CN106148283A - 一种造血干细胞贴壁性好的培养基 - Google Patents
一种造血干细胞贴壁性好的培养基 Download PDFInfo
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Abstract
本发明公开了一种造血干细胞贴壁性好的培养基,包括RPMI‑1640培养基,还包括:透明质酸5~8μg/L,海藻糖3.2~3.8g/L,乙醇胺20~30mg/L,碳酸氢钾0.7~1g/L,蛋氨酸螯合硒6~9μg/L,β‑巯基乙醇2~5μg/L,转铁蛋白2~3g/L,干细胞因子200~260mg/L,胎盘生长因子50~80mg/L,纤维细胞生长因子30~50mg/L,谷胱甘肽80~100mg/L,维生素80~110mg/L,植物提取物180~220mg/L。本发明细胞贴壁性好,增殖速度快,提高培养基的抗病原微生物作用,同时无动物源成分,不会造成残留或者污染,降低了培养基成本。
Description
技术领域
本发明涉及干细胞培养基技术领域,尤其涉及一种造血干细胞贴壁性好的培养基。
背景技术
现有技术中的干细胞培养基往往采用胎牛血清,由于使用动物源的胎牛血清,导致干细胞培养基中掺入非人源物质,造成残留或者污染,而且现有的干细胞培养基存在细胞贴壁性差,细胞增殖速度不够理想等问题。
发明内容
基于背景技术存在的技术问题,本发明提出了一种造血干细胞贴壁性好的培养基,细胞贴壁性好,增殖速度快,提高培养基的抗病原微生物作用,同时无动物源成分,不会造成残留或者污染,降低了培养基成本。
本发明提出的一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基;还包括:透明质酸5~8μg/L,海藻糖3.2~3.8g/L,乙醇胺20~30mg/L,碳酸氢钾0.7~1g/L,蛋氨酸螯合硒6~9μg/L,β-巯基乙醇2~5μg/L,转铁蛋白2~3g/L,干细胞因子200~260mg/L,胎盘生长因子50~80mg/L,纤维细胞生长因子30~50mg/L,谷胱甘肽80~100mg/L,维生素80~110mg/L,植物提取物180~220mg/L。
优选地,维生素按重量份包括:维生素E 6~8份,维生素C 12~14份,辅酶Q10 3~5份,氯化胆碱1~2份,硫胺素3~6份,核黄素2~4份,烟酸1~2份,泛酸1~2份,吡哆醇1~2份,氰钴胺0.8~1.5份,叶酸0.5~1份,生物素0.5~0.8份。
优选地,透明质酸、海藻糖、乙醇胺、碳酸氢钾、蛋氨酸螯合硒、β-巯基乙醇的重量比为6~7:3.4×106~3.6×106:2.2×104~2.8×104:8×105~9×105:7~8:3~4。
优选地,转铁蛋白、干细胞因子、胎盘生长因子、纤维细胞生长因子、谷胱甘肽的重量比为2500~2800:220~240:60~70:35~45:85~95。
优选地,还包括:透明质酸6~7μg/L,海藻糖3.4~3.6g/L,乙醇胺22~28mg/L,碳酸氢钾0.8~0.9g/L,蛋氨酸螯合硒7~8μg/L,β-巯基乙醇3~4μg/L,转铁蛋白2.5~2.8g/L,干细胞因子220~240mg/L,胎盘生长因子60~70mg/L,纤维细胞生长因子35~45mg/L,谷胱甘肽85~95mg/L,维生素90~100mg/L,植物提取物190~210mg/L。
优选地,植物提取物按重量份包括:绿原酸1~3份,白藜芦醇5~7份,大枣多糖13~15份,人参皂苷14~16份,黄精多糖23~25份。
本发明采用RPMI-1640培养基、透明质酸、海藻糖、乙醇胺、碳酸氢钾、蛋氨酸螯合硒、β-巯基乙醇、转铁蛋白、干细胞因子、胎盘生长因子、纤维细胞生长因子、谷胱甘肽、维生素相互配合,能滋养造血干细胞,促进造血干细胞保持细胞的活性;其中RPMI-1640培养基、透明质酸、海藻糖、乙醇胺、转铁蛋白、干细胞因子、胎盘生长因子、纤维细胞生长因子、谷胱甘肽、维生素为造血干细胞提供营养成分;而谷胱甘肽和维生素相互配合,可以清理细胞内自由基,保护许多蛋白质和酶等分子中的巯基,有利于酶活性的发挥,并且能恢复已被破坏的酶分子中-SH基的活性功能,使酶重新恢复活性,降低细胞发生凋亡的概率;透明质酸、海藻糖、乙醇胺、碳酸氢钾、蛋氨酸螯合硒、β-巯基乙醇、转铁蛋白、干细胞因子、胎盘生长因子、纤维细胞生长因子相互配合,维持细胞的渗透平衡;转铁蛋白、干细胞因子、胎盘生长因子、纤维细胞生长因子相互配合,能使造血干细胞在增殖的过程中进行缓慢修复,维持细胞活性;同时透明质酸、海藻糖、乙醇胺、谷胱甘肽和植物提取物相互配合,可以部分替代特殊抗体因子,增强细胞的新陈代谢,促进造血干细胞快速大量增殖,降低了培养基成本;而采用绿原酸、白藜芦醇、大枣多糖、人参皂苷、黄精多糖作为植物提取物,不仅提高培养基的抗病原微生物作用,提高干细胞的存活率,而且进一步提高造血干细胞的增殖速度,提高造血干细胞的活性。
本发明培养造血干细胞,使造血干细胞的贴壁率达到94%以上,死细胞较少,大大提高了细胞的数量。本发明培养造血干细胞生长至平台期需要49~52h,而现有技术中的培养基最快需要54h,常规干细胞培养基则需要至少66h,由此可知本发明能够提高造血干细胞的生长速率。同时本发明经流式细胞表型检测,发现本发明所得造血干细胞与常规培养基所得造血干细胞的细胞表型无统计学差异。
具体实施方式
下面,通过具体实施例对本发明的技术方案进行详细说明。
实施例1
本发明提出的一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基;还包括:透明质酸5μg/L,海藻糖3.8g/L,乙醇胺20mg/L,碳酸氢钾1g/L,蛋氨酸螯合硒6μg/L,β-巯基乙醇5μg/L,转铁蛋白2g/L,干细胞因子260mg/L,胎盘生长因子50mg/L,纤维细胞生长因子50mg/L,谷胱甘肽80mg/L,维生素110mg/L,植物提取物180mg/L。
实施例2
本发明提出的一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基;还包括:透明质酸8μg/L,海藻糖3.2g/L,乙醇胺30mg/L,碳酸氢钾0.7g/L,蛋氨酸螯合硒9μg/L,β-巯基乙醇2μg/L,转铁蛋白3g/L,干细胞因子200mg/L,胎盘生长因子80mg/L,纤维细胞生长因子30mg/L,谷胱甘肽100mg/L,维生素80mg/L,植物提取物220mg/L。
维生素按重量份包括:维生素E 6份,维生素C 14份,辅酶Q10 3份,氯化胆碱2份,硫胺素3份,核黄素4份,烟酸1份,泛酸2份,吡哆醇1份,氰钴胺1.5份,叶酸0.5份,生物素0.8份。
实施例3
本发明提出的一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基;还包括:透明质酸6μg/L,海藻糖3.6g/L,乙醇胺22mg/L,碳酸氢钾0.9g/L,蛋氨酸螯合硒7μg/L,β-巯基乙醇4μg/L,转铁蛋白2.5g/L,干细胞因子240mg/L,胎盘生长因子60mg/L,纤维细胞生长因子45mg/L,谷胱甘肽85mg/L,维生素100mg/L,植物提取物190mg/L。
维生素按重量份包括:维生素E 8份,维生素C 12份,辅酶Q10 5份,氯化胆碱1份,硫胺素6份,核黄素2份,烟酸2份,泛酸1份,吡哆醇2份,氰钴胺0.8份,叶酸1份,生物素0.5份。
植物提取物按重量份包括:绿原酸2份,白藜芦醇6份,大枣多糖14份,人参皂苷15份,黄精多糖24份。
实施例4
本发明提出的一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基;还包括:透明质酸6μg/L,海藻糖3.6g/L,乙醇胺22mg/L,碳酸氢钾0.9g/L,蛋氨酸螯合硒7μg/L,β-巯基乙醇4μg/L,转铁蛋白2.5g/L,干细胞因子240mg/L,胎盘生长因子60mg/L,纤维细胞生长因子45mg/L,谷胱甘肽85mg/L,维生素100mg/L,植物提取物190mg/L。
维生素按重量份包括:维生素E 7份,维生素C 13份,辅酶Q10 4份,氯化胆碱1.5份,硫胺素5份,核黄素3份,烟酸1.5份,泛酸1.5份,吡哆醇1.5份,氰钴胺1.2份,叶酸0.8份,生物素0.7份。
植物提取物按重量份包括:绿原酸3份,白藜芦醇5份,大枣多糖15份,人参皂苷14份,黄精多糖25份。
实施例5
本发明提出的一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基;还包括:透明质酸6.5μg/L,海藻糖3.5g/L,乙醇胺25mg/L,碳酸氢钾0.85g/L,蛋氨酸螯合硒7.5μg/L,β-巯基乙醇3.5μg/L,转铁蛋白2.6g/L,干细胞因子230mg/L,胎盘生长因子65mg/L,纤维细胞生长因子40mg/L,谷胱甘肽90mg/L,维生素95mg/L,植物提取物200mg/L。
植物提取物按重量份包括:绿原酸1份,白藜芦醇7份,大枣多糖13份,人参皂苷16份,黄精多糖23份。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种造血干细胞贴壁性好的培养基,包括RPMI-1640培养基,其特征在于,还包括:透明质酸5~8μg/L,海藻糖3.2~3.8g/L,乙醇胺20~30mg/L,碳酸氢钾0.7~1g/L,蛋氨酸螯合硒6~9μg/L,β-巯基乙醇2~5μg/L,转铁蛋白2~3g/L,干细胞因子200~260mg/L,胎盘生长因子50~80mg/L,纤维细胞生长因子30~50mg/L,谷胱甘肽80~100mg/L,维生素80~110mg/L,植物提取物180~220mg/L。
2.根据权利要求1所述造血干细胞贴壁性好的培养基,其特征在于,维生素按重量份包括:维生素E 6~8份,维生素C 12~14份,辅酶Q10 3~5份,氯化胆碱1~2份,硫胺素3~6份,核黄素2~4份,烟酸1~2份,泛酸1~2份,吡哆醇1~2份,氰钴胺0.8~1.5份,叶酸0.5~1份,生物素0.5~0.8份。
3.根据权利要求1或2所述造血干细胞贴壁性好的培养基,其特征在于,透明质酸、海藻糖、乙醇胺、碳酸氢钾、蛋氨酸螯合硒、β-巯基乙醇的重量比为6~7:3.4×106~3.6×106:2.2×104~2.8×104:8×105~9×105:7~8:3~4。
4.根据权利要求1-3任一项所述造血干细胞贴壁性好的培养基,其特征在于,转铁蛋白、干细胞因子、胎盘生长因子、纤维细胞生长因子、谷胱甘肽的重量比为2500~2800:220~240:60~70:35~45:85~95。
5.根据权利要求1-4任一项所述造血干细胞贴壁性好的培养基,其特征在于,还包括:透明质酸6~7μg/L,海藻糖3.4~3.6g/L,乙醇胺22~28mg/L,碳酸氢钾0.8~0.9g/L,蛋氨酸螯合硒7~8μg/L,β-巯基乙醇3~4μg/L,转铁蛋白2.5~2.8g/L,干细胞因子220~240mg/L,胎盘生长因子60~70mg/L,纤维细胞生长因子35~45mg/L,谷胱甘肽85~95mg/L,维生素90~100mg/L,植物提取物190~210mg/L。
6.根据权利要求1-5任一项所述造血干细胞贴壁性好的培养基,其特征在于,植物提取物按重量份包括:绿原酸1~3份,白藜芦醇5~7份,大枣多糖13~15份,人参皂苷14~16份,黄精多糖23~25份。
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