CN107686827B - 人脂肪干细胞特异性培养基 - Google Patents
人脂肪干细胞特异性培养基 Download PDFInfo
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Abstract
本发明提供一种人脂肪干细胞特异性培养基，由高糖型DMEM基础培养基、碱性成纤维细胞生长因子、人表皮细胞生长因子、重组人胰岛素、左旋卡尼丁、还原型谷胱甘肽、细胞增殖促进肽、亚油酸、α‑酮戊二酸组成。该培养基不仅能提供加速的人脂肪干细胞培养时间，同时培养得到人脂肪干细胞均一性较好，并且非常有利于后续的分化。
Description
技术领域
本发明涉及生物技术领域，特别涉及一种人脂肪干细胞特异性培养基。
背景技术
脂肪干细胞(adipose-derived stem cells，ADSCs)、ADSC多能细胞是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞。主要恢复组织细胞的修复功能，促进细胞的再生，恢复年轻面容的同时，身体机能也得到充分改善，有效改善亚健康、早衰等疾病，由内而外真正的有效抵抗衰老。
研究发现ADSCs细胞能够在体外稳定增殖且衰亡率低，同时它具有取材容易、少量组织即可获取大量干细胞，适宜大规模培养，对机体损伤小等优点，而且其来源广泛，体内储备量大，适宜自体移植，逐渐成为近年来新的研究热点之一。
传统的脂肪移植手段由于存在免疫排斥、炎症反应等缺陷难以得到让人满意的疗效。据统计，自体脂肪组织移植到缺损部位后，通常其中40％～60％会被吸收。通过患者自身的脂肪组织内的干细胞，来构建具有完整生物学结构和功能工程脂肪组织无疑将是解决这一难题的最佳方案。如何实现从干细胞到脂肪细胞的分化，是构建工程北京丽都脂肪组织不可回避的问题。早在2001年，Zuk等人发现脂肪干细胞以来，就已经证明脂肪干细胞具有向脂肪、软骨、成骨、成肌等多向分化潜能。传统的成脂诱导分化采用的方法多为混合诱导剂法。成脂诱导剂的不足是带有毒性，对人体有较大危害。
除了以上通过诱导剂进行体外诱导分化外，用成熟体细胞与干细胞共培养同样可以使干细胞定性诱导分化。2000年，Maurin等采用间接共培养的方式，研究脂肪细胞对成骨细胞扩增和活性的影响，并证明成熟脂肪细胞对人的成骨细胞的扩增有抑制作用；但对成骨基质细胞的扩增及活性，无显着影响。2003年Shigehisa等采用三维胶原凝胶中共培养脂肪细胞和内皮细胞的方式研究二者间的相互作用，得出结论：内皮细胞参与到脂肪组织形成和扩增的过程当中。2007年，Wang等通过实验证明，鼠来源脂肪干细胞在胶原凝胶三维体系中，与OECs(Olfactory ensheathing cells-嗅鞘细胞)共培养过程中，能够分化成为OECs-like(嗅鞘样细胞)。
人脂肪干细胞在现有技术中有多种培养方式。比如：CN 102732477 B公开了一种人脂肪干细胞无血清基础培养基。本培养基使用血清替代剂，由高糖型DMEM基础培养基、人血清白蛋白、转铁蛋白、牛磺酸、还原型谷胱甘肽、铜蓝蛋白、L-抗坏血酸-2-硫酸酯、α-生育酚琥珀酸单酯、亚油酸、α-酮戊二酸和硒组成。无血清基础培养基因能免除常规含血清培养基中动物血清给人体健康带来的潜在威胁，其培养的脂肪干细胞更适于临床应用。
CN 104877962 A提供一种无血清的脂肪干细胞培养基，属于干细胞技术领域，包括IMDM基础培养基，还包括如下组分：重组人胰岛素、重组转铁蛋白、维生素C、人血清白蛋白、成纤维细胞生长因子、血小板衍生生长因子、表皮生长因子、胰岛素样生长因子、转化生长因子、纤维连接蛋白、胎球蛋白、茶多酚和干细胞生长因子。
CN 102757936 B公开了一种人脂肪干细胞增殖促进剂及其应用方法。所述的人脂肪干细胞增殖促进剂由胰岛素、氢化可的松、甲状旁腺激素、雌二醇、睾酮、血小板衍生生长因子-AB、表皮生长因子、碱性成纤维细胞生长因子、L-谷氨酰胺、2-巯基乙醇、白血病抑制因子、L-抗坏血酸、生物素、地塞米松、IGF-I和SCF组成。
申请人发现以上的细胞培养基不仅成分复杂，有些组分成本高昂，而且细胞的培养一致性上还有改进的空间。
发明内容
本发明的目的是提供一种人脂肪干细胞特异性培养基，克服现有技术的培养基的缺点和风险。
本发明另外提供一种人脂肪干细胞特异性培养基，该培养基不仅能提供加速的人脂肪干细胞培养时间，同时培养得到人脂肪干细胞均一性较好，并且非常有利于后续的分化。
一种人脂肪干细胞特异性培养基，由以下几种成分组成：由高糖型DMEM基础培养基、碱性成纤维细胞生长因子、人表皮细胞生长因子、重组人胰岛素、左旋卡尼丁、还原型谷胱甘肽、细胞增殖促进肽、亚油酸、α-酮戊二酸组成。
本发明另外提供一种人脂肪干细胞特异性培养基，其中各组分的具体使用量为：DMEM/F12细胞培养液，碱性成纤维细胞生长因子20ng/ml，人表皮细胞生长因子25ng/ml，重组人胰岛素2mg/ml，左旋卡尼丁5mM，还原型谷胱甘肽浓度为10-12μg/mL，细胞增殖促进肽30-50ng/ml，亚油酸80-100mg/ml、α-酮戊二酸40-50mg/ml组成。
本发明另外提供一种细胞增殖促进肽，其序列为QLHYTRIPCFFDKQRGHCGWPTKRGHR(SEQIDNO：1)。该肽为申请人前期研究获得的能够显著促进细胞增殖的活性小肽。
该培养基提供各种丰富的因子和蛋白、多肽和微量元素参与细胞代谢，可以提供丰富的营养，维持细胞的高活性加速传代。同时该培养基不含动物血清，不含动物血清内潜在动物源性内毒素或病毒，方便应用于临床。
附图说明
图1是本发明人脂肪干细胞特异性培养基与常规含血清基础培养基培养人脂肪干细胞生长曲线图；对照是采用实施例1常规的含血清基础培养基培养；培养基A和培养基B为采用本发明实施例2和3进行培养的结果。
具体实施方式
下述实施例中的实验方法，如无特别说明，均为常规方法。实验所用器具仪器试剂皆可通过商业途径获得。
实施例1:普通人脂肪干细胞基础培养基配制:
取900mL的DMEM/F12细胞培养液(从TAKARA BIO INC.购得)加抗生素:青霉素90000U，链霉素90000U。
调pH值:用质量分数5％NaHCO3调节pH到7.2。过滤除菌。
加小牛血清:临用前将加入小牛血清定容至1OOOmL(从TAKARA BIO INC.购得)。
实施例2:制备本发明的人脂肪干细胞特异性培养基A
预定量为1OOOmL则取900mL的DMEM/F12细胞培养液(TAKARA BIO INC.)，加抗生素:青霉素90000U，链霉素90000U。
调pH值:用质量分数5％NaHCO3调节pH到7.2。
加入各组分:按碱性成纤维细胞生长因子20ng/ml，人表皮细胞生长因子25ng/ml，重组人胰岛素2mg/ml，左旋卡尼丁5mM，还原型谷胱甘肽浓度为10μg/mL，细胞增殖促进肽(序列如SEQ ID NO：1所示)50ng/ml，亚油酸100mg/ml、α-酮戊二酸40mg/ml所示成分与量称量并依次加入到上述配制好的DMEM/F12细胞培养液中，振荡或超声助溶，不加热助溶。过滤除菌:采用孔径0.45um和0.22um滤膜各一张，上层为0.45um,下层为0.22um,以保证过滤效果。
实施例3:制备本发明的人脂肪干细胞特异性培养基B
预定量为1OOOmL则取900mL的DMEM/F12细胞培养液(TAKARA BIO INC.)，加抗生素:青霉素90000U，链霉素90000U。
调pH值:用质量分数5％NaHCO3调节pH到7.2。
加入各组分:按碱性成纤维细胞生长因子20ng/ml，人表皮细胞生长因子25ng/ml，重组人胰岛素2mg/ml，左旋卡尼丁5mM，还原型谷胱甘肽浓度为12μg/mL，细胞增殖促进肽(序列如SEQ ID NO：1所示)30ng/ml，亚油酸80mg/ml、α-酮戊二酸50mg/ml所示成分与量称量并依次加入到上述配制好的DMEM/F12细胞培养液中，振荡或超声助溶，不加热助溶。过滤除菌:采用孔径0.45um和0.22um滤膜各一张，上层为0.45um,下层为0.22um,以保证过滤效果。
实施例4细胞培养增殖实验
将1*106个的脂肪干细胞分别接种到三组盛有不同的培养基的直径100mm培养皿中，每组10个培养皿，混匀；
加入培养基:
第一组10个培养皿，加入实施例1制备的培养基5mL；
第二组10个培养皿，加入实施例2制备的培养基A5mL；
第三组10个培养皿，加入实施例3制备的培养基B5mL。
将细胞放在在37℃，5％CO2的培养箱中进行培养；置于倒置式显微镜观察，每隔3天用移液管吸去上层培养基，加入新的培养基，继续培养。
细胞计数:每天在各组中取一个培养皿放入洁净工作台，然后用移液管吸弃培养基。PBS洗涤2次，加入1ml0.25％Trypsin-EDTA，倒置显微镜下发现贴壁细胞分离后，加入4ml含有10％FBS的基础培养基终止胰酶作用。台盼蓝(Typan Blue)染色血细胞计数板计数活细胞数。
实验结果如图1所示，实施例1-3的培养基同样能有效地培养人脂肪干细胞。但是本发明的培养基A和B培养的细胞在生长速率、数量上都能够超过常规有血清培养基培养的细胞，具有较好的培养效果。
表面抗原分析，取用本发明的培养基培养的原代脂肪干细胞，去掉培养液，用1:1的2.5％的胰蛋白酶溶液和0.02％EDTA溶液混合消化，用含1％牛血清白蛋白(BSA)的PBS洗涤后制成每100ul含有2*106个单细胞悬液，等分为7份，分别加入7个Eppendorf管中并编号，一号管加入共20μL的FITC Mouse IgGl、APC_CY7Mouse IgG2b和染色缓冲液用于检测由于抗体非特异性结合产生的背景作为对照，其他试管分别加入CD29、CD34、CD44、CD45、CD105和HLA.DR单克隆抗体各20μL，每管分别加入细胞悬液100μL(含2*1O6个细胞)，室温孵育25min，用含1％BSA的PBS洗涤后，流式细胞仪检测。
分析结果:流式细胞仪分析原代人脂肪干细胞六种表面抗原,结果表明使用无血清培养基培养出的细胞具有人脂肪干细胞特性。HLA.DR阴性，排除该类细胞是成纤维细胞，流式细胞仪分析结果见表1。
| 表面抗原 | CD29 | CD34 | CD44 | CD45 | CD105 | HLA.DR |
| 流式表达 | + | - | + | - | + | - |
表1
实施例5细胞分化实验-脂肪干细胞的成脂诱导
1.将培养的干细胞以1.5*106每孔的密度接种在六孔板中，进行成脂诱导实验。具体方法是：向基础培养基(DMEM+10％胎牛血清)中分别加入终浓度分别为1μmol/L地塞米松、10μmol/L胰岛素、200μmol/L吲哚美辛和0.5mmol/L的异丁基甲基黄噪呤，配制成脂诱导培养基。每周换2次液，直到进行成脂染色观察为止，以上组均做平行实验(η＝3)。
2.油红O染色
先小心轻缓倒去培养液，用D-hanks轻缓漂洗，加10％中性甲醛固定细胞膜30min。加入0.5％油红0(六孔板加1.5ml)，染色1h左右。
3.脱色，75％酒精/60％异丙醇漂洗，除去多余的染料。
4.复染，淡苏木染色1min，PBS漂洗。
5.甘油明胶封片，显微镜观察
每组样本随机选取5-10个视野进行拍照观察以考察共培养方法的成脂诱导分化效果。脂肪呈鲜红色，细胞核呈蓝色，间质无色。
6.结果见下表
表2
从表2的结果可以看出，本发明培养基制备得到人脂肪干细胞具有较好的诱导分化能力，具有显著地优势。
以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明造构思的前提下，还可以做出若干改变和改进，这些都属于发明的保护范围。
序列表
<110> 洛阳轩智生物科技有限公司
<120> 人脂肪干细胞特异性培养基
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Claims (2)
1.一种人脂肪干细胞特异性培养基，由以下几种成分组成：DMEM/F12细胞培养液，碱性成纤维细胞生长因子20ng/ml，人表皮细胞生长因子25ng/ml，重组人胰岛素2mg/ml，左旋卡尼丁5mM，还原型谷胱甘肽浓度为10-12μg/mL，细胞增殖促进肽30-50ng/ml，亚油酸80-100mg/ml、α-酮戊二酸40-50mg/ml组成；其中，细胞增殖促进肽其序列为SEQ ID NO：1所示。
2.权利要求1所示的培养基在培养人脂肪干细胞中的用途。
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