CN112089733A - 一种改造的脐带干细胞在制备抗衰老的药物组合物或者保健品中的用途 - Google Patents
一种改造的脐带干细胞在制备抗衰老的药物组合物或者保健品中的用途 Download PDFInfo
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Abstract
本发明涉及一种改造的脐带干细胞在制备抗衰老的药物组合物或者保健品中的用途。本发明将miR‑375转染脐带间充质干细胞发现，所述转染后的脐带间充质干细胞一方面提高干细胞本身的抗衰老功能，另外一方面在提高干细胞本身活性的同时提高对人抗衰老的功能，而且所述的抗衰老特性是在高糖环境实现的，适于糖尿病患者的抗衰老治疗，具有极大的市场价值。
Description
技术领域
本发明涉及药物和保健品领域，具体的涉及一种改造的脐带干细胞在制备抗衰老的药物组合物或者保健品中的用途。
背景技术
衰老是机体随年龄增长而呈现渐进性结构退行性变和机能减退的过程和现象。人口老龄化已经成为严重的政治、社会、经济和健康问题，寻找有效提高老年人的生活质量、逆转或延缓衰老的技术是目前迫在眉睫的问题。干细胞具有自我更新、多向分化潜能，在理论上具有参与和促进组织细胞再生、损伤修复、逆转人体衰老的可行性。
间充质干细胞（mesenchymal stem cells，MSCs）是一种多能干细胞，来源于发育早期的中胚层和外胚层，MSCs 最初在骨髓中发现，因其具有多向分化潜能、造血支持和免疫调控等特点而日益受到人们的关注。间充质干细胞在体内或体外特定的诱导条件下可分化多种组织细胞，可作为理想的种子细胞用于衰老和病变引起的组织器官损伤修复。王宏兰等通过研究发现，骨髓间充质干细胞（bone marrow mesenchymal stem cells，BM-MSCs）对D- 半乳糖衰老模型小鼠具有抗衰老作用。随着MSCs 研究的进展，科学家发现脐带间充质干细胞（umbilical cord mesenchymal stem cells，UC-MSCs）相对于BM-MSCs 具有无肿瘤污染、增殖能力强、采集方便、免疫活性低、无需配型等许多优点，并且脐带作为医疗废弃物，对供者无不利影响，不受伦理问题的限制，使得UC-MSCs 极有可能成为BM-MSCs 的理想替代物。
但是干细胞本身也存在衰老的问题。细胞衰老是细胞对各种各样的应激源作出应答后的一种细胞状态，应激源包括基因毒性因子，营养元素缺乏，缺氧，线粒体功能障碍和致癌基因激活等。研究表明，衰老会影响MSCs 的功能，包括增殖能力、克隆形成能力、分化潜能、免疫特性、端粒酶活性、细胞迁移能力和粘附性等，突出体现在细胞增殖能力和分化潜能的衰减。文献表明，衰老后MSCs 表面特异性标志物CD44、CD90、CD105 和Stro-1 等表达有所降低; 细胞形态逐渐扁平肥大，胞膜成分改变，如膜内在蛋白caveolin-1 含量上调;胞质pH 改变，颗粒固缩; 溶酶体间隔增大且活性增加，β-半乳糖苷酶( β-galactosidase，β-gal) 产量升高;溶酶体内脂褐质累积，细胞自体荧光水平上升; 线粒体较大但功能异常，导致过量的活性氧自由基( reactive oxygen species，ROS) 产生; 同时因核纤层蛋白B1 的缺失，胞核完整性遭到破坏，胞质内出现染色质碎片，端粒酶明显缩短等。总之，衰老MSCs 最终的结局是停止增殖，导致干细胞用于治疗衰老的作用停止。
MSCs 体外培养易衰老特性使MSCs 扩增数量受限，从而限制其在临床的应用。如何使衰老的MSCs 再现年轻化状态而延长其利用价值成为再生医学的研究热点之一。Romanov 等率先利用人乳腺上皮细胞探索细胞衰老状态是否可以被逆转。此后，越来越多的学者尝试通过添加生物活性物质或药物等方法延缓MSCs 衰老进程。溶血磷脂酸(lysobisphosphatidic acids，LPA) 是膜磷脂合成中的一种代谢产物，研究表明LPA 参与并维持机体的正常生理功能。MSCs 优先表达LPA受体亚型1，通过添加该受体拮抗物Ki16425，细胞衰老相关的p16Ink4a、Rb、p53 和p21Cip1在hBMSCs 中的表达均受到抑制，明显减缓了体外连续培养的hBMSCs 衰老状态。生物活性分子载体丝蛋白与姜黄素结合可延缓BMSCs 衰老进程，且延缓程度与姜黄素剂量和刺激时间有一定关系，但具体机制还有待进一步探究。雷帕霉素( rapamycin，RAPA) 是一种mTOR 抑制剂，已被证实具有延缓细胞衰老的作用，机制可能是通过对AKT /mTOR 的抑制作用从而调节胞内ROS 的产生，多能性基因Nanog 和Oct-4 的表达及DNA 损伤累积而产生相应效应。曹玉林等研究发现复制性衰老的UCMSCs 经RAPA 处理后，细胞体积变小，β-gal 染色阳性细胞减少，p53 表达降低，血管相关形成因子表达增高; 小鼠体内实验表明RAPA 治疗组较对照组能增强缺血区域血管新生，证明RAPA 可以成为延缓MSCs 衰老状态的新手段。曾毅等利用Ku0063794 和RAPA 作用于衰老的BMSCs，也得到了同样的结果。RAPA 还可以通过抑制mTOR 信号通路，明显改善来自系统性红斑狼疮患者的BMSCs 的衰老表型和免疫调节功能，为治愈此类疾病带来新的希望。但是这些都是药物的作用，药物本身对于人体还有不确定的副作用或肝脏伤害，导致其推广应用存在诸多障碍。
发明内容
本发明克服现有的技术的缺陷，一方面通过提高干细胞的抗衰老活性来提高干细胞本身的抗衰老功能，另外一方面在提高干细胞本身活性的同时提高对人抗衰老的功能。
本发明一个方面，提供一种脐带间充质干细胞在高糖环境下衰老前后miRNA 差异表达与功能分析的方法，所述方法包括将所属脐带间充质干细胞通过在用于模拟糖尿病患者体内高糖微环境高糖培养基中连续培养16代模拟糖尿病患者细胞衰老状态，通过对细胞的miRNA 表达进行检测，对衰老前后的核内miRNA 表达情况进行统计分析，筛选出差异倍数大于2 倍且P ＜0.001 的miRNA 为表达显著差异的miRNA，进而对miRNA 表达谱数据进行差异表达分析。进一步的，本发明筛选出了2条差异最显著的miRNA作为研究基础用于抗衰老的研究。
本发明一方面，请求保护转miR-375的脐带间充质干细胞在制备药物组合物中的应用；所述药物组合物的功能为如下：
(a)延缓衰老；
(b)延缓细胞衰老；
(c)延缓间充质干细胞衰老。
本发明保护一种转miR-375的脐带间充质干细胞的制备方法，包括如下步骤：
（1）提高脐带间充质干细胞中miR-375的含量；或
（2）降低脐带间充质干细胞中抑制miR-375表达的编码基因的表达。
本发明进一步的，提供了所述转miR-375的脐带间充质干细胞在制备抗衰老的保健品中的用途。
进一步的，所述药物或者保健品中还可以含有其他的抗衰老的药物。
在另一优选例中，所述的药物组合物的剂型是：口服剂，注射剂，输液剂，片剂，粉剂，胶囊剂，丸剂；较佳地为口服剂。
本发明还提供一种抗衰老冻干剂，由本发明所述的抗衰老组合物、冻干保护剂、稳定剂组成。
一些实施方案中，所述稳定剂为冻干保护剂、酸碱调节剂、乳化剂中的一种或多种。
其中，所述冻干保护剂为海藻糖、甘氨酸、甘露糖醇、葡聚糖、乳糖中 的一种或多种；
所述酸碱调节剂为磷酸氢二钠、磷酸二氢钠、氨基酸、柠檬酸钾中的一 种或多种。
所述乳化剂为聚山梨醇酯-20、聚山梨醇酯-60、聚山梨醇酯-80中的一种 或多种。
有益效果：本发明通过针对脐带间充质干细胞在高糖环境下衰老前后miRNA 差异表达与功能进行分析筛选出差异表达显著差异的miRNA作为研究基础用于抗衰老的研究。通过将所述的miRNA转染脐带间充质干细胞发现，所述转染后的脐带间充质干细胞一方面提高干细胞本身的抗衰老功能，另外一方面在提高干细胞本身活性的同时提高对人抗衰老的功能，而且所述的抗衰老特性是在高糖环境实现的，适于糖尿病患者的抗衰老治疗，具有极大的市场价值。
附图说明
图1 miR-375 表达水平结果图
图2 CCK-8 法检测转基因干细胞的活力结果图
图3 细胞抗衰老检测结果图
具体实施方式
为了更进一步的说明本发明的目的、技术方案和优点，我们结合以下具体实施例来阐述本发明，这些实施例仅为了更好的说明本发明专利，而不用于限制本发明范围。基于本发明中的实施例，本领域的技术人员在没有做出创造性的情况下所得到的其他所有实施方式均属于本发明所保护的范围。
实施例1 脐带间充质干细胞的分离鉴定
无污染脐带5 cm左右，PBS 充分洗涤，分离并去除脐带外膜和血管组织，将其剪碎成1mm3 小块后PBS 清洗并以3000 r/min 离心5min，弃上清保留沉淀；PBS 洗涤3次后，以1×106/mL 的细胞密度接种于T-75 培养瓶中，置37℃，5%二氧化碳的饱和湿度环境下，于体积分数为10%FBS的DMEM培养。5 d 后首次全量换液，弃去未贴壁细胞，以后每3-4 d 换液1次。观察细胞达85％融合后，0.25%胰酶消化5min，按1:3 的比例传代，继续培养。培养过程中，采用流式细胞术鉴定细胞表面标志。收集脐带间充质干细胞，取处于对数生长期的5代细胞，去掉培养液，PBS 洗2遍用0.25%的胰蛋白酶消化液消化，去消化液后加入PBS，制成浓度为1×106/ml 的单细胞悬液，每个Eppendorf 管加100μL的细胞悬液。分别加入抗人的20μLFITCCD90、FITC-CD34、PE- CD45、PE- HLA-DR、FITCCD105、PE-CD73，对照管分别加入同量的抗人FITC-IgG1 和PE-IgG1，25℃避光孵育30 min，PBS洗2次，1500 r/min离心5 min，重新制成细胞悬液，流式细胞仪检测。经流式细胞术检测，结果如表1所示。
表1人脐带MSCs标志物表达结果
从表1的结果可以看出，人脐带MSCs 高表达CD73（98.34%）、CD90（99.87%）和CD105（97.42%），低表达造血系干细胞标志CD34（0.05%）和CD45（0.15%），符合国际上对间充质干细胞鉴定，表明干细胞分离成功。
实施例2 脐带间充质干细胞在高糖环境下衰老前后miRNA 差异表达与功能分析
取前述鉴定得到的干细胞，分为3组: 一组细胞继续培养于DMEM/NG( 含糖5.5 mmol/L) 培养基中作为正常糖对照组(NG组)培养16代；另一组细胞培养于DMEM/HG (含糖25mmol/L) 培养基中作为HG 组(用于模拟糖尿病患者体内高糖微环境) ，连续培养16代。第三组细胞培养于DMEM/HG (含糖25mmol /L) 培养基中作为对照组(用于模拟糖尿病患者体内高糖微环境) ，连续培养2代。将三种细胞分别采用RNA提取试剂盒提取RNA。
miRNA 表达谱检测使用Megaplex Pools miRNA 表达检测系统( AppliedBiosystems 公司，美国) 并按照说明书操作，主要步骤包括: 使用Megaplex RT Primers特异性引物将RNA 样本逆转录为cDNA; 使用TaqManMicroRNA Arrays 试剂盒( AppliedBiosystems ) 通过ABI7900HT 荧光定量PCR 系统( Applied Biosystems)对miRNA 表达进行检测; 最后结果以U6 为内参对照进行归一化处理。使用DEGseq 工具对衰老前后的核内miRNA 表达情况进行统计分析，筛选出差异倍数大于2 倍且P ＜0.001 的miRNA 为表达显著差异的miRNA。结果显示RNA，利用miRNA 定量PCR 芯片对miRNA 的表达情况进行了检测。对miRNA 表达谱数据进行差异表达分析结果显示，在高糖培养16代环境下，与高糖培养2代和无糖培养16代MSCs 相比，高糖培养环境有243个miRNA 的表达出现差异( 差异倍数＞2 倍且P＜0.001) ，其中187个miRNA 呈显著上调， 56个miRNA 呈显著下调。其中差异最显著的5个miRNA的结果如下表2所示。
表2 高糖脐带间充质干细胞差异表达miRNA
| ID | 倍数变化（Log2） |
| miR-375 | -10.986201 |
| miR-16 | -9.855023 |
| miR-330-5p | 9.221345 |
| miR-362-3p | 8.521436 |
| miR-609 | -7.253691 |
从表2的结果初步判定，最显著的这些miRNA与干细胞的衰老相关。发明人通过将下调明显的二个miRNA进行上调，预期能够提高所述细胞的抗衰老特性。
实施例3 miR-375转染脐带间充质干细胞的建立
以人DNA为模板，以扩增引物为：5'-AAGCTTGGCTGATGCTGAGAAG-3' 和5'-TCTAGACGGCCCCGGGTCTTC-3'进行miR-375的扩增，回收纯化后与pMD18-T 相连，得到质粒pMD18-T-miR-375, 将目的片段插入质粒pEGFP-N1-MCS 的多克隆位点中，构建了质粒pEGFP-N1-miR-375。 miR-375 inhibitor 购买自广州锐博生物科技公司。
分别转染pEGFP-N1-miR375 质粒以及miR-375 inhibitor至脐带间充质干细胞培养细胞：具体的，待实施例1分离的脐带间充质干细胞密度80%时,按转染试剂说明将2ugpEGFP-N1-miR375 质粒和2ug miR-375 inhibitor分别和10uL lipofectamine2000 在无血清培养基中混匀，室温静置20min，然后再缓慢加入到培养基中，37℃，5% CO2 培养，24h后收获细胞。
检测miR-375 表达水平：细胞转染24h 后，收获细胞，用Ambion 公司的mirVanaTMqRT-PCR miRNA Detection Kit 提取mRNA 逆转录后做RT-PCR 检测，U6 snRNA作为内参基因；反应体系为：10 μL SYBR Green supermix；0.5 μL mir VanaPCR primers；0.5 μL product from TR reaction；9 μL Nuclease-free Water；反应条件：95℃ for 3min；95℃ for 15 sec，60℃for 30 sec(40cycles)，运用LightCycler 480 系统分析数据。结果如图1所示。
从图1可以看出，转染了miR-375的脐带间充质干细胞中的miR-375表达水平显著提高，而转染了miR-375 inhibitor的干细胞中相应的miR-375表达显著降低，这表明相应的转基因干细胞构建成功。
实施例4 CCK-8 法检测转基因干细胞的活力
将转基因的干细胞按每孔3000个细胞接种于96孔板，CCK-8 法检测分别转染miR-375、miR-375 inhibitor 和空白对照后48 h 的细胞在高糖环境下随着时间的活力的变化。具体操作参照CCK-8 试剂盒说明书，用酶标仪测定样本450 nm 波长处吸光度( A) 值。结果如图2所示。
本实验用CCK-8 法检测miR-375 在高糖条件下与脐带间充质干细胞活力的关系，用CCK-8 法检测转染了miR-375和miR-375 inhibitor 48 h之后的干细胞的活力，结果显示转染后相应的第4天，在高糖环境下，转染miR-375的细胞活力A值达到了1.61，显著比转染miR-375-inhibitor的0.42和未转基因对照的0.55的活力要强。充分说明，miR-375能够提高脐带间充质干细胞在高糖下的细胞活力。
实施例5 细胞抗衰老检测
采用衰老相关β-半乳糖苷酶染色法检测细胞的衰老情况。各组细胞转染后在高糖培养基中继续培养48h后，经PBS 冲洗，加入β-半乳糖苷酶染色固定液，室温固定15 min，PBS 再次冲洗后，加入染色工作液， 37 ℃无CO2条件下孵育过夜;普通光学显微镜下观察，每个样本计数800个细胞，胞浆蓝染者为衰老细胞，计数阳性细胞占观察细胞总数的百分比。结果如图3所示。
从图3的结果可以看出，我们检测转染了miR-375和miR-375 inhibitor 48 h之后的干细胞的衰老水平，结果表明，HG + miR-375组的β-半乳糖苷酶阳性染色细胞数明显降低只有23个，HG + miR-375 inhibitor 组的β-半乳糖苷酶阳性染色细胞数明显增加达到了670。这表明miR-375能够增强脐带间充质干细胞在高糖环境下的抗衰老特性。
实施例6 糖尿病衰老小鼠模型试验
雌性Wistar 大鼠, 由中国科学院上海实验动物中心提供, 体重 140g左右。
将Wistar 大鼠适应性喂养1 周后, 按下列方式制备动物模型, 具体操作方法如下:
A 组( 正常对照组) : 大鼠先腹腔注射生理盐水0.01 mL/100 ( g·d- 1) 40 d,后给予生理盐水灌胃( 1 mL/100 g·d- 1) 2 周, 同时给予自由饮水及饲料;
B组( 衰老糖尿病模型对照组) : 大鼠给予D- 半乳糖腹腔注射( 50 mg/kg,每天1次) 40 d；同时从第11 天起灌以高脂高糖乳剂( 1 mL/100 g 体重, 每天1 次, 含100 g/L蔗糖、100 g/L 猪油、10 g/L 胆固醇) , 连续4 周后,腹腔注射STZ( 30 mg/kg,STZ 现配现用, 冰浴条件下, 用pH4.2 柠檬酸缓冲液配成0.25%溶液) , 4 d后筛选禁食不禁水12h 后血糖≥11.1 mmol/L 为衰老糖尿病成模鼠。继续给予生理盐水灌胃2 周, 0.01 mL/100 ( g·d) ;
治疗给药方式：每次连续3 d 对模型组小鼠尾静脉注射实施例3的转基因脐带充质干细胞悬液0.3mL/ 只，细胞浓度为1×106/mL，间隔时间7 d，共计注射9 次。模型对照组和正常对照组则于尾静脉给予等量细胞培养液。各组其余饲养条件同正常对照组。实验结束后乙醚麻醉,腹主动脉取血,检测各项指标。血糖、MDA、SOD、CAT检测分别采用葡萄糖氧化酶法、TBA 法、黄嘌呤氧化酶法, 过氧化氢酶（CAT）活力测定：钼酸铵比色法法检测, 严格按试剂盒要求操作。结果如下表3所示。
表3 转基因脐带间充质干细胞对血糖、CAT、SOD、MDA含量的影响
| 组别 | 血糖（mmol/L） | CAT（U/ml） | SOD（nmol/ml） | MDA(U/ml) |
| 正常对照组 | 3.59±0.62 | 95.61±9.58 | 162.33±14.17 | 18.11±3.59 |
| 衰老糖尿病模型组 | 29.40±2.57* | 30.44±10.03* | 120.54±11.36* | 36.17±4.83* |
| 衰老糖尿病模型治疗组 | 22.01±1.49* | 79.89±11.29** | 151.29±13.02** | 19.47±2.23** |
与相应对照组比较*P<0.05,**P﹤0.01。
从表3的结果可以看出，经过检测，模型组经D- 半乳糖注射以及高糖饲喂后后小鼠体内SOD、CAT 活力值明显下降，MDA 含量明显上升，血糖含量相比正常对照组也显著的上升说明模型构建较为成功。而经过转基因干细胞的治疗后，比较模型组与治疗组CAT、SOD活力值以及MDA 含量发现，经输注转基因脐带间充质干细胞后的小鼠CAT、SOD 活力较模型组均明显上升，而MDA 含量则明显下降，说明其抗衰老作用显著。而且从血糖的结果也可以看出，初步也具有下调血糖浓度的效果。
Claims (10)
1.转miR-375的脐带间充质干细胞在制备药物组合物中的应用；所述药物组合物的功能为如下：
(a)延缓高糖环境下的人体的衰老；
(b)延缓高糖环境下细胞的衰老；
(c)延缓高糖环境下脐带间充质干细胞的衰老。
2.一种转miR-375的脐带间充质干细胞的制备方法，包括如下步骤：
（1）提高脐带间充质干细胞中miR-375的含量；或
（2）降低脐带间充质干细胞中抑制miR-375表达的编码基因的表达。
3.转miR-375的脐带间充质干细胞在制备保健品中的应用；所述保健品的功能为如下：
(a)延缓高糖环境下的人体的衰老；
(b)延缓高糖环境下细胞的衰老；
(c)延缓高糖环境下脐带间充质干细胞的衰老。
4.如权利要求1或3所述的应用，其特征在于：所述药物或者保健品中还可以含有其他的抗衰老的药物。
5.如权利要求1所述的应用，其特征在于所述的药物组合物的剂型是：口服剂，注射剂，输液剂，片剂，粉剂，胶囊剂，丸剂。
6.如权利要求1所述的应用，其特征在于所述的药物组合物为冻干剂，所述冻干剂由转miR-375的脐带干细胞以及冻干保护剂和稳定剂以及酸碱调节剂组成。
7.如权利要求6所述的应用，其特征在于：所述稳定剂为冻干保护剂、酸碱调节剂、乳化剂中的一种或多种。
8.如权利要求6所述的应用，其特征在于：所述冻干保护剂为海藻糖、甘氨酸、甘露糖醇、葡聚糖、乳糖 的一种或多种。
9.如权利要求6所述的应用，其特征在于：所述酸碱调节剂为磷酸氢二钠、磷酸二氢钠、氨基酸、柠檬酸钾中的一种或多种。
10.如权利要求7所述的应用，其特征在于所述乳化剂为聚山梨醇酯-20、聚山梨醇酯-60、聚山梨醇酯-80中的一种或多种。
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