CN110819587A - 一种间充质干细胞无支架三维凝胶干性鉴定方法 - Google Patents

一种间充质干细胞无支架三维凝胶干性鉴定方法 Download PDF

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CN110819587A
CN110819587A CN201911188845.7A CN201911188845A CN110819587A CN 110819587 A CN110819587 A CN 110819587A CN 201911188845 A CN201911188845 A CN 201911188845A CN 110819587 A CN110819587 A CN 110819587A
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mesenchymal stem
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张剑慧
谭毅
马贺然
张慧慧
韩镇
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Shandong Qilu Cell Therapy Engineering Technology Co Ltd
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Abstract

本发明涉及干细胞和再生医学领域，具体涉及一种间充质干细胞无支架三维凝胶干性研究方法。间充质干细胞经过凝胶诱导培养7天获得间充质干细胞无支架三维凝胶，分别接种于成脂、成骨或成软骨诱导培养基中诱导培养14天，每72‑96h换液，培养环境为37℃、5%CO2培养箱，分化诱导后油红O进行成脂染色鉴定、茜素红进行成骨鉴定、阿利新蓝进行成软骨染色鉴定。本发明提供了一种验证药物诱导获得的间充质干细胞凝胶仍保持间充质干细胞多向分化能力具有干性的方法。本发明的方法对脐带、脂肪、骨髓、脐血来源的间充质干细胞凝胶干性均可验证。

Description

一种间充质干细胞无支架三维凝胶干性鉴定方法
技术领域
本发明涉及干细胞和再生医学领域，具体涉及一种间充质干细胞无支架三维凝胶干性研究方法。
背景技术
间充质干细胞是存在于体内多种组织中的一种具有自我更新和多向分化潜能的多能干细胞。由于具有容易分离扩增、自我更新、多向分化和低免疫原性等特性，而成为了目前组织工程和再生医学中理想的种子细胞，为一些传统药物治疗困难的疾病带来新希望。
对工程软骨移植术来讲，选择合适的间充质干细胞为种子细胞，建立恰当的细胞增殖和分化培养体系构建生物工程软骨，是治疗关节损伤的关键步骤。许多研究都将焦点集中到寻找合适的培养骨架上，而这些骨架往往是合成高分子材料或者生物材料，而其在人体内长期存在，对机体是否有不良作用还不明朗。目前，几种骨架材料已经进入临床应用，而其长期存在体内的安全性仍然需要跟踪。除此之外，关节软骨细胞由于其独特的矩阵结构，使软骨具有抗粘附的特性，此特性也成为临床中工程软骨移植治疗软骨损伤的一个难点。为了克服这一难点，临床上通过用酶处理软骨基质表面增加其粘着性，或者通过缝合将移植组织强制固定，或者用可溶性针将其固定。然而，这些方式都会对软骨造成二次损伤，并有动物实验证明这些缝合处的软骨并不会自动愈合，并且会引起周围基质组织的退行降解。因此，移植组织对软骨的粘附能力成为治疗软骨损伤的一个重要部分。
一种间充质干细胞无支架三维凝胶可以用于替代组织工程软骨，并可以很好解决上述问题。然而，凝胶中的间充质干细胞是否仍具有干细胞的特性成为重点。如何研究间充质干细胞凝胶中间充质干细胞的干性成为本发明主要解决的问题。
发明内容
本发明的目的在于提供一种间充质干细胞凝胶干性的鉴定方法，凝胶干性研究方法主要研究其中间充质干细胞的成脂、成骨、成软骨分化能力。
为实现上述发明目的，本发明采用以下技术方案：
采用如下步骤：
间充质干细胞经过凝胶诱导培养7天获得间充质干细胞无支架三维凝胶，分别接种于成脂、成骨和成软骨诱导培养基中诱导培养14天，每三天换液，培养环境为37℃、5% CO2培养箱。
进一步的，所述的染色鉴定为：采用油红O进行成脂分化鉴定，当细胞内油滴被染为亮红色时为间充质干细胞保持向脂肪细胞分化能力；采用茜素红进行成骨分化鉴定，当钙沉淀被染为红色或产生钙结节时为间充质干细胞能够诱导分化为骨细胞；采用阿利辛蓝染色鉴定进行成软骨分化鉴定，当软骨细胞微球被染为亮蓝色时为间充质干细胞保持分化为软骨细胞的能力。
进一步的，间充质干细胞在上述2-3种染色成阳性反应时，说明间充质干细胞保持干性。
进一步的，间充质干细胞的来源为脐带、脂肪、骨髓或脐血；
进一步的，成脂诱导培养基配方为含5% ultroGRO +10mg/mL 胰岛素 + 1µM 地塞米松+ 100µM 吲哚美辛 + 500µM IBMX的达优MSCBM培养基；
进一步的，所述成骨诱导培养基的配方为含5% ultroGRO +0.1µM 地塞米松 + 50µM抗坏血酸 + 10mM β-甘油磷酸的达优MSCBM培养基；
进一步的，成软骨诱导培养基的配方为含110µg/mL丙酮酸钠+ 10ng/mL TGF-β3 + 0.1µM地塞米松+ 50µg/mL L-抗坏血酸-2-磷酸盐+ 40µg/mL L-脯氨酸+ 1%ITS+Premix 100*的达优MSCBM培养基。
有益效果
本发明提供了一种验证药物诱导获得的间充质干细胞凝胶仍保持间充质干细胞多向分化能力具有干性的方法。本发明的方法对脐带、脂肪、骨髓、脐血来源的间充质干细胞凝胶干性均可验证。
附图说明
图1为间充质干细胞三维凝胶分别进行成脂、成骨、成软骨分化诱导后油红O、茜素红、阿利新蓝染色鉴定。
具体实施方式
为了进一步了解本发明的特征、技术手段以及所达到的具体目的、功能，解析本发明的优点和精神，通过下面的实施例和对比例对本发明的上述内容在作进一步的详细说明。但是，不应该将此理解为本发明上述主体范围仅仅局限于以下实施例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1
1、脂肪间充质干细胞无支架三维凝胶诱导培养：脂肪组织去除筋膜，清洗掉血细胞，向含有脂肪组织的离心管中以体积比1:1的比例加入浓度为0.1%的I型胶原酶/PBS消化液中，以90rpm振荡消化60min；用移液管吸出离心管中的下层液体，通过100µm滤网过滤到一个新的50mL离心管中，离心收集基质细胞。以3×106个细胞/mL的密度接种在含5%ultroGRO无血清添加剂的MSCBM培养基中，将培养瓶放入37℃ 5%CO2培养箱中；培养最初3-4d，保持培养瓶物理静止。第4天后观察细胞贴壁情况对细胞进行换液，以后每周两次。待细胞生长至约80%汇合时进行细胞传代培养。获得P5代以内的脂肪间充质干细胞，调整间充质干细胞铺板密度为4×105/cm2 ，6cm培养皿的底面积为21 cm2，共8.2×106个细胞，接种于6cm培养皿中。添加含5%ultroGRO血清替代品、200uM ASC2P的达优MSCBM培养基的凝胶诱导培养基，于37℃、5% CO2培养箱，培养7天获得水凝胶养无支架三维凝胶。
2、脂肪间充质干细胞凝胶成脂、成骨、成软骨诱导：将培养7天的脂肪间充质干细胞培养液换为成脂、成骨、成软骨诱导培养基，分别进行诱导培养，37℃ 5%CO2培养箱中培养14天，成脂诱导培养基配方为含5% ultroGRO +10mg/mL 胰岛素 + 1µM 地塞米松 + 100µM 吲哚美辛 + 500 µM IBMX的达优MSCBM培养基；成骨诱导培养基的配方为含5%ultroGRO+0.1µM 地塞米松 + 50µM抗坏血酸 + 10mM β-甘油磷酸的达优MSCBM培养基；成软骨诱导培养基的配方为含110µg/mL丙酮酸钠+ 10ng/mL TGFβ-3 + 0.1µM地塞米松+ 50µg/mL L-抗坏血酸-2-磷酸盐+ 40µg/mL L-脯氨酸+ 1%ITS+Premix 100*的达优MSCBM培养基。
3、诱导结果鉴定：将获得的三维凝胶分别进行成脂、成骨、成软骨分化诱导，切片进行染色鉴定，对照组a1、b1、 c1均为未经分化诱导的间充质干细胞凝胶。油红O进行成脂分化鉴定，图A.对照组a1只有少量油滴，诱导分化组a2脂肪细胞中的油滴被染成亮红色；茜素红进行成骨分化鉴定，图B.对照组b1未见向骨细胞分化，诱导分化组b2产生的钙沉积被染成红色；阿利辛蓝染色鉴定进行成软骨分化鉴定，图C.对照组c1阿利新蓝染色未着色，说明未见向软骨分化，诱导分化组c2被阿利新蓝染成亮蓝色。经鉴定脂肪间充质干细胞在体外能成功诱导成功分化为脂肪、骨和软骨细胞，经过凝胶诱导脂肪间充质干细胞仍具有分化能力，保持干性。

Claims (7)

1.一种间充质干细胞凝胶干性的鉴定方法，其特征在于，采用如下步骤：
间充质干细胞经过凝胶诱导培养7天获得间充质干细胞无支架三维凝胶，分别接种于成脂、成骨和成软骨诱导培养基中诱导培养14天，每72-96h换液，培养环境为37℃、5% CO2培养箱，分化诱导后油红O进行成脂染色鉴定、茜素红进行成骨鉴定、阿利新蓝进行成软骨染色鉴定。
2.根据权利要求1所述的鉴定方法，其特征在于，所述油红O进行成脂染色鉴定时当细胞内油滴被油红O染为亮红色时为间充质干细胞保持向脂肪细胞分化能力；所述茜素红进行成骨鉴定时当钙沉淀被茜素红染为红色时为间充质干细胞保持分化为骨细胞的能力；所述阿利新蓝进行成软骨染色鉴定时当软骨细胞微球被阿利辛蓝染为亮蓝色时为间充质干细胞保持分化为软骨细胞的能力。
3.根据权利要求1或2所述的鉴定方法，其特征在于，间充质干细胞在上述2-3种染色成阳性反应时，说明间充质干细胞保持干性。
4.根据权利要求1-3任一项所述的培养方法，其特征在于，所述间充质干细胞的来源为脐带、脂肪、骨髓或脐血。
5.根据权利要求1所述的培养方法，其特征在于，所述成脂诱导培养基配方为含5%ultroGRO+10mg/mL 胰岛素+ 1µM 地塞米松 + 100µM 吲哚美辛 + 500µM IBMX的达优MSCBM培养基。
6.根据权利要求1所述的培养方法，其特征在于，所述成骨诱导培养基的配方为含5%ultroGRO +0.1µM 地塞米松 + 50µM抗坏血酸 + 10mM β-甘油磷酸的达优MSCBM培养基。
7.根据权利要求1所述的培养方法，其特征在于，所述成软骨诱导培养基的配方为含110µg/mL丙酮酸钠+ 10ng/mL TGF-β3 + 0.1µM地塞米松+ 50µg/mL L-抗坏血酸-2-磷酸盐+ 40µg/mL L-脯氨酸+ 1%ITS+Premix 100*的达优MSCBM培养基。
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