CN115595303A - 一种促进间充质干细胞分化的培养基及应用 - Google Patents
一种促进间充质干细胞分化的培养基及应用 Download PDFInfo
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- CN115595303A CN115595303A CN202211321466.2A CN202211321466A CN115595303A CN 115595303 A CN115595303 A CN 115595303A CN 202211321466 A CN202211321466 A CN 202211321466A CN 115595303 A CN115595303 A CN 115595303A
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Abstract
本发明公开了一种促进间充质干细胞分化的培养基及应用。所述培养基包括以下原料制备而成:基础培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β‑甘油磷酸钠、胰岛素、磺丁基‑β‑环糊精、甜菜碱、乙酸铟、谷胱甘肽,其中基础培养基为α‑MEM/HG‑DMEM培养基或者DMEM/F12培养基。本发明提供的促进间充质干细胞分化的培养基,配比合理,可显著促进包括人骨髓间充质干细胞、人脐带间充质干细胞、人脂肪间充质干细胞在内的多种人间充质干细胞向骨细胞的诱导分化,成骨定向分化特异性高,专一性强;且分化时间短,大幅度提高人间充质干细胞成骨分化的效率。
Description
技术领域
本发明属于干细胞培养技术领域,具体涉及一种促进间充质干细胞分化的培养基及应用。
背景技术
间充质干细胞是一类多能干细胞,具有强大的自我更新和多向分化的潜能,广泛存在于骨髓、脂肪、脐带等组织中,易于分离、培养、传代扩增,且多次传代扩增后仍具有干细胞特性,在特定条件下可诱导分化为脂肪细胞、骨细胞、神经细胞等多种细胞的能力。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。
中国专利CN103667182B公开了一种骨髓间充质干细胞在体外向成骨细胞分化的诱导方法及诱导培养基,诱导培养基的组成如下:地塞米松1×10-8mol/L,抗坏血酸50μmol/L,β-甘油磷酸钠10mmol/L,溶剂为所述骨片培养72~96小时的上清液,该诱导培养基只针对骨髓间充质干细胞在体外向成骨细胞分化的诱导,具有很大的局限性。
发明内容
本发明的目的在于克服现有技术的不足,提供一种促进间充质干细胞分化的培养基,能够诱导促进人骨髓间充质干细胞、人脐带间充质干细胞、人脂肪间充质干细胞多种人间充质干细胞分化为骨细胞,分化时间短。
为了实现上述目的,本发明采用的技术方案如下:
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:基础培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
优选地,白术内酯Ⅱ的含量为10-50μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为1×10-7-3×10-7mol/L,抗坏血酸的含量为30-50μg/L,β-甘油磷酸钠的含量为5-10mmol/L,胰岛素的含量为15-25μg/L,磺丁基-β-环糊精的含量为15-25μg/mL,甜菜碱的含量为碱45-55μg/mL,乙酸铟的含量为5-15ng/mL,谷胱甘肽的含量为30-50μg/mL。
优选地,所述基础培养基包括α-MEM/HG-DMEM(高浓度葡萄糖)培养基、DMEM/F12培养基中的任一种。
α-MEM培养基是一种改良的MEM培养基,与MEM培养基相比,α-MEM培养基营养更为丰富,在MEM培养基的基础上又添加了NEAA(非必需氨基酸)、丙酮酸钠、硫酸锌、VB12、生物素、抗坏血酸等成分,广泛应用于各种哺乳动物悬浮和贴壁细胞的培养;
DMEM是一种含各种氨基酸和葡萄糖的培养基,是在MEM培养基的基础上研制的,分为高糖型和低糖型,HG-DMEM培养基为高糖型DMEM培养基;
F12培养基起初是作为一种无血清配方设计的,常补加血清用于支持各种正常的和转化细胞的增殖,F12培养基常和DMEM培养基以质量比1:1结合,称为DMEM/F12培养基。
优选地,间充质干细胞为人间充质干细胞;
所述人间充质干细胞选自人骨髓间充质干细胞、人脐带间充质干细胞、人脂肪间充质干细胞中的任一种。
优选地,促进间充质干细胞分化的培养基,包括如下步骤制备而成:
向基础培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基。
优选地,所述滤膜的孔径为0.22μm。
优选地,所述杀菌处理采用紫外线杀菌灯进行杀菌处理,紫外线杀菌灯的功率为300-400W。
优选地,一种人间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取人间充质干细胞进行培养,待人间充质干细胞融合度达到80-90%时,用0.25%胰酶消化传代,传代培养的比例为1:3-4;
步骤二、将步骤一中传代培养得到的人间充质干细胞接种于如权利要求1-7任一项所述的促进间充质干细胞分化的培养基上,进行诱导分化培养。
优选地,所述步骤二中,取传代培养至第3-5代的人间充质干细胞接种于促进间充质干细胞分化的培养基上。
优选地,所述步骤二中,人间充质干细胞的接种密度为1×105-4×105个/mL。
与现有技术相比,本发明具有以下有益效果:
本发明提供的促进间充质干细胞分化的培养基,可显著促进包括人骨髓间充质干细胞、人脐带间充质干细胞、人脂肪间充质干细胞在内的多种人间充质干细胞向骨细胞的诱导分化,成骨定向分化特异性高,专一性强;且分化时间短,大幅度提高人间充质干细胞成骨分化的效率。
附图说明
图1为本发明中促进间充质干细胞分化的培养基的制备工艺流程图;
图2为本发明中利用促进间充质干细胞分化的培养基对人间充质干细胞进行成骨分化培养的过程。
图3为对本发明中各实施例、对比例中制得的培养基的诱导分化培养12天的细胞进行检测得到的钙化面积柱形图。
具体实施方式
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,所描述的实施例仅仅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。
实施例1
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:α-MEM/HG-DMEM培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
其中,白术内酯Ⅱ的含量为10μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为1×10-7mol/L,抗坏血酸的含量为30μg/L,β-甘油磷酸钠的含量为5mmol/L,胰岛素的含量为15μg/L,磺丁基-β-环糊精的含量为15μg/mL,甜菜碱的含量为碱45μg/mL,乙酸铟的含量为5ng/mL,谷胱甘肽的含量为30μg/mL;
包括如下步骤制备而成:
向α-MEM/HG-DMEM培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基;
其中,滤膜的孔径为0.22μm,采用紫外线杀菌灯进行杀菌处理,紫外线杀菌灯的功率为300W。
实施例2
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:DMEM/F12培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
其中,白术内酯Ⅱ的含量为50μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为3×10-7mol/L,抗坏血酸的含量为50μg/L,β-甘油磷酸钠的含量为10mmol/L,胰岛素的含量为25μg/L,磺丁基-β-环糊精的含量为25μg/mL,甜菜碱的含量为碱55μg/mL,乙酸铟的含量为15ng/mL,谷胱甘肽的含量为50μg/mL;
包括如下步骤制备而成:
向DMEM/F12培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基;
其中,滤膜的孔径为0.22μm,采用紫外线杀菌灯进行杀菌处理,紫外线杀菌灯的功率为400W。
实施例3
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:α-MEM/HG-DMEM培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
其中,白术内酯Ⅱ的含量为30μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为2×10-7mol/L,抗坏血酸的含量为40μg/L,β-甘油磷酸钠的含量为7.5mmol/L,胰岛素的含量为20μg/L,磺丁基-β-环糊精的含量为20μg/mL,甜菜碱的含量为碱50μg/mL,乙酸铟的含量为10ng/mL,谷胱甘肽的含量为40μg/mL;
包括如下步骤制备而成:
向α-MEM/HG-DMEM培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基;
其中,滤膜的孔径为0.22μm,采用紫外线杀菌灯进行杀菌处理,紫外线杀菌灯的功率为350W。
实施例4
一种人骨髓间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取人骨髓间充质干细胞接种于基础培养基DMEM/F12中,接种密度为1×104个/mL,在37℃,5%CO2的培养箱中培养,待细胞融合度达到80%时,用0.25%胰酶消化传代,传代培养的比例为1:3;
步骤二、将步骤一中传代培养得到的的第3代骨髓间充质干细胞接种于实施例1中制得的促进间充质干细胞分化的培养基上,在37℃,5%CO2的培养箱中进行诱导分化培养,接种密度为1×105个/mL。
实施例5
一种人脐带间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取清洗后的脐带,剥离脐带动静脉保留外膜,将脐带剪成1cm3左右大小的组织块,将组织块以接种密度为5×104/cm2均匀接种于间充质干细胞培养基中,放置于37℃、5%CO2的恒温箱内培养;待贴壁细胞融合至90%时,用0.25%胰酶消化传代,将其接种于间充质干细胞无血清培养基中,于37℃、5%CO2条件下进行连续传代扩增培养,传代培养的比例为1:4;
其中,间充质干细胞无血清培养基包括以下成分:人血小板裂解液2%、L-谷氨酰胺1mmol/L、2-O-乙醚抗坏血酸12μg/mL、香菇多糖0.6μg/mL、DMEM/F12培养基补足至1L;
步骤二、将步骤一中传代培养得到的人脐带间充质干细胞接种于实施例2中制得的促进间充质干细胞分化的培养基上,在37℃,5%CO2的培养箱中进行诱导分化培养,接种密度为4×105个/mL。
实施例6
一种人脂肪间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取健康人腹部脂肪抽脂术中抽取的脂肪组织,脂肪组织用PBS溶液冲洗3次,再用体积分数0.25%Ⅰ型胶原酶于37℃水浴消化30min,期间反复震荡3次,使胶原酶溶液与脂肪组织充分混合接触;用与胶原酶消化后的混悬液等体积的完全培养液(高糖DMEM+体积含量10%胎牛血清+1%青链霉素)终止胶原酶的消化,200目滤网过滤混悬液,以去除未消化的纤维结缔组织,1200×g离心5min以去除悬浮的脂肪细胞和脂滴等,去上清;将细胞沉淀用1mL的完全培养基重悬后,加入6倍体积的红细胞裂解液,混匀后室温下解育6min,1200×g离心5min,去上清,显微镜下计数细胞数,再以适量完全培养基重悬沉淀,以1×106/皿的密度接种于直径为10cm的培养皿,再加入适量完全培养基培养,细胞每隔3d换1次培养基,每周以1:3的比例传代培养;
待85%的细胞变圆后,加入完全培养基以终止消化,用加样枪反复吹打培养皿的底部,使贴壁的细胞脱落,将细胞混悬液移入离心管中,1200×g离心5min,去上清,以适量完全培养基重悬沉淀,接种于培养皿,再加入适量完全培养基培养;
步骤二、将步骤一中传代培养得到的第3代人脂肪间充质干细胞接种于实施例3中制得的促进间充质干细胞分化的培养基上,在37℃,5%CO2的培养箱中进行诱导分化培养,接种密度为2.5×105个/mL。
对比例1
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:α-MEM/HG-DMEM培养基、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
其中,胎牛血清的体积含量为10%,地塞米松的含量为1×10-7mol/L,抗坏血酸的含量为30μg/L,β-甘油磷酸钠的含量为
5mmol/L,胰岛素的含量为15μg/L,磺丁基-β-环糊精的含量为15μg/mL,甜菜碱的含量为碱45μg/mL,乙酸铟的含量为5ng/mL,谷胱甘肽的含量为30μg/mL;
包括如下步骤制备而成:
向α-MEM/HG-DMEM培养基中加入胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基;
其中,滤膜的孔径为0.22μm,采用紫外线杀菌灯进行杀菌处理,紫外线杀菌灯的功率为300W。
对比例2
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:DMEM/F12培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
其中,白术内酯Ⅱ的含量为50μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为3×10-7mol/L,抗坏血酸的含量为50μg/L,胰岛素的含量为25μg/L,磺丁基-β-环糊精的含量为25μg/mL,甜菜碱的含量为碱55μg/mL,乙酸铟的含量为15ng/mL,谷胱甘肽的含量为50μg/mL;
包括如下步骤制备而成:
向DMEM/F12培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基。
对比例3
一种促进间充质干细胞分化的培养基,包括以下原料制备而成:α-MEM/HG-DMEM培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
其中,白术内酯Ⅱ的含量为30μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为2×10-7mol/L,抗坏血酸的含量为40μg/L,β-甘油磷酸钠的含量为7.5mmol/L,磺丁基-β-环糊精的含量为20μg/mL,甜菜碱的含量为碱50μg/mL,乙酸铟的含量为10ng/mL,谷胱甘肽的含量为40μg/mL;
包括如下步骤制备而成:
向α-MEM/HG-DMEM培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基。
对比例4
一种人骨髓间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取人骨髓间充质干细胞接种于基础培养基DMEM/F12中,接种密度为1×104个/mL,在37℃,5%CO2的培养箱中培养,待细胞融合度达到80%时,用0.25%胰酶消化传代,传代培养的比例为1:3;
步骤二、将步骤一中传代培养得到的的第3代骨髓间充质干细胞接种于对比例1中制得的促进间充质干细胞分化的培养基上,在37℃,5%CO2的培养箱中进行诱导分化培养,接种密度为1×105个/mL。
对比例5
一种人脐带间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取清洗后的脐带,剥离脐带动静脉保留外膜,将脐带剪成1cm3左右大小的组织块,将组织块以接种密度为5×104/cm2均匀接种于间充质干细胞培养基中,放置于37℃、5%CO2的恒温箱内培养;待贴壁细胞融合至90%时,用0.25%胰酶消化传代,将其接种于间充质干细胞无血清培养基中,于37℃、5%CO2条件下进行连续传代扩增培养,传代培养的比例为1:4;
其中,间充质干细胞无血清培养基包括以下成分:人血小板裂解液2%、L-谷氨酰胺1mmol/L、2-O-乙醚抗坏血酸12μg/mL、香菇多糖0.6μg/mL、DMEM/F12培养基补足至1L;
步骤二、将步骤一中传代培养得到的人脐带间充质干细胞接种于对比例2中制得的促进间充质干细胞分化的培养基上,在37℃,5%CO2的培养箱中进行诱导分化培养,接种密度为4×105个/mL。
对比例6
一种人脂肪间充质干细胞成骨分化的方法,包括以下步骤:
步骤一、取健康人腹部脂肪抽脂术中抽取的脂肪组织,脂肪组织用PBS溶液冲洗3次,再用体积分数0.25%Ⅰ型胶原酶于37℃水浴消化30min,期间反复震荡3次,使胶原酶溶液与脂肪组织充分混合接触;用与胶原酶消化后的混悬液等体积的完全培养液(高糖DMEM+体积含量10%胎牛血清+1%青链霉素)终止胶原酶的消化,200目滤网过滤混悬液,以去除未消化的纤维结缔组织,1200×g离心5min以去除悬浮的脂肪细胞和脂滴等,去上清;将细胞沉淀用1mL的完全培养基重悬后,加入6倍体积的红细胞裂解液,混匀后室温下解育6min,1200×g离心5min,去上清,显微镜下计数细胞数,再以适量完全培养基重悬沉淀,以1×106/皿的密度接种于直径为10cm的培养皿,再加入适量完全培养基培养,细胞每隔3d换1次培养基,每周以1:3的比例传代培养;
待人脂肪间充质干细胞融合度达到85%时后,加入完全培养基以终止消化,用加样枪反复吹打培养皿的底部,使贴壁的细胞脱落,将细胞混悬液移入离心管中,1200×g离心5min,去上清,以适量完全培养基重悬沉淀,接种于培养皿,再加入适量完全培养基培养;
步骤二、将步骤一中传代培养得到的第3代人脂肪间充质干细胞接种于对比例3中制得的促进间充质干细胞分化的培养基上,在37℃,5%CO2的培养箱中进行诱导分化培养,接种密度为2.5×105个/mL。
上述实施例与对比例中的DMEM/F12培养基由武汉普诺赛生命科技有限公司提供,货号:PM150310B,α-MEM/HG-DMEM培养基是由α-MEM培养基与HG-DMEM培养基按质量比1:1配制而成,HG-DMEM培养基由广东环凯微生物科技有限公司提供,货号:XB01,α-MEM培养基由上海慧颖生物科技有限公司提供,货号:SH30265.01B。
试验例
分别取实施例4-6、对比例4-6中诱导分化培养12天的细胞,弃去培养基,用6℃预冷的PBS清洗后,用4%的多聚甲醛在常温下固定10min,弃去多聚甲醛,再用PBS清洗三遍,用0.5%的茜素红染色10min,弃去染色液,水洗3遍,在显微镜线观察染色情况,统计钙化面积百分比,结果如表1所示:
表1
成骨细胞会出现钙化现象,钙化后的成骨细胞会被茜红素染成红色,根据被染色细胞的面积可以计算出钙化面积百分比。由表1可知,本发明制得的促进间充质干细胞分化的培养基,用于人骨髓间充质干细胞、人脐带间充质干细胞、人脂肪间充质干细胞的诱导分化培养,均能诱导分化得到较多的成骨细胞。
对比例4中,采用的是对比例1中制得的没有加入白术内酯Ⅱ的促进间充质干细胞分化的培养基,由于白术内酯Ⅱ可以促进骨髓间充质干细胞的成骨分化,对比例4中钙化面积明显降低;对比例5中,采用的是对比例2中制得的没有加入β-甘油磷酸钠的促进间充质干细胞分化的培养基,由于β-磷酸甘油钠则作为细胞磷酸盐来源,具有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的功效,对比例5中钙化面积亦明显降低;对比例6中,采用的是对比例3中制得的没有加入胰岛素的促进间充质干细胞分化的培养基,由于胰岛素中含有胰岛素生长因子,具有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,对比例6中钙化面积下降较多。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (10)
1.一种促进间充质干细胞分化的培养基,其特征在于,包括以下原料制备而成:基础培养基、白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽。
2.根据权利要求1所述的促进间充质干细胞分化的培养基,其特征在于,白术内酯Ⅱ的含量为10-50μmol/L,胎牛血清的体积含量为10%,地塞米松的含量为1×10-7-3×10-7mol/L,抗坏血酸的含量为30-50μg/L,β-甘油磷酸钠的含量为5-10mmol/L,胰岛素的含量为15-25μg/L,磺丁基-β-环糊精的含量为15-25μg/mL,甜菜碱的含量为碱45-55μg/mL,乙酸铟的含量为5-15ng/mL,谷胱甘肽的含量为30-50μg/mL。
3.根据权利要求1所述的促进间充质干细胞分化的培养基,其特征在于,所述基础培养基包括α-MEM/HG-DMEM培养基、DMEM/F12培养基中的任一种。
4.根据权利要求1所述的促进间充质干细胞分化的培养基,其特征在于,间充质干细胞为人间充质干细胞;
所述人间充质干细胞选自人骨髓间充质干细胞、人脐带间充质干细胞、人脂肪间充质干细胞中的任一种。
5.如权利要求1-4任一项所述的促进间充质干细胞分化的培养基,其特征在于,包括如下步骤制备而成:
向基础培养基中加入白术内酯Ⅱ、胎牛血清、地塞米松、抗坏血酸、β-甘油磷酸钠、胰岛素、磺丁基-β-环糊精、甜菜碱、乙酸铟、谷胱甘肽,混合均匀,通过滤膜过滤后,杀菌处理,制得促进间充质干细胞分化的培养基。
6.根据权利要求5所述的促进间充质干细胞分化的培养基,其特征在于,所述滤膜的孔径为0.22μm。
7.根据权利要求5所述的促进间充质干细胞分化的培养基,其特征在于,所述杀菌处理采用紫外线杀菌灯进行杀菌处理,紫外线杀菌灯的功率为300-400W。
8.一种人间充质干细胞成骨分化的方法,其特征在于,包括以下步骤:
步骤一、取人间充质干细胞进行培养,待人间充质干细胞融合度达到80-90%时,用0.25%胰酶消化传代,传代培养的比例为1:3-4;
步骤二、将步骤一中传代培养得到的人间充质干细胞接种于如权利要求1-7任一项所述的促进间充质干细胞分化的培养基上,进行诱导分化培养。
9.根据权利要求8所述的人间充质干细胞成骨分化的方法,其特征在于,所述步骤二中,取传代培养至第3-5代的人间充质干细胞接种于促进间充质干细胞分化的培养基上。
10.根据权利要求8所述的人间充质干细胞成骨分化的方法,其特征在于,所述步骤二中,人间充质干细胞的接种密度为1×105-4×105个/mL。
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