CN112143708B - 一种脐带间充质干细胞、干细胞精华因子和在抗皮肤衰老方面的应用 - Google Patents
一种脐带间充质干细胞、干细胞精华因子和在抗皮肤衰老方面的应用 Download PDFInfo
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Abstract
本发明公开了一种脐带间充质干细胞、干细胞精华因子和在抗皮肤衰老方面的应用。本发明发现，miR‑626高表达人脐带间充质干细胞不仅具有更强的体外增殖活性，其分泌的外泌体还具有更强的抗皮肤衰老的活性，该外泌体可以用于制备抗皮肤衰老的药物，或者用于制备抗皮肤衰老的化妆品。
Description
技术领域
本发明属于干细胞领域，涉及干细胞的应用，具体涉及一种脐带间充质干细胞、干细胞精华因子和在抗皮肤衰老方面的应用。
背景技术
随着人口老龄化日趋明显，衰老相关疾病的防治日益受到关注。皮肤衰老是一个复杂的生理过程，其内源性因素包括组织结构及功能、机体激素水平等的变化，外源性因素包括紫外线辐照、化学物质损伤等刺激，造成DNA损伤修复能力下降、基因组稳定性降低，组织细胞凋亡，细胞外基质成分降解及胶原紊乱，进一步导致皮肤变薄、弹性下降及皱纹形成、再生修复功能受损、皮肤屏障功能下降、色素沉积等表现。在此基础上，可发生皮肤癌前病变、皮肤肿瘤及其他衰老相关皮肤疾病。为促进皮肤健康、提升生活质量，抗皮肤衰老产品市场逐年扩大，其中间充质干细胞及相关衍生物因其修复再生潜力，在皮肤年轻化及美容领域被寄予厚望(黄健华等，干细胞抗皮肤衰老研究进展，老年医学与保健，2018年)。
间充质干细胞是一类多能干细胞，具有高度自我更新的能力以及向多种细胞分化的潜能。其中，人脐带来源间充质干细胞因其具备的综合优点受到广泛关注。研究表明，它们可以在体外和体内通过不同方式诱导分化为脂肪组织细胞、软骨组织细胞、结缔组织细胞和骨组织细胞以及神经干细胞。研究还表明，脐带间充质干细胞分泌的生物活性因子对其在治疗多种疾病的过程中起到了非常重要的作用。
可见，包括脐带间充质干细胞在内的间充质干细胞及相关产品在面部年轻化领域展现出良好前景和潜力，但是将间充质干细胞及相关产品进行应用、产业化时，对细胞数量要求很高。而间充质干细胞的来源决定了不可能全部从源头获取，这就对间充质干细胞的体外增殖扩增能力提出了很高的要求。
目前，如何提高间充质干细胞的体外增殖、获得具有高增殖活性的干细胞对间充质干细胞应用于面部年轻化领域具有重要意义。
发明内容
本发明是为了克服现有技术中存在的不足，提供一种脐带间充质干细胞、干细胞精华因子和在抗皮肤衰老方面的应用。
技术方案：
一种人脐带间充质干细胞，为一种miR-626高表达的人脐带间充质干细胞。miR-626高表达的人脐带间充质干细胞具有更强的体外增殖活性。
一种促进人脐带间充质干细胞体外增殖的方法，提高其miR-626表达。
一种干细胞精华因子，为上述miR-626高表达人脐带间充质干细胞分泌的外泌体。miR-626高表达的人脐带间充质干细胞分泌的外泌体具有更强的抗皮肤细胞衰老的活性。
上述干细胞精华因子在制备抗皮肤衰老的化妆品中的应用。
上述干细胞精华因子在制备抗皮肤衰老的药物中的应用。
技术效果：
本发明发现，miR-626高表达人脐带间充质干细胞不仅具有更强的体外增殖活性，其分泌的外泌体还具有更强的抗皮肤衰老的活性，该外泌体可以用于制备抗皮肤衰老的药物，或者用于制备抗皮肤衰老的化妆品。
附图说明
图1为人脐带间充质干细胞倒置显微镜观察结果；可见细胞呈梭形，呈漩涡状生长，符合人脐带间充质干细胞的生长特点。
图2为人脐带间充质干细胞免疫表型的流式测定结果；CD34、CD45、HLA-DR表达水平很低，CD73、CD90、CD105表达水平很高，符合人脐带间充质干细胞的免疫表型特点。
图3为各组人脐带间充质干细胞中miR-626相对表达水平；与空白对照组相比，过表达组人脐带间充质干细胞中miR-626表达水平显著升高(P＜0.05)，说明miR-626过表达慢病毒已经成功将miR-626转染进入人脐带间充质干细胞中。与空白对照组相比，阴性对照组miR-626表达水平无显著差异(P＞0.05)。
图4为各组人脐带间充质干细胞培养24h、48h、72h细胞数量(×105个/孔)；过表达组人脐带间充质干细胞数量显著高于空白对照组(P＜0.05)，阴性对照组人脐带间充质干细胞数量与空白对照组无显著差异(P＞0.05)。
图5为Western blot检测结果；过表达Exo、阴性对照Exo和空白对照Exo均强表达外泌体标志物蛋白CD63和CD81，符合外泌体的表型特点。
图6为各组β半乳糖苷酶染色阳性率；与对照组相比，衰老模型组β半乳糖苷酶染色阳性率显著升高(P＜0.05)，说明衰老模型建模成功；与衰老模型组相比，过表达Exo、阴性对照Exo、空白对照Exo组β半乳糖苷酶染色阳性率均显著降低(P＜0.05)，且过表达Exo组β半乳糖苷酶染色阳性率显著低于阴性对照Exo组(P＜0.05)、空白对照Exo组(P＜0.05)。
具体实施方式
一、实验材料
人脐带间充质干细胞购自上海传秋生物科技有限公司。
人真皮成纤维细胞(HDFs)购自ATCC。
胎牛血清、DMEM/F12(1:1)培养基购自购自美国Gibco公司。
碱性成纤维生长因子(b-FGF)购自碧云天生物。
miR-626过表达慢病毒及其阴性对照慢病毒、RT-PCR引物序列由吉凯基因构建。
ExoQuick-TCTM外泌体提取试剂盒购自美国System Biosciences公司。
DMEM-LG培养基、DMEM-HG培养基购自美国Gibco公司。
β半乳糖苷酶检测试剂盒购自美国Gibco公司。
二、实验方法
1、人脐带间充质干细胞的培养、传代、形态观察和表型检测
将冻存的人脐带间充质干细胞按常规方法复苏后，用含10％胎牛血清和10ng/mLb-FGF的DMEM/F12培养基培养于T25培养瓶中，培养条件为37℃、5％CO2、饱和湿度。2～3d换一次液，待细胞长满瓶底面积80％～90％时传代。传代比例为1:5，步骤为：将0.25％胰酶-0.53mM EDTA消化液与DPBS置于37℃预热，倒掉培养瓶中的培养基，往培养瓶中加入3-5mLDPBS，轻晃洗涤后弃去。使用DPBS将胰酶稀释4倍，往瓶中加2mL稀释后的胰酶，置于室温孵育消化，消化好后加入2mL含血清的完全培养基或其他胰酶中和液终止消化。用移液枪轻轻吹打瓶壁上的细胞，使之完全脱落，然后收集细胞悬液并吹打成单个细胞悬液，1200rpm离心5min，弃上清，加入完全培养基重悬细胞，进行传代。
取传代3次后的人脐带间充质干细胞用于后续实验。
形态观察：在倒置显微镜下观察人脐带间充质干细胞的形态和生长情况，拍照。
表型检测：将人脐带间充质干细胞用0.25％胰酶消化，PBS重悬，制备1×106/mL的细胞悬液。然后取100μL细胞悬液与20μL标记一抗(CD73-PE、CD90-FITC、CD105-APC、CD34-PE、CD45-APC、HLA-DR-FITC)4℃避光孵育1h，通过流式细胞仪检测细胞的免疫荧光。
2、细胞转染、培养和计数
取生长状态良好的人脐带间充质干细胞，以1×105个/孔的密度接种于6孔板中，待细胞生长至70％～80％融合度时，按照病毒感染比率值(MOI)50:1分别加入miR-626过表达慢病毒及其阴性对照慢病毒感染人脐带间充质干细胞，同时设置不做任何处理的人脐带间充质干细胞作为空白对照，记为过表达组、阴性对照组和空白对照组，每组2个复孔，8块6孔板平行操作。培养6h后，洗涤、收集其中2块6孔板上的各组细胞用于miR-626相对表达水平检测，其余6块6孔板用PBS洗涤细胞并更换新鲜的完全培养液，继续培养24h、48h、72h时，分别取出2块6孔板采用细胞计数板法分别对各组每孔细胞数量进行计数，每组4个孔。
3、人脐带间充质干细胞转染后表型检测和miR-626相对表达水平检测
转染培养6h后，miR-626相对表达水平检测的方法为：根据试剂盒说明书用Trizol试剂提取细胞总RNA，用1μg总RNA和PrimeScriptTM RT试剂盒通过反转录反应合成cDNA，用SYBR Premix Ex Taq在RT-PCR仪上进行RT-PCR，以GAPDH为内参，通过2－ΔΔCt法分析各组人脐带间充质干细胞中miR-626的相对表达水平，并以空白对照组计为1.00。
RT-PCR引物序列如下：
miR-626正向序列：5'-GTGCGATCGTAAACCGTC-3'
miR-626反向序列：5'-CCGTGAAATGCTAGCTGA-3'
GAPDH正向序列：5'-CGGAGTCAACGGATTTGGTCGTAT-3'
GAPDH反向序列：5'-AGCCTTCTCCATGGTGGTGAAGAC-3'
将上述“2、细胞转染、培养和计数”步骤中，继续培养24h、48h、72h并计数后的细胞收集，PBS洗涤，取少量细胞按照“1、人脐带间充质干细胞的培养、传代、形态观察和表型检测”中的表型检测方法检测免疫表型。
4、外泌体的收集和检测
取生长状态良好的人脐带间充质干细胞，用完全培养基制备成浓度为1×106/mL的细胞悬液，接种于T25培养瓶中，待细胞生长至70％～80％融合度时，按照病毒感染比率值(MOI)50:1分别加入miR-626过表达慢病毒及其阴性对照慢病毒感染人脐带间充质干细胞，同时设置不做任何处理的人脐带间充质干细胞作为空白对照。培养6h后，PBS洗涤细胞，用DMEM/F12培养基制备成浓度为1×106/mL的细胞悬液，继续培养24h后，收集培养基，3000r/min离心30min去除细胞碎片，0.22μm微孔过滤器过滤，收集滤液，再用微孔离心过滤装置100000r/min离心2h浓缩，得到浓缩液，使用ExoQuick-TCTM外泌体提取试剂盒根据说明书方法提取浓缩液中的外泌体。用PBS将外泌体重悬后，BCA法测定其浓度，置于-80℃保存备用，分别标记为过表达Exo、阴性对照Exo和空白对照Exo。
各组取等量外泌体采用Western blot法检测外泌体标志物蛋白CD63和CD81的表达。
5、人脐带间充质干细胞外泌体的抗皮肤衰老活性测试
使用DMEM-HG培养基(葡萄糖浓度为26mmol/L)培养制备HDFs衰老模型，依据的原理为高糖环境加速HDFs衰老。细胞衰老β-半乳糖苷酶染色实验步骤如下：
取生长状态良好的HDFs，用含10％FBS、1％双抗的DMEM-LG培养基(葡萄糖浓度为5.5mmol/L)制成浓度2×105/mL的细胞悬液，接种于24孔板中，每孔1mL，培养条件为37℃、5％CO2、饱和湿度。24h后，吸弃培养基，对照组更换为新的含10％FBS、1％双抗的DMEM-LG培养基继续培养，衰老模型组更换为含10％FBS、1％双抗的DMEM-HG培养基继续培养，外泌体组更换为含10％FBS、1％双抗、50ng/mL外泌体(过表达Exo、阴性对照Exo或空白对照Exo)的DMEM-HG培养基继续培养，每组3个复孔。继续培养7d，期间每2d使用各自对应的培养基更换一次培养基。参照衰老β半乳糖苷酶检测试剂盒说明书进行染色，倒置显微镜下观察细胞染色情况。未老化细胞不着色，而老化细胞核周围呈蓝绿色，统计各孔每个视野中染色细胞百分比，各孔取3个不同的视野，计算各组β半乳糖苷酶染色阳性率。
6、统计学处理
数据采用GraphPad Prism 5.0软件进行统计处理，以x±s表示，两组间比较采用t检验，P＜0.05表示具有显著性差异。
三、实验结果
1、人脐带间充质干细胞形态观察和表型检测结果
倒置显微镜观察结果如图1所示，细胞呈梭形，呈漩涡状生长，符合人脐带间充质干细胞的生长特点。表型检测结果如图2所示，CD34、CD45、HLA-DR表达水平很低，CD73、CD90、CD105表达水平很高，符合人脐带间充质干细胞的免疫表型特点。
2、转染、计数和表型检测检测结果
转染培养6h后，各组人脐带间充质干细胞中miR-626相对表达水平如表1和图3所示，与空白对照组相比，过表达组人脐带间充质干细胞中miR-626表达水平显著升高(P＜0.05)，说明miR-626过表达慢病毒已经成功将miR-626转染进入人脐带间充质干细胞中。与空白对照组相比，阴性对照组miR-626表达水平无显著差异(P＞0.05)。
表1各组人脐带间充质干细胞中miR-626相对表达水平
| 组别 | miR-626/GAPDH的相对表达水平 |
| 空白对照组 | 1.00±0.05 |
| 过表达组 | 2.95±0.09 |
| 阴性对照组 | 1.02±0.06 |
过表达组、阴性对照组和空白对照组人脐带间充质干细胞培养24h、48h、72h细胞计数结果如表2和图4所示，过表达组人脐带间充质干细胞数量显著高于空白对照组(P＜0.05)，阴性对照组人脐带间充质干细胞数量与空白对照组无显著差异(P＞0.05)。
表2各组人脐带间充质干细胞培养24h、48h、72h细胞数量(×105个/孔)
| 组别 | 24h | 48h | 72h |
| 空白对照组 | 1.130±0.012 | 1.258±0.014 | 1.375±0.011 |
| 过表达组 | 1.304±0.021 | 1.612±0.025 | 1.883±0.027 |
| 阴性对照组 | 1.128±0.018 | 1.252±0.019 | 1.369±0.022 |
过表达组、阴性对照组和空白对照组人脐带间充质干细胞培养24h、48h、72h后，CD34、CD45、HLA-DR表达率均在4％以下，CD73、CD90、CD105表达率均在95％以上，依然维持较高的干细胞特性。
上述研究结果表明，转染miR-626后高表达miR-626的人脐带间充质干细胞依然维持较高的干细胞特性，且具有更强的细胞增殖活性，可以体外获得更大量的干细胞作为种子细胞。
3、Western blot法检测外泌体的标志物蛋白
Western blot结果如图5所示，过表达Exo、阴性对照Exo和空白对照Exo均强表达外泌体标志物蛋白CD63和CD81，符合外泌体的表型特点。
4、活性测试结果
在β半乳糖苷酶染色实验中，未老化细胞不着色，而老化细胞核周围呈蓝绿色。各组β半乳糖苷酶染色阳性率如表3和图6所示，与对照组相比，衰老模型组β半乳糖苷酶染色阳性率显著升高(P＜0.05)，说明衰老模型建模成功；与衰老模型组相比，过表达Exo、阴性对照Exo、空白对照Exo组β半乳糖苷酶染色阳性率均显著降低(P＜0.05)，且过表达Exo组β半乳糖苷酶染色阳性率显著低于阴性对照Exo组(P＜0.05)、空白对照Exo组(P＜0.05)。该结果表明，人脐带间充质干细胞外泌体可以抑制HDFs衰老的活性，miR-626高表达人脐带间充质干细胞分泌的外泌体具有更强的抑制HDFs衰老的活性。
表3各组β半乳糖苷酶染色阳性率
上述研究结果表明，miR-626高表达人脐带间充质干细胞分泌的外泌体具有更强的抑制人真皮成纤维细胞衰老的活性。
综上可见，miR-626高表达人脐带间充质干细胞不仅具有更强的体外增殖活性，其分泌的外泌体还具有更强的抗皮肤衰老的活性，该外泌体可以用于制备抗皮肤衰老的药物，或者用于制备抗皮肤衰老的化妆品。
Claims (2)
1.一种人脐带间充质干细胞，其特征在于：为miR-626高表达的人脐带间充质干细胞。
2.一种促进人脐带间充质干细胞体外增殖的方法，其特征在于：提高其miR-626表达。
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