F12培养基和胎牛血清，GIBCO公司。MTT试剂和单抗，碧云天。
二、实验方法
1、脐带MSCs分离培养和表型鉴定
无菌条件下采用传统贴壁法从正常分娩产妇的脐带中分离培养脐带MSCs，具体步骤为：将脐带剪成3-5cm小段，PBS洗去残存血液，剔除静脉和动脉，剪成0.5-1.0mm3大小组织块，均匀平铺于培养瓶中，置于37℃、5％CO2、饱和湿度条件约1～2h待组织块紧密贴壁于培养瓶内壁后，加入含10％胎牛血清和1％青链霉素的DMEM/F12培养基于37℃、5％CO2、饱和湿度条件培养，5～7d换液1次，待长至80％-90％融合时消化传代。选取第5代脐带MSCs，消化，PBS洗涤、离心、重悬，取少量于倒置显微镜下观察细胞形态，取适量分装于EP管中，分别加入5μL CD34、CD45、CD73、CD90、CD105单克隆抗体及同型对照，4℃孵育30min，PBS洗涤、离心、重悬细胞，上流式细胞仪检测，鉴定表型。
2、MTT法检测脐带MSCs的体外增殖活性
将生长状态良好的第5代脐带MSCs消化重悬后按5×103细胞/孔接种于96孔培养板中，分为对照组、低浓度多肽组和高浓度多肽组，每组6个孔。培养24h后，更换培养基继续培养：对照组更换为新鲜的含10％胎牛血清和1％青链霉素的DMEM/F12培养基，低浓度多肽组更换为含10％胎牛血清、1％青链霉素和10μg/mL多肽K的DMEM/F12培养基，高浓度多肽组更换为含10％胎牛血清、1％青链霉素和20μg/mL多肽K的DMEM/F12培养基。继续培养48、72h后分别取出一块培养板，每孔加入20μL 5mg/mL MTT溶液，继续培养4h后，弃上清液，每孔加入150μL DMSO，振荡5min，自动酶标仪系统上测定波长490nm处吸光度值。
3、干细胞特性检测
将生长状态良好的脐带MSCs消化重悬后按3×104细胞/cm2的细胞密度接种于25cm2细胞培养瓶中，培养24h后，更换为含10％胎牛血清、1％青链霉素和20μg/mL多肽K的DMEM/F12培养基。继续培养72h后，消化收集细胞，PBS洗涤、离心、重悬细胞，并分装于EP管中，分别加入5μL CD34、CD45、CD73、CD90、CD105单克隆抗体及同型对照，4℃孵育30min，PBS洗涤、离心、重悬细胞，上流式细胞仪检测。
将生长状态良好的脐带MSCs消化重悬后按4×105/孔接种于6孔培养板中，分为对照组、低浓度多肽组和高浓度多肽组，每组2个孔。培养24h后，更换培养基继续培养：对照组更换为新鲜的含10％胎牛血清和1％青链霉素的DMEM/F12培养基，多肽组更换为含10％胎牛血清、1％青链霉素和10、20μg/mL多肽K的DMEM/F12培养基。继续培养72h后，消化收集细胞，PBS洗涤，裂解细胞，提取细胞总蛋白，采用BCA法测定蛋白浓度，在10％的十二烷基硫酸钠-聚丙烯酰胺凝胶中进行蛋白电泳分离，以β-actin作为内参，蛋白上样量为40μg。90V恒压流转膜，将凝胶蛋白转至硝酸纤维素(NC)膜上。将NC膜放入含5％脱脂奶粉的磷酸盐吐温缓冲液(PBST)溶液中室温封闭2h。然后分别加入稀释的SOX2、OCT4和β-actin一抗溶液，4℃孵育过夜。PBST溶液洗膜3次，每次10min，加入稀释的辣根过氧化物酶标记的二抗室温孵育2h。PBST溶液洗膜3次后加入ECL发光液进行显色、拍照分析。
4、统计学分析
应用SPSS19.0统计软件进行统计学分析，多组均数比较用单因素方差分析(ANOVA)，两两组间比较用独立样本t检验，P<0.05为差异有显著性意义。
三、实验结果
1、脐带MSCs分离培养和表型鉴定
倒置显微镜观察结果如图2中A所示，细胞形态呈长梭形；流式检测结果如图2中B所示，高表达CD73、CD90、CD105，低表达CD34和CD45，符合脐带MSCs特点。
2、体外增殖活性检测结果
各组吸光度值如表1所示，低浓度多肽组和高浓度多肽组脐带MSCs的增殖活性明显强于对照组(与对照组比差异有统计学意义，*P<0.05)，且多肽K浓度越高脐带MSCs增殖活性越强(与低浓度多肽组比差异有统计学意义，#P<0.05)。
表1各组吸光度值
3、干细胞特性检测结果
流式检测结果显示CD73、CD90、CD105表达率分别为99.17％、99.35％、98.08％，CD34、CD45表达率分别为2.73％、2.09％。干细胞特性蛋白检测结果如图3所示，多肽组脐带MSCs的干细胞特性蛋白SOX2、OCT4的表达水平与对照组无明显降低。该结果表明，脐带MSCs在多肽K干预下增殖活性提高的同时依然维持干细胞特性。
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<110> 陈飞
<120> 一种多肽及其促进脐带MSCs增殖、制备脐带MSCs培养基的用途
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<170> SIPOSequenceListing 1.0
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Val Phe Gly Trp Met Thr Glu Asp
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Claims (3)
1.一种氨基酸序列为VFGWMTED的多肽。
2.权利要求1所述的多肽在促进人脐带间充质干细胞体外增殖中的应用。
3.权利要求1所述的多肽在制备促进人脐带间充质干细胞体外增殖的培养基中的应用。
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- 2022-05-10 CN CN202210504830.2A patent/CN114716511A/zh active Pending
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Application publication date: 20220708 |