CN116716255A - 用于改善色素沉着的基因修饰外泌体及其制备方法与应用 - Google Patents
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Abstract
本发明涉及外泌体生物技术领域，尤其是涉及一种用于改善色素沉着的基因修饰外泌体及其制备方法与应用；一种用于改善色素沉着的基因修饰外泌体，基因修饰外泌体为过表达miR‑330‑5p基因修饰的间充质干细胞外泌体，基因修饰外泌体的制备方法包括以下步骤：构建miR‑330‑5p基因的过表达载体；将miR‑330‑5p基因的过表达载体转染293T细胞，进行慢病毒包装，收集上清；将收集的上清转染间充质干细胞；筛选转染阳性的间充质干细胞，并扩大培养，收集上清，提取得到过表达miR‑330‑5p基因修饰的间充质干细胞外泌体；本申请中采用慢病毒转染MSC，细胞核内的miR‑330‑5p基因可随MSC的扩增长期存在，形成稳定的工业化大量生产。
Description
技术领域
本发明涉及外泌体生物技术领域，尤其是涉及一种用于改善色素沉着的基因修饰外泌体及其制备方法与应用。
背景技术
皮肤的色素沉着是表皮黑色素细胞数量增多和活性增强导致的，黑色素细胞的胞浆内合成黑色素的量在miRNA水平受精确的调控。现有文献表明miR-330-5p可与酪氨酸酶家族的TYR-mRNA靶向结合，抑制其分泌黑色素的能力。现有文献中报道了miR-330-5p对小鼠黑色素细胞的抑制效果，利用脂质体将miR-330-5p导入小鼠角质形成细胞，从培养上清中分离过表达miR-330-5p的外泌体；该研究利用角质形成细胞外泌体可以大量被黑素细胞摄取的特性，提出了一条皮肤美白的创新性思路。
现有技术中在研究miR-330-5p对小鼠黑色素细胞的抑制效果时，治疗目标使用的是小鼠黑色素细胞，治疗药物使用的是miR-330-5p过表达的小鼠角质形成细胞来源的外泌体，虽然疗效确切，但是否适用于人类仍有待验证。
现有技术中在研究miR-330-5p对小鼠黑色素细胞的抑制效果，所用的细胞为角质形成细胞，属于成熟体细胞，缺点是：
（1）获取不易：需要切割大量皮肤才能获取；
（2）分离方法繁琐：需要在无实验室用胶原酶消化，对设备要求较高；
（3）体外扩增困难：已近分化末期，不能像干细胞一样传代数十次。
因此，从培养上清中提取基因修饰外泌体在动物层面做机制研究尚可，但是工业化量产用于人类使用难以实现；另外，如果使用表皮干细胞系，类似肿瘤细胞，与原代人脂肪MSC相比有携带肿瘤基因的风险。
现有技术中只做了细胞学研究，没有做动物研究，离临床转化仍有较远距离；因此，如何实现基因修饰外泌体工业化量产成为迫切需要解决的问题。
发明内容
针对现有技术存在的不足，本申请提供用于改善色素沉着的基因修饰外泌体及其制备方法与应用。
第一方面，本申请提供一种用于改善色素沉着的基因修饰外泌体，采用如下的技术方案：
一种用于改善色素沉着的基因修饰外泌体，所述基因修饰外泌体为过表达miR-330-5p基因修饰的间充质干细胞外泌体。
优选的，间充质干细胞为脂肪间充质干细胞。
第二方面，本申请提供一种用于改善色素沉着的基因修饰外泌体的制备方法，采用如下的技术方案：
一种用于改善色素沉着的基因修饰外泌体的制备方法，包括以下步骤：
（1）构建miR-330-5p基因的过表达载体；
（2）将miR-330-5p基因的过表达载体转染293T细胞，进行慢病毒包装，收集上清；
（3）将收集的上清转染间充质干细胞；
（4）用嘌呤霉素筛选转染阳性的间充质干细胞，并扩大培养，收集上清，提取得到过表达miR-330-5p基因修饰的间充质干细胞外泌体。
优选的，步骤（1）中miR-330-5p基因的过表达载体的构建方法如下：将miR-330-5p基因、绿色荧光蛋白筛选标记和嘌呤霉素抗性基因连接在慢病毒载体上，得到miR-330-5p基因的过表达载体。
优选的，miR-330-5p基因的过表达载体的核苷酸序列如SEQ ID No.1所示。
优选的，绿色荧光蛋白筛选标记的核苷酸序列如SEQ ID No.2所示。
优选的，嘌呤霉素抗性基因的核苷酸序列如SEQ ID No.3所示。
本申请中公开了一种用于改善色素沉着的基因修饰外泌体的制备方法，通过将miR-330-5p基因通过慢病毒转染MSC后，从培养上清中提取外泌体；与脂质体或电转染相比，本申请中采用的慢病毒转染MSC属于稳定转染，细胞核内的miR-330-5p基因可随MSC的扩增长期存在，形成稳定的工业化大量生产，从而可以实现工业化制备外泌体。
第三方面，本申请提供一种基因修饰外泌体在制备用于改善色素沉着的药物中的应用，采用如下的技术方案：
上述的基因修饰外泌体（过表达miR-330-5p基因修饰的间充质干细胞外泌体）在制备用于改善色素沉着的药物中的应用。
第四方面，本申请提供一种基因修饰外泌体在制备用于改善色素沉着的化妆品中的应用，采用如下的技术方案：
上述的基因修饰外泌体（过表达miR-330-5p基因修饰的间充质干细胞外泌体）在制备用于改善色素沉着的化妆品中的应用。
在一个具体的可实施方案中，一种用于改善色素沉着的基因修饰外泌体的制备方法，包括以下步骤：
（1）从整形美容机构获取无菌的脂肪抽取物，在实验室用胶原酶溶解，贴壁培养3天获取脂肪MSC，用细胞工厂大量扩增；
（2）将miR-330-5p基因序列（如SEQ ID No.4）、绿色荧光蛋白筛选标记（如SEQ IDNo.2）和嘌呤霉素抗性基因（如SEQ ID No.3）的序列都连接在慢病毒载体上，得到miR-330-5p基因的过表达载体（如SEQ ID No.1所示），将得到的miR-330-5p基因的过表达载体在293T细胞中进行慢病毒包装，从293T细胞培养上清获取包装好的携带miR-330-5p的慢病毒，-20℃保存；
（3）将携带miR-330-5p的慢病毒加入MSC培养体系，感染MSC使其胞浆过表达miR-330-5p，持续用嘌呤霉素筛选直至全部MSC都表达绿色荧光，得到过表达miR-330-5p的MSC，将得到过表达miR-330-5p的MSC与未转染的MSC在mRNA水平上表达强度进行比对，当过表达miR-330-5p的MSC为高强度表达时，再继续扩大培养，并开始持续收集培养上清，-20℃保存；
（4）将10L左右的培养上清，统一从-20℃取出并融化，用北京华龛公司生产的3DFloTrix vivaEXO外泌体收获系统将培养上清浓缩至500mL的外泌体溶液，即为Exo-miR-330-5p（过表达miR-330-5p基因修饰的间充质干细胞外泌体）；将外泌体溶液按每支1 mL分装，-80℃保存。
经纳米颗粒跟踪分析（NTA）检测外泌体的浓度为3×1011个/mL，粒径值范围为54nm-140nm，平均粒径值为60nm。
综上所述，本申请包括以下至少一种有益技术效果：
本申请中公开了一种用于改善色素沉着的基因修饰外泌体的制备方法，通过将miR-330-5p基因通过慢病毒转染MSC后，从培养上清中提取外泌体；本申请中采用慢病毒转染MSC，细胞核内的miR-330-5p基因可随MSC的扩增长期存在，形成稳定的工业化大量生产。
附图说明
图1为miR-330-5p基因的过表达载体结构示意图；
图2为过表达miR-330-5p的MSC、普通脂肪MSC的mRNA表达水平；
图3为过表达miR-330-5p基因修饰的MSC外泌体（Exo-miR-330-5p）的粒径分布图；
图4为过表达miR-330-5p基因修饰的MSC外泌体（Exo-miR-330-5p）、天然脂肪MSC外泌体（MSC-Exo）在黑色素细胞培养体系中效果比对图；
图5为过表达miR-330-5p基因修饰的MSC外泌体（Exo-miR-330-5p，实验组）、天然脂肪MSC外泌体（MSC-Exo，对照组）对黑鼠的美白效果比对图；
图6为过表达miR-330-5p基因修饰的MSC外泌体（Exo-miR-330-5p）对人的美白效果比对图。
具体实施方式
本申请涉及的原料均采用市售产品，其中：
miR-330-5p基因序列、绿色荧光蛋白筛选标记序列、嘌呤霉素抗性基因的序列、miR-330-5p基因的过表达载体均由南京斑马鱼生物科技有限公司提供。
细胞工厂购自于康宁公司（Corning Inc.）。
本实施例中涉及的缩略语和关键术语定义：
MSC：间充质干细胞。
以下结合实施例和对比例对本申请作进一步详细说明。
实施例1：
过表达miR-330-5p基因修饰的MSC外泌体的制备方法，步骤如下：
1、脂肪MSC的获得
从整形美容机构获取无菌的脂肪抽取物，用0.01 mol/L无菌PBS冲洗脂肪组织至无血色，将组织用无菌眼科剪刀和镊子清理干净后剪成约1mm3大小，加入适量体积浓度0.1%Ⅰ型胶原酶（Ⅰ型胶原酶的加入量为抽取物的体积的0.1%），37℃下振荡消化30 min；用含体积分数10%胎牛血清的培养基终止消化并离心(1000r/min，15min)；弃掉离心后的混悬液上清，将未消化完全的组织块及下层的细胞团重悬，混匀后接种至培养皿，放入37℃，体积分数5% CO2培养箱中培养；贴壁培养3天获取脂肪MSC，用细胞工厂在37℃，体积分数5%CO2下大量扩增。
2、构建miR-330-5p基因的过表达载体
将miR-330-5p基因序列（如SEQ ID No.4所示）、绿色荧光蛋白筛选标记（如SEQ IDNo.2所示）和嘌呤霉素抗性基因的序列（如SEQ ID No.3）都连接在慢病毒载体上，得到miR-330-5p基因的过表达载体（如SEQ ID No.1所示），见图1。
上述步骤由南京斑马鱼生物科技有限公司制备。
3、慢病毒包装
将得到miR-330-5p基因的过表达载体在293T细胞中进行慢病毒包装，从293T细胞培养上清获取包装好的携带miR-330-5p的慢病毒，-20℃保存。
上述步骤由南京斑马鱼生物科技有限公司制备。
4、获得过表达miR-330-5p基因修饰的MSC外泌体
4.1、将步骤1获得的人脂肪MSC加入至MSC培养基，并培养于T175瓶中，加入1mL携带miR-330-5p的慢病毒（步骤3制得）并感染MSC，于37℃继续培养24小时；更换T175瓶中的培养基为筛选培养基，用嘌呤霉素持续筛选培养，直至全部MSC都表达绿色荧光，得到过表达miR-330-5p的MSC；将得到的过表达miR-330-5p的MSC与未转染的MSC在mRNA水平上表达强度进行比对，当过表达miR-330-5p的MSC为高强度表达时，再继续扩大培养，并开始持续收集培养上清，-20℃保存。
本步骤中所用的MSC培养基为人间充质干细胞无血清基础培养基（AM-V SerumFree Medium；货号SC-2013-G-A，天津灏洋），添加质量浓度1%青链霉素。
本步骤中筛选培养基为人间充质干细胞无血清基础培养基（AM-V Serum FreeMedium；货号SC-2013-G-A，天津灏洋），添加2μg/mL嘌呤霉素。
其中，过表达miR-330-5p的MSC和普通脂肪MSC在mRNA水平上表达强度比对，具体如下：
将过表达miR-330-5p的MSC和普通脂肪MSC进行realtime-PCR检测，对比可知过表达miR-330-5p的MSC在mRNA水平上表达强度增大40-50倍（见图2）。
4.2、将步骤4.1制备得到的培养上清10L左右，统一从-20℃取出并融化，用北京华龛公司生产的3D FloTrix vivaEXO外泌体收获系统将培养上清浓缩至500mL的外泌体溶液，即为Exo-miR-330-5p（过表达miR-330-5p基因修饰的MSC外泌体）；将外泌体溶液按每支1 mL分装，-80℃保存。
用纳米颗粒跟踪分析（NTA）对制备得到的Exo-miR-330-5p进行检测，外泌体的浓度为3×1011个/mL，粒径值范围为54nm-140nm，平均粒径值为60nm（图3）。
应用例：
应用例1：
将Exo-miR-330-5p（实施例1制得）作为实验组、天然脂肪MSC外泌体（MSC-Exo）作为对照组，各取1 mL分别加入至黑色素细胞培养体系中，56小时后使用酶标仪测量培养上清颜色的深浅；相对于单纯黑色素细胞的培养基，加入MSC-Exo组颜色减淡了25%，加入Exo-miR-330-5p组颜色减淡了39%（图4）。
本实验中所用的黑色素细胞培养体系为 A-375 人恶性黑色素瘤细胞系（ATCC细胞库编号CRL-1619）；黑色素细胞培养体系中包括DMEM培养基，体积浓度10%胎牛血清，体积浓度5% L-谷氨酰胺和体积浓度1%青链霉素。
应用例2：
将Exo-miR-330-5p（实施例1制得）作为实验组、天然脂肪MSC外泌体（MSC-Exo）作为对照组，分别皮内注射黑鼠的背部固定位置的皮肤，隔天一次，每次0.2mL，从1周后开始实验组皮肤颜色逐渐变浅，1个月后显著变浅，对照组始终没有颜色改变（图5）。
应用例3：
给一位皮肤黝黑的志愿者前臂的前端注射Exo-miR-330-5p（实施例1制得）作为实验组，前臂的后端不注射，作为对照组；隔天一次，每次0.2mL，从1周后开始实验组皮肤颜色逐渐变浅，1个月后显著变浅（图6，右），对照组始终没有颜色改变（图6，左）。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (9)
1.一种用于改善色素沉着的基因修饰外泌体，其特征在于：所述基因修饰外泌体为过表达miR-330-5p基因修饰的间充质干细胞外泌体。
2.根据权利要求1所述的一种用于改善色素沉着的基因修饰外泌体，其特征在于：间充质干细胞为脂肪间充质干细胞。
3.一种制备权利要求1或2所述用于改善色素沉着的基因修饰外泌体的方法，其特征在于，包括以下步骤：
（1）构建miR-330-5p基因的过表达载体；
（2）将miR-330-5p基因的过表达载体转染293T细胞，进行慢病毒包装，收集上清；
（3）将收集的上清转染间充质干细胞；
（4）筛选转染阳性的间充质干细胞，并扩大培养，收集上清，提取得到过表达miR-330-5p基因修饰的间充质干细胞外泌体。
4.根据权利要求3所述的方法，其特征在于，步骤（1）中miR-330-5p基因的过表达载体的构建方法如下：将miR-330-5p基因、绿色荧光蛋白筛选标记和嘌呤霉素抗性基因连接在慢病毒载体上，得到miR-330-5p基因的过表达载体。
5.根据权利要求4所述的方法，其特征在于，miR-330-5p基因的过表达载体的核苷酸序列如SEQ ID No.1所示。
6.根据权利要求4所述的方法，其特征在于，绿色荧光蛋白筛选标记的核苷酸序列如SEQ ID No.2所示。
7.根据权利要求4所述的方法，其特征在于，嘌呤霉素抗性基因的核苷酸序列如SEQ IDNo.3所示。
8.权利要求1或2所述的基因修饰外泌体或权利要求3-7任一项所述方法得到的基因修饰外泌体在制备用于改善色素沉着的药物中的应用。
9.权利要求1或2所述的基因修饰外泌体或权利要求3-7任一项所述方法得到的基因修饰外泌体在制备用于改善色素沉着的化妆品中的应用。
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