CN105695407B - 一种对干细胞具有活化作用的微量元素组合物及其应用 - Google Patents
一种对干细胞具有活化作用的微量元素组合物及其应用 Download PDFInfo
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- CN105695407B CN105695407B CN201610145744.1A CN201610145744A CN105695407B CN 105695407 B CN105695407 B CN 105695407B CN 201610145744 A CN201610145744 A CN 201610145744A CN 105695407 B CN105695407 B CN 105695407B
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Abstract
本发明公开一种对干细胞具有活化作用的微量元素组合物及其应用,所述配方包括:铁0.1~2mmol、铜1~10μmol、锌1~20μmol、钴5~100μmol、锰5~50μmol、铬1~10μmol、硒1~10nmol、碘0.1~50μmol、镍0.1~10μmol、钼0.1~10μmol、钒0.1~5μmol、锡1~10nmol、硅1~30mmol、锶5~50μmol、硼0.1~10μmol、铷5~100μmol、锂1~50μmol、锗10~100nmol、钛1~10mmol、钨1~300μmol。本发明提供了一种对干细胞具有活化作用的微量元素组合物,并将所述微量元素组合物应用于干细胞的活化培养中,通过本发明实验结果发现,微量元素组合物不仅具有在体外促进造血干细胞和间充质干细胞增殖的作用,同时又不影响间充质干细胞的分化潜能,并且微量元素还具有促进类似于小鼠这种哺乳生物体内干细胞活化、增殖的作用。
Description
技术领域
本发明涉及微量元素的应用领域,尤其涉及一种对干细胞具有活化作用的微量元素组合物及其应用。
背景技术
微量元素是指一些在体内含量甚微但对个体的早期发育及后天的生命维护等一系列生物学过程起至关重要的化学元素。一方面,一个生命体从一个受精卵开始发育,历经胚胎干细胞到组织器官的形成以及成熟个体的后天维护,在形成成熟个体之后,体内干细胞的数量非常少,在成体组织中只有万分之一左右的细胞是干细胞, 正是这些极少量的干细胞在生命的后天维护起至关重要的作用,除神经细胞终身不死以外,绝大多数成体细胞寿命只有数百天,但生命有机体要维持数年甚至上百年的寿命主要依赖于干细胞的增殖、活化以及向其他成体细胞分化的潜能。另一方面,虽然微量元素对有机体的整个生命活动起很重要的作用,但目前行业内对于微量元素如何调节干细胞的功能还不清楚,导致现有技术中,还无法提供一种组合物合理的微量元素组合物来促进造血干细胞的活化。
因此,现有技术还有待改进和发展。
发明内容
鉴于上述现有技术的不足,本发明的目的在于提供一种对干细胞具有活化作用的微量元素组合物及其应用,旨在解决现有技术还没有提供一种组合物合理的微量元素组合物来促进造血干细胞的活化的问题。
本发明的技术方案如下:
一种对干细胞具有活化作用的微量元素组合物,其中,所述组合物包括:
铁 0.1~2mmol
铜 1~10μmol
锌 1~20μmol
钴 5~100μmol
锰 5~50μmol
铬 1~10μmol
硒 1~10nmol
碘 0.1~50μmol
镍 0.1~10μmol
钼 0.1~10μmol
钒 0.1~5μmol
锡 1~10nmol
硅 1~30mmol
锶 5~50μmol
硼 0.1~10μmol
铷 5~100μmol
锂 1~50μmol
锗 10~100nmol
钛 1~10mmol
钨 1~300μmol
优选地,所述的一种对干细胞具有活化作用的微量元素组合物,其中,所述组合物包括:
铁 1mmol
铜 5μmol
锌 10μmol
钴 50μmol
锰 25μmol
铬 5μmol
硒 5nmol
碘 25μmol
镍 5μmol
钼 6μmol
钒 3μmol
锡 4nmol
硅 15mmol
锶 30μmol
硼 7μmol
铷 55μmol
锂 30μmol
锗 60nmol
钛 4mmol
钨 100μmol
优选地,所述的一种对干细胞具有活化作用的微量元素组合物,其特征在于,所述微量元素为各种形式的无毒性的有机金属化合物或无机金属化合物。
所述的微量元素组合物在促进干细胞活化中的应用。
有益效果:本发明提供了一种对干细胞具有活化作用的微量元素组合物,并将所述微量元素组合物应用于干细胞的活化培养中,通过本发明发现,所述微量元素组合添加物对不同组织来源的干细胞具有增殖以及激活作用。
附图说明
图1为本发明中微量元素组合物对造血干细胞增殖的促进作用对比图。
图2为本发明中微量元素组合物对间充质干细胞增殖的促进作用对比图。
图3为本发明中微量元素组合物活化间充质干细胞的成脂能力对比图。
图4为本发明中微量元素组合物活化间充质干细胞的成骨能力对比图。
图5为本发明中微量元素组合物活化间充质干细胞的成软骨能力对比图。
图6为本发明中微量元素组合物活化造血干细胞后对肿瘤模型小鼠存活率的影响对比图。
具体实施方式
本发明提供一种对干细胞具有活化作用的微量元素配方及其应用,为使本发明的目的、技术方案及效果更加清楚、明确,以下对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
微量元素组合物对造血干细胞增殖的促进作用
本实施例中微量元素组合物为:
铁 0.5mmol
铜 2μmol
锌 8μmol
钴 37μmol
锰 42μmol
铬 6μmol
硒 7nmol
碘 42μmol
镍 5μmol
钼 8μmol
钒 0.8μmol
锡 3nmol
硅 17mmol
锶 42μmol
硼 4.5μmol
铷 62μmol
锂 31μmol
锗 49nmol
钛 6mmol
钨 118μmol
首先通过CD34磁珠分选获得的人造血干细胞,采用Life生产的造血干细胞无血清培养基培养,细胞接种培养袋后置于37℃、5% CO2培养,每过12小时温和摇动培养袋,整个培养过程培养基一直额外添加有上述组合物的微量元素组合。每隔 12 小时显微镜下目测细胞密度,72小时之后用MTT法测定细胞总密度。结果如图1所示,图1为微量元素组合物对造血干细胞增殖的促进作用对比图,研究结果表明采用本发明所描述的添加有微量元素的培养基条件下,造血干细胞密度比不添加微量元素的培养条件下获得的细胞密度提高9.4倍,数据为6次独立实验的平均值±SEM (P<0.01)。
实施例2
微量元素组合物对间充质干细胞增殖的促进作用
本实施例中微量元素组合物为:
铁 1mmol
铜 3μmol
锌 5μmol
钴 47μmol
锰 25μmol
铬 7.5μmol
硒 4.5nmol
碘 33μmol
镍 6μmol
钼 6.3μmol
钒 4.2μmol
锡 8nmol
硅 23mmol
锶 44μmol
硼 3.8μmol
铷 56μmol
锂 34μmol
锗 73nmol
钛 4mmol
钨 89μmol
本发明培养间充质干细胞的培养基配方为:将上述微量元素组合物添加到DMEM/F12基本培养基中,10% 胎牛血清(FBS),为比较本发明所描述的培养基与未添加本发明所描述的微量元素组合物培养基对人干细胞培养的扩增速度的影响,我们选用了脂肪、骨髓间以及脐带三种组织器官来源的人干细胞 (ADSC,BMSC,UMBSC) 做体外细胞培养实验,细胞接种密度控制在10-20%之间,接种后加入培养基培养(37°C,5% CO2)。每隔12小时通过MTT法测一次细胞密度。结果如图2所示,图2为微量元素组合物对间充质干细胞增殖的促进作用对比图,研究结果表明采用本发明所描述的微量元素组合物培养基对干细胞的扩增速度与常规的血清培养基相比,达到细胞密度饱和的时间大大缩短,数据为6次独立实验的平均值±SEM (P<0.01)。
实施例3
微量元素组合物活化间充质干细胞的成脂能力
本实施例中微量元素组合物为:
铁 1.6mmol
铜 5.5μmol
锌 12.5μmol
钴 74μmol
锰 43μmol
铬 4μmol
硒 5nmol
碘 31μmol
镍 3.6μmol
钼 4.5μmol
钒 2.4μmol
锡 7nmol
硅 22mmol
锶 32μmol
硼 7.5μmol
铷 63μmol
锂 38μmol
锗 71nmol
钛 6mmol
钨 170μmol
在体外适当诱导分化条件下间充质干细胞可以分化成脂肪细胞,为检测培养后间充质干细胞的成脂能力,本发明设计实验组为添加有上述微量元素组合物的培养条件下获得的间充质干细胞, 对照组为常规方法培养获得的间充质干细胞,培养的间充质干细胞来源于脐带、脐带和脂肪,因此又分为脐带间充质干细胞(UMSC)、骨髓间充质干细胞(BMSC)以及脂肪干细胞(ADSC),本发明采用如下步骤进行检测:1)首先配制分化培养基A和B,其中分化培养基A组分包括:基础培养基,胎牛血清,青霉素链霉素,谷氨酰胺,胰岛素,3-异丁基-1-甲基黄嘌呤,罗格列酮,地塞米松及吲哚美辛;分化培养基B组分包括:基础培养基,胎牛血清,青霉素链霉素,谷氨酰胺以及胰岛素,配制完毕之后混合均匀,放4℃冰箱避光保存备用;2)在六孔板中接种105个细胞/孔,继续培养细胞;3)待细胞长到100%汇合度后,吸去旧的培养基,每孔加入1.5 ml分化培养基A;4)三天后吸去分化培养基A,每孔加入1.5 ml分化培养基B;5)24小时后,吸去分化培养基B,每孔加入1.5 ml分化培养基A;6)轮换加入分化培养基A和B,重复d-e步骤4次;7)最后加入分化培养基B培养7天(中间需换液一次分化培养基B);8)诱导结束之后,吸去培养基,向每孔中加入1 ml的4%多聚甲醛固定30分钟;9)用PBS洗2遍;10)向每孔中加入1 ml油红O染色液,染色30分钟;11)用PBS洗3遍,然后置于倒置显微镜下观察拍照,统计脂肪细胞所占比率。结果如图3所示,图3为微量元素组合物活化间充质干细胞的成脂能力对比图,研究结果表明微量元素组合物培养条件下获得的不同来源的间充质干细胞相对常规培养条件下获得的间充质干细胞的形成脂肪细胞的能力完全持平,数据为6次独立实验的平均值±SEM (P<0.01)。
实施例4
微量元素组合物活化间充质干细胞的成骨能力
本实施例微量元素组合物为:
铁 1.2mmol
铜 8.5μmol
锌 13μmol
钴 75μmol
锰 36μmol
铬 4μmol
硒 7nmol
碘 33μmol
镍 7.5μmol
钼 6.4μmol
钒 3.5μmol
锡 7nmol
硅 22mmol
锶 38μmol
硼 10μmol
铷 83μmol
锂 41μmol
锗 76nmol
钛 3mmol
钨 225μmol
在体外适当诱导分化条件下间充质干细胞可以分化成骨细胞, 为检测培养后间充质干细胞的成骨能力,本发明设计实验组为微量元素组合物培养条件下获得的间充质干细胞,对照组为常规条件下培养获得的间充质干细胞,培养的间充质干细胞来源于脐带、脐带和脂肪,因此又分为脐带间充质干细胞(UMSC)、骨髓间充质干细胞(BMSC)以及脂肪干细胞(ADSC),本发明采用如下步骤进行检测:1)首先配制分化培养基,包括基础培养基,胎牛血清,青霉素链霉素,谷氨酰胺,维生素C,β-甘油磷酸以及地塞米松,混合均匀,放4℃冰箱避光保存备用;2)将六孔板用0.1%的明胶预处理,接种细胞前至少提前30分钟用明胶处理,或者加入明胶之后把六孔板用封口膜封上,放4℃冰箱备用;3)六孔板在使用前吸去多余明胶液体,然后在1%明胶预处理的六孔板中接种适量细胞(5×104个细胞/孔);4)在37℃,5%CO2培养箱中培养24小时;5)24小时后吸去培养基,换上成骨诱导分化培养基(1.5 ml/孔);6)每3天换一次成骨诱导分化培养基,诱导2-3周;7)诱导结束之后,吸去培养基,向每孔中加入1 ml的4%多聚甲醛固定30分钟;8)用PBS洗2遍;9)向每孔中加入1 ml茜素红S染色液,染色5-10分钟;9)用PBS洗3遍,然后置于倒置显微镜下观察拍照,统计骨细胞所占比率。结果如图4所示,图4为微量元素组合物活化间充质干细胞的成骨能力对比图,研究结果表明微量元素组合物培养条件下获得的间充质干细胞相对常规培养条件下获得的间充质干细胞的形成骨细胞的能力完全持平,数据为6次独立实验的平均值±SEM (P<0.01)。
实施例5
微量元素组合物活化间充质干细胞的成软骨能力
本实施例微量元素组合物为:
铁 0.8mmol
铜 4μmol
锌 12μmol
钴 65μmol
锰 24μmol
铬 5.5μmol
硒 3.4nmol
碘 42μmol
镍 6.2μmol
钼 4.5μmol
钒 2.8μmol
锡 9nmol
硅 17mmol
锶 42μmol
硼 4μmol
铷 74μmol
锂 36μmol
锗 92nmol
钛 3mmol
钨 150μmol
在体外适当诱导分化条件下间充质干细胞可以分化成软骨细胞,为检测培养后间充质干细胞的成软骨能力,本发明设计实验组为微量元素组合物培养条件下获得的间充质干细胞,对照组为常规条件下培养获得的间充质干细胞,培养的间充质干细胞来源于脐带、脐带和脂肪,因此又分为脐带间充质干细胞(UMSC)、骨髓间充质干细胞(BMSC)以及脂肪干细胞(ADSC),本发明采用如下步骤进行检测:1)首先配制分化培养基,包括基础培养基,胎牛血清,青霉素链霉素,谷氨酰胺,维生素C,β-甘油磷酸以及地塞米松,混合均匀,放4℃冰箱避光保存备用;2)将六孔板用0.1%的明胶预处理,接种细胞前至少提前30分钟用明胶处理,或者加入明胶之后把六孔板用封口膜封上,放4℃冰箱备用;3)六孔板在使用前吸去多余明胶液体,然后在1%明胶预处理的六孔板中接种适量细胞(5×104个细胞/孔);4)在37℃,5% CO2培养箱中培养24小时;5)24小时后吸去培养基,换上成骨诱导分化培养基(1.5ml/孔);6)每3天换一次成骨诱导分化培养基,诱导2-3周;7)诱导结束之后,吸去培养基,向每孔中加入1 ml的4%多聚甲醛固定30分钟;8)用PBS洗2遍;9)向每孔中加入1 ml茜素红S染色液,染色5-10分钟;9)用PBS洗3遍,然后置于倒置显微镜下观察拍照,统计软骨细胞所占比率。结果如图5所示,图5为微量元素组合物活化间充质干细胞的成软骨能力对比图,研究结果表明微量元素组合物培养条件下获得的间充质干细胞相对常规培养条件下获得的间充质干细胞的形成软骨细胞的能力完全持平,数据为6次独立实验的平均值±SEM (P<0.01)。
实施例6
微量元素组合物活化造血干细胞后对肿瘤模型小鼠存活率的影响
本实施例微量元素组合物为:
铁 2mmol
铜 3.4μmol
锌 5.6μmol
钴 74μmol
锰 38μmol
铬 3μmol
硒 6nmol
碘 9μmol
镍 4.3μmol
钼 4.8μmol
钒 3.5μmol
锡 6nmol
硅 22mmol
锶 35μmol
硼 7μmol
铷 75μmol
锂 34μmol
锗 85nmol
钛 4mmol
钨 250μmol
将急性B淋巴细胞系白血病细胞株(Nalm-6)用含青霉素100U/ml、链霉素100U/ml、15%重量百分比的灭活新生牛血清组成RPMI-1640完全培养液,在37℃含5%CO2饱和湿度(体积百分比)的培养箱中培养。用无菌PBS液将环磷酰胺浓度调整到10mg/ml,对小鼠的腹腔注射所述环磷酰胺(2 mg/只),连续注射2d(d指天数);24h后,收集处于对数生长期的Nalm-6细胞并进行1000r/min离心5min后,悬浮于无菌PBS液中,调整Nalm-6细胞密度至2.5×107个/ml,对小鼠进行尾静脉注射(5×106个/只),制备白血病动物模型,并进行放疗处理。放疗处理之后辅助以经过上述微量元素组合物活化后的造血干细胞治疗,即注射按以上方法扩增的造血干细胞2.5×105个/只。4个月之后统计动物的存活率, 结果如图6所示,图6为微量元素组合物活化造血干细胞后对肿瘤模型小鼠存活率的影响对比图,研究结果表明造血干细胞治疗前动物存活率由原来的16%提高到64%,数据为8次独立实验的平均值±SEM(P<0.01)。
综上所述,本发明提供了一种对干细胞具有活化作用的微量元素组合物,并将所述微量元素组合物应用于干细胞的活化培养中,通过本发明实验结果发现,微量元素组合物不仅具有在体外促进造血干细胞和间充质干细胞增殖的作用,同时又不影响间充质干细胞的分化潜能,并且微量元素还具有促进类似于小鼠这种哺乳生物体内干细胞活化、增殖的作用。
应当理解的是,本发明的应用不限于上述的举例,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (4)
1.一种对干细胞具有活化作用的微量元素组合物,其特征在于,所述组合物包括:
铁 0.1~2mmol
铜 1~10μmol
锌 1~20μmol
钴 5~100μmol
锰 5~50μmol
铬 1~10μmol
硒 1~10nmol
碘 0.1~50μmol
镍 0.1~10μmol
钼 0.1~10μmol
钒 0.1~5μmol
锡 1~10nmol
硅 1~30mmol
锶 5~50μmol
硼 0.1~10μmol
铷 5~100μmol
锂 1~50μmol
锗 10~100nmol
钛 1~10mmol
钨 1~300μmol。
2.根据权利要求1所述的一种对干细胞具有活化作用的微量元素组合物,其特征在于,所述组合物包括:
铁 1mmol
铜 5μmol
锌 10μmol
钴 50μmol
锰 25μmol
铬 5μmol
硒 5nmol
碘 25μmol
镍 5μmol
钼 6μmol
钒 3μmol
锡 4nmol
硅 15mmol
锶 30μmol
硼 7μmol
铷 55μmol
锂 30μmol
锗 60nmol
钛 4mmol
钨 100μmol。
3.根据权利要求1所述的一种对干细胞具有活化作用的微量元素组合物,其特征在于,所述微量元素为各种形式的无毒性的有机金属化合物或无机金属化合物。
4.一种如权利要求1~3任一项所述的微量元素组合物的应用,其特征在于,将其应用于制备促进造血干细胞的活化的药物。
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