CN109022356A - 一种提高间充质干细胞向软骨细胞分化的无血清培养基 - Google Patents
一种提高间充质干细胞向软骨细胞分化的无血清培养基 Download PDFInfo
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Abstract
本发明属于干细胞诱导分化技术领域,具体涉及一种提高间充质干细胞向软骨细胞分化的无血清培养基,以α‑MEM培养基为基础培养基,还包括人血白蛋白、亚硒酸、亚油酸、花生四烯酸、血小板源性生长因子、维生素C、L‑谷氨酰胺、血小板衍生因子、红花提取物、抗坏血酸、胰岛素、BMP‑2、碱性成纤维细胞生长因子。本发明提供的一种提高间充质干细胞向软骨细胞分化的无血清培养基,作为将间充质干细胞诱导成软骨细胞的诱导培养基,采用红花提取物联合生长因子诱导间充质干细胞分化为软骨细胞,所选用的诱导成分均无毒,并且诱导效率高、诱导时间短,安全性高。
Description
技术领域
本发明属于干细胞诱导分化技术领域,具体涉及一种提高间充质干细胞向软骨细胞分化的无血清培养基。
背景技术
干细胞的分化是部分基因选择性地被激活或差异性表达,从而控制细胞表型和蛋白质的特异性分布。间充质干细胞分化为特定细胞类型主要取决于基因的激活,而细胞外微环境中各种因子类型和浓度则是基因激活的重要因素。目前运用间充质干细胞体外诱导分化为软骨细胞方法主要有:(1)传统的化学诱导剂法;(2)生长因子诱导法;(3)生长因子与化学诱导剂联合;(4)共培养或用接近生理状态的条件培养基;(5)基因转染;传统的化学诱导剂法虽然可以高效、快速地将脂肪间充质干细胞分化为软骨细胞,但细胞死亡率较高(化学诱导剂具有一定毒性,BHA、DMSO等可使细胞突变,用于细胞移植存在致瘤性的危险);生长因子诱导方法虽然诱导后细胞存活率高,但诱导时间长、诱导效率低。基因转染诱导因为基因改变存在罹患癌症的潜在风险。
发明内容
本发明的目的是针对现有的诱导剂及诱导方法存在的缺陷,提供一种安全无毒,特别是诱导效率高,诱导时间短的间充质干细胞向软骨细胞分化的无血清培养基。
具体的技术方案为:
一种提高间充质干细胞向软骨细胞分化的无血清培养基,以α-MEM培养基为基础培养基,还包括以下物质:
浓度为4-8g/L的人血白蛋白;
浓度为2-10mg/L亚硒酸;
浓度为3-20mg/L亚油酸;
浓度为4-15mg/L花生四烯酸;
浓度为10-30μg/L的血小板源性生长因子;
浓度为10-20mg/L维生素C;
浓度为2-5v/v%L-谷氨酰胺;
浓度为5-20μg/L血小板衍生因子;
浓度为12-18mg/L红花提取物;
浓度为3-8v/v%抗坏血酸;
浓度为2~50μg/mL胰岛素;
浓度为1~40μg/mL的BMP-2;
浓度为5-20μg/L的碱性成纤维细胞生长因子。
优选的,还包括浓度为15-40μg/L的胰岛素样生长因子类。
进一步的,还包括浓度为10-30μg/L的转化生长因子α和/或浓度为10-30μg/L的转化生长因子β。
还包括浓度为30-90μg/L的骨形态形成蛋白质类。
还包括浓度为0.1-2g/L的铁传递蛋白和/或浓度为0.5-1.5g/L的白蛋白。
还包括浓度为10-20mg/L混合氨基酸类物质,混合氨基酸类物质包括以下物质:L-丙氨酸、L-丝氨酸、L-苏氨酸、L-谷氨酸、L-脯氨酸、、L-缬氨酸L-亮氨酸、L-异亮氨酸、L-甲硫氨酸、L-苯丙氨酸、L-天门冬氨酸、L-精氨酸盐酸盐、L-胱氨酸二盐酸盐、L-赖氨酸盐酸盐、L-组氨酸盐酸盐单水合物、L-天冬酰胺单水合物、L-半胱氨酸盐酸盐单水合物和甘氨酸。
其中,所述的红花提取物,由以下方法制备所得:
(1)红花先洗净后打浆,浆液经真空冷冻干燥处理,冷冻温度-70~-60℃、冷冻时间36~48h和真空度15~20Pa;
(2)然后粉碎进行超声提取,超声功率400~800W,乙醇浓度55~70%,料液比1∶120~1∶250g/mL,提取时间60~80min,提取温度55~60℃;得到粗提取液;
(3)粗提取液依次经过过滤,过滤在-0.10~-0.15MPa真空,孔径为15~20μm双层滤纸的条件下抽滤,再减压浓缩得粗提物,减压浓缩为在-0.10~-0.15MPa真空,旋转速度为100~120r/min,蒸发温度40~45℃,循环冷凝温度3~5℃的条件下浓缩;
(4)粗提物再用凝胶柱纯化,得到红花提取,物凝胶柱为羟丙基葡聚糖凝胶柱。
其中,所述的步骤(1)优选的冷冻方法如下:从常温开始在10-15分钟内降温到0摄氏度保持30-40分钟,再于10-15分钟内迅速降温到-40摄氏度保持30-60分钟,然后在-40摄氏度保持2-3小时,然后在10-15分钟内降温到-70~-60℃摄氏度,保存36~48h小时,进行冷冻干燥,这是本发明的一个发明点经过了这个方法,可以最大限度保持营养物质,对细胞分化培养具有非常好的促进作用,这也是本发明的发明点之一。
本发明提供的一种提高间充质干细胞向软骨细胞分化的无血清培养基,作为将间充质干细胞诱导成软骨细胞的诱导培养基,采用红花提取物联合生长因子诱导间充质干细胞分化为软骨细胞,所选用的诱导成分均无毒,并且诱导效率高、诱导时间短,安全性高。
具体实施方式
结合实施例说明本发明的具体技术方案。
实施例1
一种提高间充质干细胞向软骨细胞分化的无血清培养基,以α-MEM培养基为基础培养基,还包括以下物质:
浓度为5/L的人血白蛋白;
浓度为8mg/L亚硒酸;
浓度为14mg/L亚油酸;
浓度为5mg/L花生四烯酸;
浓度为24μg/L的血小板源性生长因子;
浓度为12mg/L维生素C;
浓度为2v/v%L-谷氨酰胺;
浓度为10μg/L血小板衍生因子;
浓度为12mg/L红花提取物;
浓度为5v/v%抗坏血酸;
浓度为50μg/mL胰岛素;
浓度为30μg/mL的BMP-2;
浓度为10μg/L的碱性成纤维细胞生长因子。
实施例2
一种提高间充质干细胞向软骨细胞分化的无血清培养基,以α-MEM培养基为基础培养基,还包括以下物质:
浓度为7g/L的人血白蛋白;
浓度为3mg/L亚硒酸;
浓度为18mg/L亚油酸;
浓度为12mg/L花生四烯酸;
浓度为28μg/L的血小板源性生长因子;
浓度为13mg/L维生素C;
浓度为5v/v%L-谷氨酰胺;
浓度为7μg/L血小板衍生因子;
浓度为16mg/L红花提取物;
浓度为5v/v%抗坏血酸;
浓度为30μg/mL胰岛素;
浓度为20μg/mL的BMP-2;
浓度为5-20μg/L的碱性成纤维细胞生长因子;
浓度为35μg/L的胰岛素样生长因子类;
浓度为20μg/L的转化生长因子α;
浓度为20μg/L的转化生长因子β;
浓度为50μg/L的骨形态形成蛋白质类;
浓度为1g/L的铁传递蛋白;
浓度为0.8g/L的白蛋白。
实施例3
一种提高间充质干细胞向软骨细胞分化的无血清培养基,以α-MEM培养基为基础培养基,还包括以下物质:
浓度为8g/L的人血白蛋白;
浓度为2mg/L亚硒酸;
浓度为6mg/L亚油酸;
浓度为13mg/L花生四烯酸;
浓度为10μg/L的血小板源性生长因子;
浓度为13mg/L维生素C;
浓度为2v/v%L-谷氨酰胺;
浓度为8μg/L血小板衍生因子;
浓度为13mg/L红花提取物;
浓度为3v/v%抗坏血酸;
浓度为8μg/mL胰岛素;
浓度为25μg/mL的BMP-2;
浓度为16μg/L的碱性成纤维细胞生长因子;
包括浓度为30μg/L的胰岛素样生长因子类;
包括浓度为14μg/L的转化生长因子α和;
包括浓度为60μg/L的骨形态形成蛋白质类;
包括浓度为0.15g/L的铁传递蛋白;
包括浓度为10-20mg/L混合氨基酸类物质,混合氨基酸类物质包括以下物质:L-丙氨酸、L-丝氨酸、L-苏氨酸、L-谷氨酸、L-脯氨酸、、L-缬氨酸L-亮氨酸、L-异亮氨酸、L-甲硫氨酸、L-苯丙氨酸、L-天门冬氨酸、L-精氨酸盐酸盐、L-胱氨酸二盐酸盐、L-赖氨酸盐酸盐、L-组氨酸盐酸盐单水合物、L-天冬酰胺单水合物、L-半胱氨酸盐酸盐单水合物和甘氨酸。
利用上述三个实施例的培养基对小鼠骨髓间充质干细胞进行诱导分化培养,成骨诱导分化21天后,进行细胞洗涤,首先pH值在7.2-7.4d的PBS缓冲液洗涤细胞,再用浓度为4%多聚甲醛固定细胞30分钟,然后用蒸馏水洗涤3遍;然后染色,采用浓度为0.2%的茜素红S室温细胞染色30分钟,除去染色液,蒸馏水洗涤3遍,在显微镜下观察小鼠骨髓间充质干细胞的成骨细胞分化结果。结果显示细胞密度较高,视野中几乎所有的细胞均染成红色,且有众多的钙化物和钙结节沉淀,其中实施例3的效果最为明显。
Claims (7)
1.一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,以α-MEM培养基为基础培养基,还包括以下物质:
浓度为4-8g/L的人血白蛋白;
浓度为2-10mg/L亚硒酸;
浓度为3-20mg/L亚油酸;
浓度为4-15mg/L花生四烯酸;
浓度为10-30μg/L的血小板源性生长因子;
浓度为10-20mg/L维生素C;
浓度为2-5v/v%L-谷氨酰胺;
浓度为5-20μg/L血小板衍生因子;
浓度为12-18mg/L红花提取物;
浓度为3-8v/v%抗坏血酸;
浓度为2~50μg/mL胰岛素;
浓度为1~40μg/mL的BMP-2;
浓度为5-20μg/L的碱性成纤维细胞生长因子。
2.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,还包括浓度为15-40μg/L的胰岛素样生长因子类。
3.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,还包括浓度为10-30μg/L的转化生长因子α和/或浓度为10-30μg/L的转化生长因子β。
4.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,还包括浓度为30-90μg/L的骨形态形成蛋白质类。
5.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,还包括浓度为0.1-2g/L的铁传递蛋白和/或浓度为0.5-1.5g/L的白蛋白。
6.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,还包括浓度为10-20mg/L混合氨基酸类物质,混合氨基酸类物质包括以下物质:L-丙氨酸、L-丝氨酸、L-苏氨酸、L-谷氨酸、L-脯氨酸、、L-缬氨酸L-亮氨酸、L-异亮氨酸、L-甲硫氨酸、L-苯丙氨酸、L-天门冬氨酸、L-精氨酸盐酸盐、L-胱氨酸二盐酸盐、L-赖氨酸盐酸盐、L-组氨酸盐酸盐单水合物、L-天冬酰胺单水合物、L-半胱氨酸盐酸盐单水合物和甘氨酸。
7.根据权利要求1所述的一种提高间充质干细胞向软骨细胞分化的无血清培养基,其特征在于,所述的红花提取物,由以下方法制备所得:
(1)红花先洗净后打浆,浆液经真空冷冻干燥处理,冷冻温度-70~-60℃、冷冻时间36~48h和真空度15~20Pa;
(2)然后粉碎进行超声提取,超声功率400~800W,乙醇浓度55~70%,料液比1∶120~1∶250g/mL,提取时间60~80min,提取温度55~60℃;得到粗提取液;
(3)粗提取液依次经过过滤,过滤在-0.10~-0.15MPa真空,孔径为15~20μm双层滤纸的条件下抽滤,再减压浓缩得粗提物,减压浓缩为在-0.10~-0.15MPa真空,旋转速度为100~120r/min,蒸发温度40~45℃,循环冷凝温度3~5℃的条件下浓缩;
(4)粗提物再用凝胶柱纯化,得到红花提取,物凝胶柱为羟丙基葡聚糖凝胶柱。
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