CN114774353A - 一种干细胞培养基 - Google Patents
一种干细胞培养基 Download PDFInfo
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- CN114774353A CN114774353A CN202210343646.4A CN202210343646A CN114774353A CN 114774353 A CN114774353 A CN 114774353A CN 202210343646 A CN202210343646 A CN 202210343646A CN 114774353 A CN114774353 A CN 114774353A
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Abstract
本发明公开了一种干细胞培养基，包括基础培养基、血小板裂解物、L‑谷氨酰胺和丝裂霉素c、罗汉果苷V，其中：血小板裂解物在干细胞培养基的体积百分比为5％、L‑谷氨酰胺在干细胞培养基的浓度为4mmol/L、丝裂霉素c在干细胞培养基的浓度为50ng/ml，罗汉果苷V在干细胞培养基的浓度为10μg/ml。用该干细胞培养基分离干细胞，能避免现有干细胞培养基大多含有血清而引入外源病毒的风险，而且公开的干细胞分离方法无需胶原酶消化法分离，能提高干细胞分离效率。
Description
【技术领域】
本发明涉及培养基技术领域，具体涉及一种干细胞培养基。
【背景技术】
干细胞(StemCell)是一种具有自我更新能力以及多向分化潜能的细胞。按照分化潜能，干细胞可分为三大类：全能干细胞、多能干细胞、单能干细胞。按照发育阶段，干细胞又可以分为：胚胎干细胞与成体干细胞。由于伦理学的限制，现在用于临床研究和应用的干细胞多为成体干细胞。成体干细胞来源于成年动物的许多组织和器官，包括造血干细胞、神经干细胞、脂肪干细胞、骨髓间充质干细胞、脐带干细胞、脐血干细胞等等。
造血干细胞是迄今为止唯一广泛应用于临床治疗的干细胞类型。造血干细胞移植技术能够治疗白血病、淋巴瘤等多种血液类疾病，甚至能够对代谢性疾病、先天性免疫缺陷、糖尿病等疾病产生疗效；据统计，世界范围内每年接受造血干细胞移植治疗的案例超过40000例，其中多数造血干细胞的供体来源于捐献者的骨髓和动员的外周血干细胞；尽管该技术取得了巨大的成功，但是由于人类白细胞抗原(HLA)配型的严格配对要求，70％的患者不能获得合适的供体。脐带血造血干细胞对于HLA配型的要求相对较低，且免疫原性低，再加上其获取方便、来源丰富，成为造血干细胞移植供体的一大来源。自20世纪80年代首例脐带血造血干细胞移植手术成功治愈Faconi男童患者后，脐带血造血干细胞移植案例逐年增加。据统计，世界范围内脐带血造血干细胞移植案例已超过30000例，且还在不断增加。目前，脐带血造血干细胞移植技术的瓶颈在于其细胞含量少，一份脐带血中所含的造血干细胞及祖细胞数量不足以快速恢复成人患者的免疫系统，造成机会性感染致死率的增高。目前暂行的策略是双份脐带血移植，即一位患者清髓后先后接受两份脐带血的移植，但这增加了供体的HLA配型匹配难度。
罗汉果(Siraitia grosvenorii)是一种发现于中国南部地区著名的食药用植物。罗汉果中的总苷为葫芦烷型三萜苷类化合物，包括罗汉果苷V(Mogroside V，MV)、罗汉果苷IVe(Mogroside IVe)、罗汉果苷IIIe(Mogroside IIIe)、罗汉果苷II A2(Mogroside IIA2)、罗汉果苷III A1(Mogroside III A1)、罗汉果苷IVa(Mogroside IVa)、罗汉果苷I(MogrosideVI)、赛门苷I(Siamenoside I)、11-O-罗汉果苷V(11-Oxomogroside V)等，其中的Mogroside I、III、IV和V是从罗汉果中提取的一组具有药理生物活性的三萜类成分，具有抗氧化、抗高血糖、免疫、镇咳和抗细胞毒活性。罗汉果苷V是研究最充分和最常见的形式之一，已被报道可减轻炎症和高反应性，并抑制高血糖诱导的肺癌细胞迁移和侵袭。罗汉果苷V在中枢神经系统疾病中的保护作用日益受到人们的关注。在非竞争性NMDA受体拮抗剂地佐西平MK801诱导的类精神分裂症小鼠模型中，罗汉果苷V可以通过促进神经突生长、抑制细胞凋亡和[Ca2+]i释放减少来拯救精神分裂症相关的行为表型。在体外卵母细胞衰老过程中，罗汉果苷V能够缓解卵母细胞质量的恶化，可能是通过SIRT1上调来降低氧化应激。
目前，脂肪干细胞的分离方法多为胶原酶消化法，导致细胞得率低，消化时间不好把握，容易影响细胞的活性。而且培养体系多采用含血清培养法，由于血清含有其它动物源成分，使培养体系存在引入外源性病毒的风险，从而使脂肪干细胞在临床应用中存在很大瓶颈。如果能将罗汉果苷V应用于干细胞的培养体系，必将具有很好的市场前景。
【发明内容】
针对现有技术中干细胞培养基含有血清会增加引入外源病毒风险的不足，本发明提供了一种干细胞培养基，用该干细胞培养基分离干细胞，能避免现有干细胞培养基大多含有血清而引入外源病毒的风险。
为了实现上述目的，本发明所采用以下技术方案：
一种干细胞培养基，包括基础培养基、血小板裂解物、L-谷氨酰胺和丝裂霉素c、罗汉果苷V，其中：血小板裂解物在干细胞培养基的体积百分比为5％、L-谷氨酰胺在干细胞培养基的浓度为4mmol/L、丝裂霉素c在干细胞培养基的浓度为50ng/ml，罗汉果苷V在干细胞培养基的浓度为10μg/ml。
本发明中：
所述的罗汉果苷V的纯度为95％-99％。
优选的，所述的罗汉果苷V的纯度为98％-99％。
本发明所述的干细胞培养基在干细胞分离中的应用，包括以下步骤：
获取离体组织，将离体组织与所述的干细胞培养基混合置于培养瓶中培养至离体组织中的细胞从组织中爬出来后刮除离体组织；继续培养，待培养基中细胞融合度达80％～90％时，消化后传代培养。
优选的，所述刮除离体组织，是在离体组织与所述的干细胞培养基混合置于培养瓶中培养的第9天。
优选的，所述离体组织为脂肪组织。
优选的，所述消化为EDTA-胰酶消化。
优选的，所述传代培养采用上述干细胞培养基进行传代培养。
优选的，所述离体组织与所述干细胞培养基混合前采用生理盐水清洗，所述生理盐水含有质量百分比为1％的青霉素和质量百分比为1％的链霉素。
优选的，所述离体组织与所述的干细胞培养基混合置于培养瓶中培养直至传代培养之间，每隔2-3天更换所述干细胞培养基。
和现有技术相比，本发明具有如下优点：
本发明公开干细胞培养基及干细胞分离方法，所述干细胞培养基包括基础培养基、血小板裂解物、L-谷氨酰胺和丝裂霉素c、罗汉果苷V，均可刺激脂肪干细胞上调干细胞维持因子Oct4的表达，研究表明，Oct4的作用是确保干细胞粘附在一起，而干细胞要茁壮成长必须粘附在一起，Oct4通过将干细胞互相粘连，阻止干细胞分化，从而使脂肪干细胞在培养过程中避免分化，维持干细胞的“干性”(自我更新和分化能力)。
【具体实施方式】
以下结合实施例对本发明的具体实施方式做进一步说明。
在对实施例进行具体描述前，有必要提供一些说明备注：采用不同厂家和不同批次的甘蔗糖蜜进行实验，实验结果会存在一定差异，属于正常现象。为了能够保证实验的重复性，需要将最后配制的培养基进行过滤，以便除去来自于甘蔗糖蜜的不溶性杂质的干扰。
其中，以下实施例的材料均为市售，DMEM/F12为Gibco品牌，血小板裂解物购自美国Compass Biomedical、L-谷氨酰胺、丝裂霉素c、罗汉果苷V均购自Sigma。
实施例：
干细胞培养基的配置
以DMEM/F12为基础培养基，加入血小板裂解物、L-谷氨酰胺、丝裂霉素c、罗汉果苷V，使血小板裂解物在干细胞培养基的体积百分比为5％；L-谷氨酰胺和丝裂霉素c在干细胞培养基的终浓度分别为50ng/ml、4mmol/L和50ng/ml，罗汉果苷V在干细胞培养基的浓度为10μg/ml、4℃避光保存备用。
1、脂肪干细胞分离，步骤如下：
1)脂肪组织由专业的美容机构或专业医院通过吸脂手术获得。将分离的脂肪组织50ml转移到无菌采集瓶中，2-8℃保存，12h内进行脂肪干细胞分离；
2)将脂肪组织用含有双抗(质量百分比为1％青霉素和质量百分比为1％链霉素)的生理盐水冲洗3遍，洗去血细胞直至加入生理盐水后脂肪组织悬液澄清无血色；
3)800r/min离心5min，去掉下层液体，吸取上层组织块放入T75培养瓶中，稍稍倾斜培养瓶去除吸取步骤二清洗时带进去的生理盐水，并将组织块均匀地铺在培养瓶中，置37℃、5％CO2的培养箱中培养，30min后组织块贴于培养面，轻柔地加入6-9ml上述制备的干细胞培养基浸润组织块，得到脂肪干细胞；
2、脂肪干细胞培养，步骤如下：
采用的培养基为上述配置的干细胞培养基：
将上步骤得到的脂肪干细胞在第二天进行全量换液，以后每2-3天进行半量换液；当脂肪干细胞在干细胞培养基培养到第9天，脂肪干细胞从组织中爬出来后刮除组织块，让脂肪干细胞继续培养至细胞融合度达80％-90％时，去除培养瓶中培养基，用生理盐水冲洗细胞2次，加入0.25％EDTA-胰酶进行消化，待细胞变圆时加入等体积实施例制备的干细胞培养基终止消化；离心收集后，用本发明的干细胞培养基重悬计数，并计算活率。
如表1所示，50ml组织最终获得原代脂肪干细胞6.5×107个，活率为99.9％。
表1 50ml脂肪组织的脂肪干细胞得率及活率
| 细胞数(个) | 活率(％) | |
| 原代脂肪干细胞 | 6.5±0.1×10<sup>7</sup> | 99.95±0.01 |
调整细胞密度为0.5×10个/ml进行传代；当P1代细胞融合度达到90％以上时；调整细胞密度为0.5×105个/ml进行传代，后续均按此标准进行传代培养，脂肪干细胞贴壁，成纤维状，传至P3代、P6代时细胞形态没有出现变化。
3、流式细胞技术检测脂肪干细胞表型，步骤如下：
取上步骤的P3代脂肪干细胞，将细胞转入流式管中，600g离心3min，弃上清，加入1ml PBS冲洗2遍；200μl PBS重悬细胞后，分别加入CD34、CD45、CD29、CD73、CD90、CD105和HLA-DR抗体；室温孵育30min后，PBS洗涤3次，弃上清后PBS重悬细胞，流式细胞技术检测细胞表型。
表2ADSCs细胞流式表型
| CD34 | CD45 | CD29 | CD73 | CD90 | CD105 | HLA-DR | |
| P3 | 1.00±0.48 | 0.32±0.20 | 99.78±0.13 | 98.95±0.11 | 99.88±1.42 | 99.52±1.12 | 0.71±0.02 |
| P6 | 0.14±0.04 | 0.16±0.01 | 99.98±0.42 | 99.96±0.04 | 99.95±0.02 | 99.83±0.44 | 0.08±0.01 |
结果如表2所示，采用本发明的干细胞培养基，分离培养获得的脂肪干细胞完全符合脂肪干细胞表面标志的鉴定标准，即高表达CD29，CD73，CD90，CD105，低表达或不表达CD34、CD45和HLA-DR，P3代和P6代无显著差异，说明应用本申请的干细胞培养基分离培养的脂肪干细胞多次传代后并未发生分化。
4、取步骤2的P6代脂肪干细胞，按1×106细胞/每孔铺于6孔板，每孔2ml；第二天换液，其中3个孔作为实验组，每孔2ml更换为本申请的干细胞培养基(含DMEM/F12、血小板裂解物、L-谷氨酰胺和丝裂霉素c、罗汉果苷V)，其细胞标记为含罗汉果苷V组。另外3孔作为对照组，每孔2ml更换为不含罗汉果苷V的培养基(即只含有DMEM/F12、血小板裂解物、L-谷氨酰胺和丝裂霉素c)，其细胞标记为不含罗汉果苷V组，将两组细胞于37℃，5％CO2的培养箱中继续培养24h，24h后收集细胞，Trizol裂解法提取RNA，并按系列反应体系和反应步骤反转录成cDNA：
1)配置下列反应体系，得到混合物：
2)将上述步骤1)的混合物：放入PCR仪在65℃，反应5min后迅速移至冰上，得到混合物；
3)将上述步骤2)的混合物添加4μl的5×buffer和2μl的0.1M的DTT后，轻轻混匀后置于PCR仪中，在37℃反应2min，得到混合物；
4)将步骤3)的混合物室温下加入1ul M的MLV后轻混匀，PCR仪中在37℃，反应50min后，在70℃反应15min后，在4℃反应5min，得到cDNA。
以步骤4)获得的cDNA为模板，GAPDH为内参，qPCR检测含罗汉果苷V组和不含罗汉果苷V组的Oct4的相对表达量，其引物序列分别为GAPDH的引物。采用常规的2－△△CT方法计算脂肪干细胞在不同培养条件的Oct4的相对表达量。
结果：含有罗汉果苷V组的Oct4的表达量为2.2，对照组为1.8，含有罗汉果苷V组的Oct4的表达量显著增高，是对照组的1.2倍，p<0.05。
5、脂肪干细胞成脂诱导分化，步骤如下：
采用Adipogenesis Differentiation Kit(GIBCO)试剂盒对步骤2所获取的P3代和P6代脂肪干细胞进行成脂诱导分化实验。
收集P3代和P6代脂肪干细胞，以2×104个/孔接种于12孔板，采用本发明的干细胞培养基培养脂肪干细胞，于37℃，5％CO2培养箱中培养，24h后细胞融合度达到80％时，将P3和P6代细胞分成实验组和对照组，实验组更换成诱导培养基继续培养，每3天换新鲜诱导液。对照组更换成本发明的干细胞培养基，对照组每3天更换本发明的干细胞培养基，持续培养14-21天。诱导14-21天后，油红0染色鉴定。
结果：本发明所获取的P3代和P6代脂肪干细胞具有成脂分化能力，且传代至P6代时仍能维持其干性。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。
Claims (3)
1.一种干细胞培养基，其特征在于：包括基础培养基、血小板裂解物、L-谷氨酰胺和丝裂霉素c、罗汉果苷V，其中：血小板裂解物在干细胞培养基的体积百分比为5％、L-谷氨酰胺在干细胞培养基的浓度为4mmol/L、丝裂霉素c在干细胞培养基的浓度为50ng/ml，罗汉果苷V在干细胞培养基的浓度为10μg/ml。
2.根据权利要求1所述的一种干细胞培养基，其特征在于：所述的罗汉果苷V的纯度为95％-99％。
3.根据权利要求2所述的一种干细胞培养基，其特征在于：所述的罗汉果苷V的纯度为98％-99％。
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