CN107418928B - 一种人间充质干细胞成骨诱导分化培养基及制备方法 - Google Patents
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Abstract
本发明涉及干细胞技术领域,具体涉及一种人间充质干细胞成骨诱导分化培养基及制备方法,所述人间充质干细胞成骨诱导分化培养基包括α‑MEM/HG‑DMEM培养基,还包括如下组分及其浓度:胎牛血清5‑50%体积百分比,抗坏血酸10‑100μM,磷酸甘油5‑50mM,地塞米松50‑500nM,胰岛素生长因子‑10.1‑10nM和胰岛素生长因子‑20.5‑50nM;本发明通过胰岛素生长因子‑1和胰岛素生长因子‑2增强间充质干细胞成骨分化信号活化,促进成骨细胞增殖,从而提高人间充质干细胞成骨分化效率及特异性,缩短分化时间,提高诱导效率,同时制备方法简便,使用方便,可实现稳定、高效的人间充质干细胞成骨诱导分化。
Description
技术领域
本发明涉及干细胞技术领域,具体涉及一种人间充质干细胞成骨诱导分化培养基及制备方法。
背景技术
间充质干细胞(mesenchymal stem cell,MSCs)是一类多能干细胞,最早发现于骨髓中,主要参与造血干细胞的定向归巢、功能发挥及稳态的维持,近年来间充质干细胞已被发现存在于多种组织中,如脐带、脂肪、牙髓等组织中,分离及扩增简便。大量研究证明,间充质干细胞具有很好的自我更新及多向分化潜能,在特定条件下可诱导分化成为脂肪细胞、骨细胞、软骨细胞、肌细胞、肝脏细胞、神经细胞、皮肤细胞等多种细胞类型,因此,间充质干细胞已被尝试应用于多种组织及器官功能损伤修复研究中,具有极大的临床应用潜能。其中基于间充质干细胞定向成骨分化特性的骨组织再造修复应用备受临床关注,已在动物及临床前期研究中取得令人满意的疗效。另外,近期国内外研究还发现间充质干细胞除具有很好的免疫调节特性外,还可被用于关节炎,骨损伤所造成的局部及系统性过度免疫反应及重度炎症的控制与缓解,临床转化及应用市场巨大。
间充质干细胞作为种子细胞,可通过自身分化进行以骨为代表的特定组织替代修复,但在自然条件下,分化效率有限。因此,为获得足量的间充质干细胞来源的成骨细胞进行临床应用,需通过给予特定培养条件,进行分化诱导,从而在短期内高效定向获得充足且均一的骨组织细胞进行损伤修复。现有人间充质干细胞成骨诱导分化培养基成分一般为:DMEM/F12培养基,10%胎牛血清(FBS),1%谷氨酰胺,100μM抗坏血酸,10mMβ-磷酸甘油,以及50nM地塞米松。
中国专利CN 102041245A公开了一种间充质干细胞成骨诱导培养基及使用方法。该培养基在α-MEM培养基基础上,添加10%胎牛血清、50mM维生素C溶液、1Mβ-甘油磷酸钠溶液,以及0.1mM地塞米松。经4周诱导培养后进行成骨细胞染色鉴定。该培养基只针对骨髓来源间充质干细胞进行成骨诱导分化,诱导分化效率不理想,且定向诱导成骨分化的特异性不强,对人间充质干细胞诱导特性不明确。
综上所述,现有的成骨诱导分化培养基无法高效稳定诱导人多种组织来源间充质干细胞向成骨细胞分化,仍需更为优化的分化条件提高人间充质干细胞成骨诱导分化效率,缩短诱导分化时间,从而为间充质干细胞成骨分化能力鉴定及临床治疗骨损伤提供有利条件。
发明内容
针对现有技术的不足,本发明提供了一种人间充质干细胞成骨诱导分化培养基及制备方法,高效稳定诱导多种人组织来源间充质干细胞定向成骨分化,提高成骨诱导效率,缩短诱导时间。
为实现以上目的,本发明通过以下技术方案予以实现:
一种人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清5-50%体积百分比,抗坏血酸10-100μM,磷酸甘油5-50mM,地塞米松50-500nM,胰岛素生长因子-10.1-10nM和胰岛素生长因子-20.5-50nM。
进一步的,所述人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清5-20%体积百分比,抗坏血酸10-80μM,磷酸甘油5-20mM,地塞米松50-200nM,胰岛素生长因子-10.1-5nM和胰岛素生长因子-20.5-10nM。
进一步的,所述人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清10%体积百分比,抗坏血酸500μM,磷酸甘油10mM,地塞米松100nM,胰岛素生长因子-11nM和胰岛素生长因子-22nM。
进一步的,所述磷酸甘油为β-磷酸甘油。
进一步的,分化方法包括如下步骤:使用含10%胎牛血清的低糖DMEM培养基培养人间充质干细胞,当人间充质干细胞融合度达到70-90%时,弃去低糖DMEM培养基,加入所述人间充质干细胞成骨高效诱导分化培养基,培养3-5周。
进一步的,所述人间充质干细胞选自骨髓间充质干细胞、脐带间充质干细胞或脂肪间充质干细胞。
上述人间充质干细胞成骨诱导分化培养基的制备方法,包括如下步骤:向α-MEM/HG-DMEM培养基中,按浓度依次加入胎牛血清、抗坏血酸、β-磷酸甘油、地塞米松、胰岛素生长因子-1和胰岛素生长因子-2,混合均匀,0.22μm滤膜过滤除菌即得。
由于采用上述的技术方案,本发明的有益如下:
现有成骨诱导分化培养基中的地塞米松可通过活化WNT/β-catenin信号通路上调Runx2分子磷酸化从而诱导人间充质干细胞向成骨细胞分化;抗坏血酸可促进间充质干细胞产生I型胶原,形成成骨钙化所需细胞外基质结构;β-磷酸甘油则作为细胞磷酸盐来源,辅助促进成骨分化相关重要基因磷酸化,激活下游成骨分化途径5。本发明在现有成骨诱导分化培养基成分中添加胰岛素生长因子-1(IGF-1)和胰岛素生长因子-2(IGF-2)。IGF-1通过活化MAPK/PI3K以及mTOR信号通路,从而特异性促进成骨细胞增殖;IGF-2可进一步上调成骨细胞中Akt2蛋白表达,正向调控BMP-2信号通路活化,促进间充质干细胞向成骨细胞分化使与成骨效应密切相关骨桥蛋白(OPN)表达量增高。本发明中所使用IGF-1和IGF-2通过增强间充质干细胞成骨分化信号活化,促进成骨细胞增殖,从而提高人间充质干细胞成骨分化效率及特异性,缩短分化时间。
本发明提供的人间充质干细胞成骨诱导分化培养基,可实现包括骨髓间充质干细胞、脐带间充质干细胞以及脂肪间充质干细胞在内的多种组织来源人间充质干细胞向成骨细胞的诱导分化,成骨定向分化特异性高,专一性强。与现有技术诱导人间充质干细胞成骨分化所需4周相比,本发明提供的人间充质干细胞成骨诱导分化培养基可在使用3周后,使间充质干细胞出现大量成骨钙结节,同时,使与成骨效应密切相关骨桥蛋白(OPN)基因表达水平显著上调,从而缩短人间充质干细胞成骨分化诱导时间,提高诱导效率。此外,本发明提供的人间充质干细胞成骨诱导分化培养基制备方法简便,使用方便,可实现稳定、高效的人间充质干细胞成骨诱导分化。
附图说明
图1是不同的人间充质干细胞成骨诱导分化培养基在诱导3周时对成骨诱导分化相关基因OPN的表达影响;
图2是不同的人间充质干细胞成骨诱导分化培养基在诱导4周时对成骨诱导分化相关基因OPN的表达影响;
图3是P3代人骨髓间充质干细胞在本发明成骨诱导分化培养基中诱导4周时成骨诱导分化相关基因OPN的表达水平;
图4是P3代人骨髓间充质干细胞在本发明成骨诱导分化培养基中诱导4周时成骨诱导分化茜素红染色结果;
图5是P3代人脐带间充质干细胞在本发明成骨诱导分化培养基中诱导4周时成骨诱导分化相关基因OPN的表达水平;
图6是P3代人脐带间充质干细胞在本发明成骨诱导分化培养基中诱导4周时成骨诱导分化茜素红染色结果;
图7是P3代人脂肪间充质干细胞在本发明成骨诱导分化培养基中诱导4周时成骨诱导分化相关基因OPN的表达水平;
图8是P3代人脂肪间充质干细胞在本发明成骨诱导分化培养基中诱导4周时成骨诱导分化茜素红染色结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,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作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。
实施例1:诱导分化培养基1
一种人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清10%体积百分比,抗坏血酸50μM,磷酸甘油10mM,地塞米松100nM,胰岛素生长因子-11nM和胰岛素生长因子-22nM。
制备方法包括以下步骤:
抗坏血酸配制:取10g抗坏血酸,用α-MEM培养基溶解,配成5mM的母液,-20℃保存。β-磷酸甘油配制:取10gβ-磷酸甘油,用α-MEM培养基溶解,配成1M的母液,-20℃保存。地塞米松配制:取10g地塞米松,用95%乙醇溶解,配成100μm母液,-20℃保存。IGF-1配制:取1mgIGF-1,用α-MEM培养基溶解,配成1μM的母液,-20℃保存。IGF-2配制:取2mg IGF-1,用α-MEM培养基溶解,配成2μM的母液,-20℃保存。
向α-MEM/HG-DMEM培养基中,按浓度依次加入胎牛血清、抗坏血酸、β-磷酸甘油、地塞米松、胰岛素生长因子-1和胰岛素生长因子-2,混合均匀,0.22μm滤膜过滤除菌即得。
实施例2:诱导分化培养基2
一种人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清15%体积百分比,抗坏血酸80μM,磷酸甘油20mM,地塞米松100nM,胰岛素生长因子-15nM和胰岛素生长因子-20.5nM。
制备方法同实施例1。
实施例3:诱导分化培养基3
一种人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清10%体积百分比,抗坏血酸50μM,磷酸甘油10mM,地塞米松150nM,胰岛素生长因子-10.5nM和胰岛素生长因子-210nM。
制备方法同实施例1。
实施例4:诱导分化培养基4
与诱导分化培养基1相比,诱导分化培养基4不包含胰岛素生长因子-1。
实施例5:诱导分化培养基5
与诱导分化培养基1相比,诱导分化培养基5不包含胰岛素生长因子-2。
实施例6:诱导分化培养基6
与诱导分化培养基1相比,诱导分化培养基6不包含胰岛素生长因子-1和胰岛素生长因子-2。
实施例7:
一种人间充质干细胞成骨诱导分化培养基,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清5%体积百分比,抗坏血酸10μM,磷酸甘油5mM,地塞米松50nM,胰岛素生长因子-10.1nM和胰岛素生长因子-20.5nM。
制备方法同实施例1。
实施例8:
一种人间充质干细胞成骨诱导分化培养基,其特征在于,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清50%体积百分比,抗坏血酸100μM,磷酸甘油50mM,地塞米松500nM,胰岛素生长因子-110nM和胰岛素生长因子-250nM。
制备方法同实施例1。
将2×105/孔的P3代人骨髓间充质干细胞接种于十二孔板中,用含10%(v/v)FBS低糖DMEM培养基培养至细胞融合度达80-90%,弃去培养基,分别加入诱导分化培养基1、诱导分化培养基2、诱导分化培养基3、诱导分化培养基4、诱导分化培养基5,和诱导分化培养基6,每3天换液一次。分别在诱导3周和4周时,利用realtime PCR检测成骨细胞相关基因OPN(骨桥蛋白)表达水平。结果如图1和图2所示,通过在现有人间充质干细胞成骨诱导分化培养基中同时添加一定浓度的胰岛素生长因子-1(IGF-1)和胰岛素生长因子-2(IGF-2),无论是在诱导培养后3周还是4周,都可显著上调成骨相关基因OPN的表达水平(与诱导分化培养基2、诱导分化培养基3、诱导分化培养基4、诱导分化培养基5,和诱导分化培养基6相比,p<0.01或p<0.05)。
将2×105/孔的P3代人骨髓间充质干细胞接种于十二孔板中,用含10%(v/v)FBS低糖DMEM培养基培养至细胞融合度达80-90%,弃去培养基,分别加入诱导分化培养基1和诱导分化培养基7(Thermo Fisher Scientific公司的人间充质干细胞成骨诱导分化培养基,货号A10072),每3天换液一次。诱导4周后,用realtime PCR检测成骨细胞相关基因OPN的表达水平,如图3所示本发明中的人间充质干细胞成骨诱导分化培养基所诱导分化细胞中的成骨相关基因OPN的表达水平更高(与诱导分化培养基7作用相比,p<0.01);同时利用茜素红染色对成骨程度进行鉴定,结果如图4所示,本发明中的人间充质干细胞成骨诱导分化培养基所诱导分化细胞具有更为丰富成骨钙结节(与诱导分化培养基7作用相比)。上述结果说明本发明中的人间充质干细胞成骨诱导分化培养基针对人骨髓间充质干细胞具有更好的成骨诱导分化效果。
将2×105/孔的P3代人脐带间充质干细胞接种于十二孔板中,用含10%(v/v)FBS低糖DMEM培养基培养至细胞融合度达80-90%,弃去培养基,分别加入诱导分化培养基1和诱导分化培养基7,每3天换液一次。诱导4周后,用realtime PCR检测成骨细胞相关基因OPN的表达水平,如图5所示本发明中的人间充质干细胞所诱导分化细胞中的成骨相关基因OPN的表达水平更高(与诱导分化培养基7作用相比,p<0.01);同时利用茜素红染色对成骨程度进行鉴定,结果如图6所示,本发明中的人间充质干细胞成骨诱导分化培养基所诱导分化细胞具有更为丰富成骨钙结节(与诱导分化培养基7作用相比)。上述结果说明本发明中的人间充质干细胞成骨诱导分化培养基针对人脐带间充质干细胞具有更好的成骨诱导分化效果。
将2×105/孔的P3代人脂肪间充质干细胞接种于十二孔板中,用含10%(v/v)FBS低糖DMEM培养基培养至细胞融合度达80-90%,弃去培养基,分别加入诱导分化培养基1和诱导分化培养基7,每3天换液一次。诱导4周后,用realtime PCR检测成骨细胞相关基因OPN的表达水平,如图7所示本发明中的人间充质干细胞培养基所诱导分化细胞中的成骨相关基因OPN的表达水平更高(与诱导分化培养基7作用相比,p<0.01);同时利用茜素红染色对成骨程度进行鉴定,结果如图8所示,本发明中的人间充质干细胞成骨诱导分化培养基所诱导分化细胞具有更为丰富成骨钙结节(与诱导分化培养基7作用相比)。上述结果说明本发明中的人间充质干细胞成骨诱导分化培养基针对人脂肪间充质干细胞具有更好的成骨诱导分化效果。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (5)
1.一种人间充质干细胞成骨诱导分化培养基,其特征在于,包括α-MEM/HG-DMEM培养基,还包括如下组分及其浓度:胎牛血清10%体积百分比,抗坏血酸50μM,磷酸甘油10mM,地塞米松100nM,胰岛素生长因子-1 1nM和胰岛素生长因子-2 2nM。
2.如权利要求1所述的人间充质干细胞成骨诱导分化培养基,其特征在于,所述磷酸甘油为β-磷酸甘油。
3.如权利要求1-2任一项所述的人间充质干细胞成骨诱导分化培养基,其特征在于,分化方法包括如下步骤:使用含10%胎牛血清的低糖DMEM培养基培养人间充质干细胞,当人间充质干细胞融合度达到70-90%时,弃去低糖DMEM培养基,加入所述人间充质干细胞成骨高效诱导分化培养基,培养3-5周。
4.如权利要求3所述的人间充质干细胞成骨诱导分化培养基,其特征在于,所述人间充质干细胞选自骨髓间充质干细胞、脐带间充质干细胞或脂肪间充质干细胞。
5.如权利要求1-2任一项所述的人间充质干细胞成骨诱导分化培养基的制备方法,其特征在于,包括如下步骤:向α-MEM/HG-DMEM培养基中,按浓度依次加入胎牛血清、抗坏血酸、β-磷酸甘油、地塞米松、胰岛素生长因子-1和胰岛素生长因子-2,混合均匀,0.22μm滤膜过滤除菌即得。
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