CN113957042A - 一种干细胞零蛋白培养基及干细胞生长因子的制备方法 - Google Patents
一种干细胞零蛋白培养基及干细胞生长因子的制备方法 Download PDFInfo
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Abstract
本发明公开了一种干细胞零蛋白培养基及干细胞生长因子的制备方法，涉及生物领域。本发明的干细胞零蛋白培养基包括氨基酸、维生素及矿物质，成分明确，且可维持干细胞的生长和繁殖，促进分泌更多的生长因子。不含有外源血清、蛋白和因子，因此不会改变干细胞分泌的生长因子的种类及浓度比。
Description
技术领域
本发明属于生物领域，具体涉及一种干细胞零蛋白培养基及干细胞培养细胞生长因子的方法。
背景技术
干细胞是一类具有自我更新，高度增殖和多向分化能力的的细胞群体，根据个体发育过程中出现的先后次序不同，干细胞可分为胚胎干细胞和成体干细胞，胚胎干细胞可以分化为特定功能的干细胞，如皮肤干细胞可分化为多种皮肤细胞，造血干细胞分化为红细胞、白细胞和血小板等。成体干细胞是一种从已经分化组织中提取的未分化的干细胞，这种细胞可以自我更新，在一定条件下可以分化为该类组织的细胞。
脂肪干细胞(ADSCs)来源于脂肪组织的成体干细胞，是一种处于静止状态，未分化的干细胞，其体外扩增和自我增殖能力很强。对组织细胞具有修复功能，可以促进细胞的再生。由于脂肪组织易获得，患者痛苦小、供应充足，可反复取材，无论理道德上的争议，因此脂肪干细胞在临床治疗上具有巨大的潜能。脂肪干细胞在培养过程中能够分泌大量的生长因子,细胞生长因子是一种多功能强力细胞因子，对促进成纤维细胞的代谢和胶原蛋白的形成发挥着重要功能,能够促进细胞的生长、修复并补充营养。
人脂肪干细胞有多重的培养方式，传统干细胞的培养基通常采用添加血清或者添加生长因子的无血清培养基，这些培养基及都是含蛋白的培养基，这些添加的蛋白可以促进细胞生长，但同时因为异种蛋白的加入导致后期生长因子纯化过程中难于除掉，干扰干细胞自身分泌的蛋白比例和数量，降低了产品的安全性。
CN103898049A公开了一种干细胞无血清基础培养基，其中以DMEM/F12为基础，添加了碱性纤维细胞生长因子、表皮生长因子、抗生素、谷氨酸盐、非必须氨基酸、抗氧化剂。无血清的培养基能避免异源动物血清给人体带来的潜在威胁，其培养的细胞生长因子更适用于人的皮肤。但是由于此发明中添加了碱性纤维细胞生长因子、表皮生长因子这次重组多肽类的因子，导致后期在纯化的过程中难于清楚干净，影响后期细胞分泌的因子的数量和后期的检测。
WO2018165997A1提供了一种人类过潜能干细胞的无血清培养基，添加了无机盐成分、有机成分、氨基酸及氨基酸盐等能量物质，及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽等，是一种可以用制药领域的高端无血清培养基。但此培养基的添加了蛋白质及多肽等多种物质，含有多种蛋白和因子类物质，造价昂贵。
发明内容
为了克服现有技术中的缺陷，本发明提供一种脂肪干细胞培养基及利用该培养基培养干细胞生长因子的制备方法。本发明所提供的培养基中不添加蛋白质类物质，能够维持干细胞的生长，并分泌更多的生长因子，且不影响分泌的生长因子之间的浓度和种类。
为了实现上述目的，本发明提供如下技术方案：
本发明提供了一种干细胞零蛋白培养基，每升零蛋白培养基中包含以下质量组分：
甘氨酸14-19mg/L、L-丙氨酸3-5mg/L、L-精氨酸盐酸盐100-200mg/L、L-天冬酰胺6-10mg/L、L-天冬氨酸4-11mg/L、L-半胱氨酸盐酸盐11-20mg/L、L-胱氨酸30-49mg/L、L-谷氨酸6-10mg/L、L-谷氨酰胺300-400mg/L、L-谷氨酸盐酸盐30-50mg/L、L-异亮氨酸50-70mg/L、L-亮氨酸55-65mg/L、L-赖氨酸90-104mg/L、L-蛋氨酸15-21mg/L、L-苯丙氨酸25-36mg/L、L-脯氨酸14-18mg/L、L-丝氨酸25-34mg/L、L-苏氨酸48-58mg/L、L-色氨酸5-12mg/L、L-缬氨酸49-55mg/L；
D-葡萄糖3000-5000mg/L、生物素0.002-0.015mg/L、氯化胆碱8.04-9.05mg/L、D-泛酸钙2.0-2.5mg/L、烟酰胺1.8-2.0mg/L、维生素B6 2.0-5mg/L、核黄素0.15-0.3mg/L、硫胺素2.0-2.3mg/L、维生素B12 0.6-0.7mg/L、肌醇10-15mg/L；
氯化钙110-150mg/L、五水硫酸铜0.0005-0.002mg/L、硫酸镁40-49mg/L、七水合硫酸铁0.04-0.07mg/L、氯化钾250-320mg/L、碳酸氢钠1100-1200mg/L、氯化钠7000-8000mg/L、磷酸氢二钠71-75mg/L、一水合磷酸二氢钠55-63mg/L；
次黄嘌呤2.0-2.8mg/L、亚油酸0.03-0.07mg/L、硫辛酸0.01-0.02mg/L、丙酮酸钠55-62mg/L、胸腺嘧啶0.34-0.38mg/L。
作为一种优选的实施方式，本发明每升零蛋白培养基中包含以下质量组分：
甘氨酸14-16mg/L、L-丙氨酸3.5-4.5mg/L、L-精氨酸盐酸盐120-140mg/L、L-天冬酰胺6.5-7.0mg/L、L-天冬氨酸4-6mg/L、L-半胱氨酸盐酸盐11-13mg/L、L-胱氨酸34-40mg/L、L-谷氨酸7-9mg/L、L-谷氨酰胺370-390mg/L、L-谷氨酸盐酸盐38-40mg/L、L-异亮氨酸54-60mg/L、L-亮氨酸60-65mg/L、L-赖氨酸99-102mg/L、L-蛋氨酸17-20mg/L、L-苯丙氨酸26-29mg/L、L-脯氨酸16-18mg/L、L-丝氨酸27-30mg/L、L-苏氨酸50-56mg/L、L-色氨酸7-9mg/L、L-缬氨酸50-52mg/L；
D-葡萄糖3000-4000mg/L、生物素0.002-0.009mg/L、氯化胆碱8.4-8.5mg/L、D-泛酸钙2.3-2.5mg/L、烟酰胺1.9-2.0mg/L、维生素B6 4-5mg/L、核黄素0.15-0.2mg/L、硫胺素2.0-2.1mg/L、维生素B12 0.65-0.69mg/L、肌醇12-13mg/L；
氯化钙120-130mg/L、五水硫酸铜0.0008-0.001mg/L、硫酸镁45-49mg/L、七水合硫酸铁0.05-0.07mg/L、氯化钾290-320mg/L、碳酸氢钠1150-1200mg/L、氯化钠7800-8000mg/L、磷酸氢二钠71-72mg/L、一水合磷酸二氢钠59-60mg/L；
次黄嘌呤2.0-2.4mg/L、亚油酸0.04-0.06mg/L、硫辛酸0.015-0.018mg/L、丙酮酸钠57-60mg/L、胸腺嘧啶0.37-0.38mg/L。
其中，本发明所述干细胞为脂肪干细胞、脐带间充质干细胞、牙髓干细胞或骨髓干细胞。
本发明还提供了一种利用上述零蛋白培养基培养干细胞生长因子的方法，包括以下步骤：
(1)从离体组织中提取原代干细胞；
(2)将原代干细胞过细胞筛接种到含血清培养基中培养，得到4-6代传代干细胞；
(3)在细胞汇合率为70-95％的时候，更换权利要求1或2所述的零蛋白培养基，5％二氧化碳低氧条件下培养，离心得到的上清液为干细胞生长因子提取物。
其中，作为一种可选的实施方式，步骤(1)具体为：将含有干细胞的离体组织清洗、酶解后采用培养基终止酶解消化，然后经重悬和离心分离后，得到原代干细胞。
进一步的，所述酶解采用胰酶和/或胶原酶在35-38℃下酶解20-60min。
进一步的，所述胰酶和/或胶原酶与所述组织的体积比为1:3-5。
作为一种可选的实施方式，步骤(2)所述原代干细胞过50-100μm的细胞筛接种到10％胎牛血清的MEM培养基中进行培养。
作为一种可选的实施方式，步骤(3)所述低氧条件培养的时间为3-4d。
其中，本发明上述方式得到的干细胞生长因子提取物包括肝细胞生长因子，血小板来源生长因子，表皮生长因子，转化生长因子β，碱性成纤维细胞生长因子，吲哚胺2,3-双加氧酶，白细胞介素，角质细胞生长因子，血管内皮细胞生长因子，胰岛素样生长因子。
本发明带来的有益效果：
本发明的干细胞零蛋白培养基包括氨基酸、维生素及矿物质，成分明确，且可维持细胞的生长和繁殖，促进分泌更多的生长因子。氨基酸类是组成蛋白质的基本单位。不同种类的细胞对氨基酸的要求各异，但有几种氨基酸细胞自身不能合成，必须依靠培养液提供。其中谷氨酰胺是细胞合成核酸和蛋白质必需的氨基酸，在缺少谷氨酰胺时，细胞生长不良而死亡。维生素是维持细胞生长的生物活性物质，在细胞代谢中起调节及控制作用。在细胞培养基中添加了各种维生素以适合更多的细胞系生长。许多维生素参与构成各种酶的活性基团的成分，没有它们，酶便没有活性，代谢活动将无法进行。D-葡萄糖和丙酮酸钠在细胞培养基中是提供细胞能量代谢的能源，其中葡萄糖相对于其他己醛糖来说具有非特异性与蛋白质的胺基反应的的倾向，丙酮酸对过氧化氢也具有保护作用；次黄嘌呤以核苷肌苷的形式存在于tRNA的反密码子，在零蛋白培养基中作为干细胞必须添加的底物和氮源。
传统的生长因子培养基添加了动物来源的血清、蛋白和因子，均会影响因子的分泌。本发明所提供用于干细胞的培养基不含有外源血清、蛋白和因子，因此不会改变干细胞分泌的生长因子的种类及浓度比，能够维持干细胞的生长增殖。
附图说明
为使本发明的目的、技术方案和有点更加清楚，下面结合附图对本发明的技术方案进行详细描述。
图1为脂肪干细胞生长因子的制备方法流程图；
图2为脂肪干细胞在本发明零蛋白培养基上低氧的条件下第一天生长状况的图片。
图3为脂肪干细胞在本发明零蛋白培养基上低氧的条件下第四天生长状况的图片。
具体实施方式
下面将结合本发明的实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例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
用于干细胞培养细胞生长因子的零蛋白培养基
称取一定量下列组分溶于蒸馏水中至目标浓度，得到干细胞零蛋白培养基。
甘氨酸14mg/L、L-丙氨酸3.5mg/L、L-精氨酸盐酸盐120mg/L、L-天冬酰胺6.5mg/L、L-天冬氨酸4mg/L、L-半胱氨酸盐酸盐11mg/L、L-胱氨酸34mg/L、L-谷氨酸7mg/L、L-谷氨酰胺370mg/L、L-谷氨酸盐酸盐38mg/L、L-异亮氨酸54mg/L、L-亮氨酸60mg/L、L-赖氨酸99mg/L、L-蛋氨酸17mg/L、L-苯丙氨酸26mg/L、L-脯氨酸16mg/L、L-丝氨酸27mg/L、L-苏氨酸50mg/L、L-色氨酸7mg/L、L-缬氨酸50mg/L；
D-葡萄糖3000mg/L、生物素0.002mg/L、氯化胆碱8.4mg/L、D-泛酸钙2.3mg/L、烟酰胺1.9mg/L、维生素B6 4.0mg/L、核黄素0.15mg/L、硫胺素2.0mg/L、维生素B12 0.65mg/L、肌醇12mg/L；
氯化钙120mg/L、五水硫酸铜0.0008mg/L、硫酸镁45mg/L、七水合硫酸铁0.05mg/L、氯化钾290mg/L、碳酸氢钠1150mg/L、氯化钠7700mg/L、磷酸氢二钠71mg/L、一水合磷酸二氢钠59mg/L；
次黄嘌呤2.0mg/L、亚油酸0.04mg/L、硫辛酸0.015mg/L、丙酮酸钠57mg/L、胸腺嘧啶0.37mg/L。
实施例2
用于干细胞培养细胞生长因子的零蛋白培养基
称取一定量下列组分溶于蒸馏水中至目标浓度，得到干细胞零蛋白培养基。
甘氨酸15mg/L、L-丙氨酸4.0mg/L、L-精氨酸盐酸盐130mg/L、L-天冬酰胺6.8mg/L、L-天冬氨酸5mg/L、L-半胱氨酸盐酸盐12mg/L、L-胱氨酸37mg/L、L-谷氨酸8mg/L、L-谷氨酰胺380mg/L、L-谷氨酸盐酸盐39mg/L、L-异亮氨酸57mg/L、L-亮氨酸63mg/L、L-赖氨酸100mg/L、L-蛋氨酸18mg/L、L-苯丙氨酸27mg/L、L-脯氨酸17mg/L、L-丝氨酸28mg/L、L-苏氨酸52mg/L、L-色氨酸8mg/L、L-缬氨酸51mg/L；
D-葡萄糖3500mg/L、生物素0.004mg/L、氯化胆碱8.4mg/L、D-泛酸钙2.4mg/L、烟酰胺1.9mg/L、维生素B6 4.0mg/L、核黄素0.17mg/L、硫胺素2.0mg/L、维生素B12 0.68mg/L、肌醇12mg/L；
氯化钙125mg/L、五水硫酸铜0.0009mg/L、硫酸镁46mg/L、七水合硫酸铁0.06mg/L、氯化钾300mg/L、碳酸氢钠1170mg/L、氯化钠7900mg/L、磷酸氢二钠71mg/L、一水合磷酸二氢钠59mg/L；
次黄嘌呤2.2mg/L、亚油酸0.05mg/L、硫辛酸0.016mg/L、丙酮酸钠58mg/L、胸腺嘧啶0.37mg/L。
实施例3
用于干细胞培养细胞生长因子的零蛋白培养基
称取一定量下列组分溶于蒸馏水中至目标浓度，得到干细胞零蛋白培养基。
甘氨酸16mg/L、L-丙氨酸4.5mg/L、L-精氨酸盐酸盐140mg/L、L-天冬酰胺7.0mg/L、L-天冬氨酸6mg/L、L-半胱氨酸盐酸盐13mg/L、L-胱氨酸40mg/L、L-谷氨酸9mg/L、L-谷氨酰胺390mg/L、L-谷氨酸盐酸盐40mg/L、L-异亮氨酸60mg/L、L-亮氨酸65mg/L、L-赖氨酸102mg/L、L-蛋氨酸20mg/L、L-苯丙氨酸29mg/L、L-脯氨酸18mg/L、L-丝氨酸30mg/L、L-苏氨酸56mg/L、L-色氨酸9mg/L、L-缬氨酸52mg/L；
D-葡萄糖4000mg/L、生物素0.009mg/L、氯化胆碱8.5mg/L、D-泛酸钙2.5mg/L、烟酰胺2.0mg/L、维生素B6 5.0mg/L、核黄素0.2mg/L、硫胺素2.1mg/L、维生素B12 0.69mg/L、肌醇13mg/L；
氯化钙130mg/L、五水硫酸铜0.001mg/L、硫酸镁49mg/L、七水合硫酸铁0.07mg/L、氯化钾320mg/L、碳酸氢钠1200mg/L、氯化钠8000mg/L、磷酸氢二钠72mg/L、一水合磷酸二氢钠60mg/L；
次黄嘌呤2.4mg/L、亚油酸0.06mg/L、硫辛酸0.018mg/L、丙酮酸钠60mg/L、胸腺嘧啶0.38mg/L。
实施例4
请参见图1所示，一种以干细胞的零蛋白培养基培养生长因子的方法，包括以下步骤：
将盛有25ml脂肪组织的容器消毒后，用生理盐水清洗脂肪组织，静置分层后移除上层，反复清洗至底层液体清亮为止，得到洁净的脂肪组织以备用。
将5ml的0.25％胶原酶加入人体脂肪中36℃消化40min，终止消化，2500rpm离心5min，收集细胞，过100μm的细胞筛，得到原代脂肪干细胞沉淀物。
将得到的原代脂肪组织干细胞加入到10％胎牛血清的MEM培养基中，放入培养箱中，37℃，5％二氧化碳湿润环境中培养1-5d。在汇合率为70-95％的时候，用胰酶消化，离心，传代获得第2-6代脂肪干细胞。将纯化后的脂肪干细胞液氮保存为种子细胞。
取出冻存的脂肪干细胞，复苏到T150的培养瓶中，37℃，5％二氧化碳湿润环境中培养40-72h，至汇合率为70-95％；
去除培养瓶中的培养基，用PBS作为缓冲液清洗1-4次；
将实施例2中的零蛋白培养基加入培养瓶中，37℃，5％二氧化碳低氧环境中培养48-96h，得到含有细胞生长因子的混合物；
将细胞分离，即得到含生长因子的上清液。
通过图2和图3可以看出传代的脂肪干细胞在零蛋白培养基上低氧条件下，依然能够很好的增殖。
用ELISA试剂盒检测生长因子浓度：HGF肝细胞生长因子；PDGF血小板来源生长因子；EGF表皮生长因子；TGF-β转化生长因子β；bFGF碱性成纤维细胞生长因子；I DO吲哚胺2,3-双加氧酶；IL-10白细胞介素；KGF角质细胞生长因子；VEGF血管内皮细胞生长因子；IGF-1胰岛素样生长因子的浓度，检测合格后冻存于-20℃冷冻保存。
下表为生长因子的最低浓度，低于此值则不合格。
实施例5
一种以骨髓干细胞的零蛋白培养基培养生长因子的方法，包括以下步骤：
将盛有骨髓组织的容器消毒后，用生理盐水清洗骨髓组织，静置分层后移除上层，反复清洗至底层液体清亮为止，得到洁净的骨髓组织以备用。
将胶原酶加入骨髓组织中37℃消化30min，终止消化，1800rpm离心10min，收集细胞，过50μm的细胞筛，将得到骨髓干细胞；
将骨髓干细胞加入到10％胎牛血清的MEM培养基中，放入培养箱中，37℃，5％二氧化碳湿润环境中培养1-5d。在汇合率为70-95％的时候，用胰酶消化，离心，传代获得第2-6代骨髓干细胞。将纯化后的骨髓干细胞液氮保存为种子细胞；
取出冻存的骨髓干细胞，复苏到T150的培养瓶中，37℃，5％二氧化碳湿润环境中培养40-72h，至汇合率为70-95％；
去除培养瓶中的培养基，用生理盐水清洗瓶1-4次；
将实施例3中的零蛋白培养基加入培养瓶中，37℃，5％二氧化碳低氧环境中培养48-96h，得到含有细胞生长因子的混合物；
将细胞分离，即得到含生长因子的上清液。
实施例6
实施例5中的零蛋白培养基中添加10ml 10％胎牛血清，内毒素含量小于10EU/ml，血红蛋白含量小于10mg/dl，其余制备方法与实施例5相同。
通过ELISA试剂盒分别检测零蛋白添加培养基和添加血清的培养基制备的各种生长因子的浓度，得出结果如表1所示。
表1不同培养基培养脂肪干细胞得到的细胞生长因子含量
注：HGF指肝细胞生长因子；PDGF指血小板来源生长因子；EGF指表皮生长因子；TGF-β指转化生长因子β；bFGF指碱性成纤维细胞生长因子；I DO指吲哚胺2,3-双加氧酶；IL-10指白细胞介素；KGF指角质细胞生长因子；VEGF指血管内皮细胞生长因子；IGF-1指胰岛素样生长因子。
从表1可以看出，本发明的零蛋白培养基培养的脂肪干细胞能够分泌更多的细胞因子，并且不会改变干细胞本身所分泌的生长因子的种类和浓度比例。
实施例7
实施例5中的零蛋白培养基中的B族维生素去除，内毒素含量小于10EU/ml，血红蛋白含量小于10mg/dl，其余制备方法与实施例5相同。
通过ELISA试剂盒分别检测零蛋白添加培养基和无B族维生素的零蛋白培养基制备的各种生长因子的浓度，得出结果如表2所示。
表2不同培养基培养脂肪干细胞得到的细胞生长因子含量
注：HGF指肝细胞生长因子；PDGF指血小板来源生长因子；EGF指表皮生长因子；TGF-β指转化生长因子β；bFGF指碱性成纤维细胞生长因子；I DO指吲哚胺2,3-双加氧酶；IL-10指白细胞介素；KGF指角质细胞生长因子；VEGF指血管内皮细胞生长因子；IGF-1指胰岛素样生长因子。
从表2可以看出，本发明的零蛋白培养基未添加B族维生素，培养的脂肪干细胞分泌的细胞因子量减少。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种干细胞零蛋白培养基，其特征在于，所述零蛋白培养基中包含以下浓度的下列组分：
甘氨酸14-19mg/L、L-丙氨酸3-5mg/L、L-精氨酸盐酸盐100-200mg/L、L-天冬酰胺6-10mg/L、L-天冬氨酸4-11mg/L、L-半胱氨酸盐酸盐11-20mg/L、L-胱氨酸30-49mg/L、L-谷氨酸6-10mg/L、L-谷氨酰胺300-400mg/L、L-谷氨酸盐酸盐30-50mg/L、L-异亮氨酸50-70mg/L、L-亮氨酸55-65mg/L、L-赖氨酸90-104mg/L、L-蛋氨酸15-21mg/L、L-苯丙氨酸25-36mg/L、L-脯氨酸14-18mg/L、L-丝氨酸25-34mg/L、L-苏氨酸48-58mg/L、L-色氨酸5-12mg/L、L-缬氨酸49-55mg/L；
D-葡萄糖3000-5000mg/L、生物素0.002-0.015mg/L、氯化胆碱8.04-9.05mg/L、D-泛酸钙2.0-2.5mg/L、烟酰胺1.8-2.0mg/L、维生素B6 2.0-5mg/L、核黄素0.15-0.3mg/L、硫胺素2.0-2.3mg/L、维生素B12 0.6-0.7mg/L、肌醇10-15mg/L；
氯化钙110-150mg/L、五水硫酸铜0.0005-0.002mg/L、硫酸镁40-49mg/L、七水合硫酸铁0.04-0.07mg/L、氯化钾250-320mg/L、碳酸氢钠1100-1200mg/L、氯化钠7000-8000mg/L、磷酸氢二钠71-75mg/L、一水合磷酸二氢钠55-63mg/L；
次黄嘌呤2.0-2.8mg/L、亚油酸0.03-0.07mg/L、硫辛酸0.01-0.02mg/L、丙酮酸钠55-62mg/L、胸腺嘧啶0.34-0.38mg/L。
2.根据权利要求1所述的零蛋白培养基，其特征在于，所述零蛋白培养基中包含以下浓度的下列组分：
甘氨酸14-16mg/L、L-丙氨酸3.5-4.5mg/L、L-精氨酸盐酸盐120-140mg/L、L-天冬酰胺6.5-7.0mg/L、L-天冬氨酸4-6mg/L、L-半胱氨酸盐酸盐11-13mg/L、L-胱氨酸34-40mg/L、L-谷氨酸7-9mg/L、L-谷氨酰胺370-390mg/L、L-谷氨酸盐酸盐38-40mg/L、L-异亮氨酸54-60mg/L、L-亮氨酸60-65mg/L、L-赖氨酸99-102mg/L、L-蛋氨酸17-20mg/L、L-苯丙氨酸26-29mg/L、L-脯氨酸16-18mg/L、L-丝氨酸27-30mg/L、L-苏氨酸50-56mg/L、L-色氨酸7-9mg/L、L-缬氨酸50-52mg/L；
D-葡萄糖3000-4000mg/L、生物素0.002-0.009mg/L、氯化胆碱8.4-8.5mg/L、D-泛酸钙2.3-2.5mg/L、烟酰胺1.9-2.0mg/L、维生素B6 4-5mg/L、核黄素0.15-0.2mg/L、硫胺素2.0-2.1mg/L、维生素B12 0.65-0.69mg/L、肌醇12-13mg/L；
氯化钙120-130mg/L、五水硫酸铜0.0008-0.001mg/L、硫酸镁45-49mg/L、七水合硫酸铁0.05-0.07mg/L、氯化钾290-320mg/L、碳酸氢钠1150-1200mg/L、氯化钠7800-8000mg/L、磷酸氢二钠71-72mg/L、一水合磷酸二氢钠59-60mg/L；
次黄嘌呤2.0-2.4mg/L、亚油酸0.04-0.06mg/L、硫辛酸0.015-0.018mg/L、丙酮酸钠57-60mg/L、胸腺嘧啶0.37-0.38mg/L。
3.根据权利要求1或2所述的干细胞零蛋白培养基，其特征在于，所述干细胞为脂肪干细胞、脐带间充质干细胞、牙髓干细胞或骨髓干细胞。
4.一种制备干细胞生长因子提取物的方法，其特征在于，包括以下步骤：
(1)从离体组织中提取原代干细胞；
(2)将原代干细胞过细胞筛接种到含血清培养基中培养，得到4-6代传代干细胞；
(3)在细胞汇合率为70-95％的时候，更换权利要求1或2所述的零蛋白培养基，5％二氧化碳低氧条件下培养，离心得到的上清液为干细胞生长因子提取物。
5.根据权利要求4所述的方法，其特征在于，步骤(1)具体为：将含有干细胞的离体组织清洗、酶解后采用培养基终止酶解消化，然后经重悬和离心分离后，得到原代干细胞。
6.根据权利要求5所述的方法，其特征在于，所述酶解采用胰酶和/或胶原酶在35-38℃下酶解20-60min。
7.根据权利要求6所述的方法，其特征在于，所述胰酶和/或胶原酶与所述组织的体积比为1:3-5。
8.根据权利要求4所述的方法，其特征在于，步骤(2)所述原代干细胞过50-100μm的细胞筛接种到10％胎牛血清的MEM培养基中进行培养。
9.根据权利要求4所述的方法，其特征在于，步骤(3)所述低氧条件培养的时间为3-4d。
10.根据权利要求4所述的方法，其特征在于，所述干细胞生长因子提取物包括肝细胞生长因子，血小板来源生长因子，表皮生长因子，转化生长因子β，碱性成纤维细胞生长因子，吲哚胺2,3-双加氧酶，白细胞介素，角质细胞生长因子，血管内皮细胞生长因子，胰岛素样生长因子。
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