CN111876390A - 负载化合物的转基因干细胞外泌体在制备药物或美白化妆品中的用途 - Google Patents
负载化合物的转基因干细胞外泌体在制备药物或美白化妆品中的用途 Download PDFInfo
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Abstract
本发明涉及负载化合物的转基因干细胞外泌体在制备药物或美白化妆品中的用途，本发明将miR‑27b‑3p转染表皮干细胞并收获了所述转基因干细胞来源的外泌体，同时负载阿霉素于外泌体上，通过实验验证了所述负载了阿霉素的外泌体能够比不负载阿霉素的外泌体能够更显著抑制黑色素细胞的增殖与迁移，具有较好的药用和化妆品应用前景。
Description
技术领域
本发明涉及生物领域，具体的涉及负载化合物的转基因干细胞外泌体在制备药物或美白化妆品中的用途。
背景技术
外泌体的分泌是一种普遍的细胞功能。外泌体被证实由独特的脂质和蛋白质组成，可递送各种生物活性分子，如蛋白质、膜受体、mRNA和microRNA等，参与细胞间通讯、调节免疫系统和遗传物质的转运。
近年来，研究人员将外泌体与皮肤疾病联系起来，发现外泌体不仅参与皮肤生理、病理过程，如调节皮肤微环境中促炎细胞因子分泌，促进皮肤缺损处血管新生及胶原沉积，以及调节皮肤成纤维细胞增殖分化，同时还在皮肤微环境发生病变时发挥特异性信息传递作用，进而促使增生性瘢痕、皮肤硬化及皮肤黑色素瘤等皮肤疾病的发生。因此，在防治多种皮肤疾病时，不仅可利用外泌体作为药物载体包裹药物，有选择性地渗入病灶，如炎症组织或肿瘤部位，还可将其作为生物标志物用于疾病的预测及诊断，或设计药物进行信息传递阻断，从而达到防治皮肤疾病的作用。
CN105483081B提供了高表达microRNA145-5p的脐带间充质干细胞所分泌的外泌体(umsc-exosome)的制备方法及其加速皮肤全层缺损愈合同时拮抗瘢痕挛缩的各种生物制剂中的应用。其外泌体是通过以下方法制备：(1)采用新鲜脐带培养人脐带间质干细胞；(2)制备高表达miRNA145-5p的间充质干细胞；(3)条件培养基的储备；(4)外泌体的抽提与纯化。其高表达microRNA145-5p的人脐带间充质干细胞来源的外泌体作为生物制剂转入大鼠背部全层创面模型中可有效促进皮肤肉芽组织增生，加快创口愈合速度并能拮抗瘢痕挛缩。但是该方法并不能抑制黑色素细胞的增殖。
黑素瘤(melanoma)是一种黑素细胞来源的恶性程度极高的肿瘤，是起源于胚胎期神经嵴的恶性肿瘤，其恶性度极高，预后较差。可发生于皮肤、眼球、消化道、生殖系统等部位，但其中以皮肤恶性黑色素瘤最常见。由于恶性黑色素瘤恶性度高，易于早期转移，即使早期进行根治手术，患者5年生存率也低于70％。因此，近几年人们将研究方向转向研究肿瘤微环境，探索黑素瘤的发病机制，从免疫靶向治疗上寻找新的突破口。
研究发现负责将黑素瘤细胞募集至前哨淋巴结的转移因子会被黑素瘤外泌体上调，黑素瘤外泌体增加的细胞募集、细胞外基质和血管增殖因子的基因表达，能在前哨淋巴结微环境中产生有利于黑素瘤细胞募集、诱捕和生长的小生境。基本上，前哨淋巴结是黑素瘤转移的“土壤”，而黑素瘤外泌体充当了“种子”。王磊对黑素瘤细胞系B16-外泌体进行分离、鉴定和染色。通过荧光显微术，免疫印迹，细胞增殖实验以及细胞迁移实验，发现B16-外泌体能够进入间充质基质细胞(MSC)，显著促进其增殖与迁移能力，并上调α-SMA的表达量，而且TGF-β受体的激活参与了上述变化。因此，针对外泌体参与黑色素瘤病理过程中的信息传递这一特点，不仅可以将外泌体作为生物标志物用于黑素瘤患者的诊断及预测，还可设计药物进行信息传递阻断，如TGF-β受体的阻断剂SB431542，将有希望控制黑素瘤的病理发展，并在此基础上研制新型的抗肿瘤疗法。
另外，外泌体携带的大量miRNA能参与细胞间通讯，选择性靶向黑素细胞的mRNAs可以通过改变基因的表达和酶活性，调节黑素细胞的色素状态。已有研究表明，miR145、miR675、miR340、miR218、miR330-5p、miR211、miR-27a-3p、miR25、miR155、miR-21a-5p等miRNAs通过多种机制影响黑素生成过程。
作为天然的纳米级药物投递载体，可用其包裹化疗药物并靶向作用于黑色素瘤细胞，提高抗肿瘤效能同时有效降低传统化疗的副作用。如赵莉娟等针对恶性黑色素瘤细胞表面αν整合素的高水平表达，在体外培养人肾上皮细胞系293T细胞，待细胞融合率达80％-90％时，体外转染iRGD，提取外泌体，通过电穿孔包裹阿霉素，利用流式细胞仪检测iRGD-外泌体-阿霉素对人恶性黑色素瘤细胞系A375细胞的亲和力，并对iRGD-外泌体-阿霉素抑制A375细胞的增殖能力进行分析。结果显示iRGD-外泌体-阿霉素可明显抑制A375细胞的增殖，而空白转染-外泌体-阿霉素未观察到明显的抑制作用，说明表达iRGD蛋白的外泌体携载阿霉素能更有效地抑制细胞增殖，起到抗肿瘤作用。
虽然目前已经有针对其他细胞来源的外泌体用于黑色素细胞的抑制研究，但是针对表皮干细胞外泌体用于黑色素细胞的抑制研究还不够，更没有针对表皮干细胞进行化合物负载的相关报道。由于皮肤的表皮干细胞具有较为活跃的细胞形态，其外泌体对于皮肤的修复和帮助作用理论上具有较好的效果，因此，选择表皮干细胞作为研究对象，需要大力开发。
发明内容
本发明的目的在于一种具有抗黑色素瘤作用的外泌体，由过表达miR-27b-3p基因的表皮干细胞来源的外泌体经化合物负载制备而成，所述化合物为本领域熟知的可用于治疗黑色素瘤的化合物。
优选地，通过电转染法或脂质体转染法将化合物导入外泌体内制备化合物负载的外泌体。
上述外泌体在制备治疗黑色素瘤的药物中的应用。
上述外泌体在制备用于美白的化妆品中的应用。
一种药物制剂，含有上述外泌体和一种或多种药学上可以接受的载体或赋形剂，通过药学上可以接受的制剂工艺制备而成。
一种美白化妆品，含有上述外泌体和一种或多种化妆品赋形剂。
本发明另外一方面提供一种过表达miR-27b-3p基因的表皮干细胞外泌体及其制备方法和应用。
本发明提供的过表达miR-27b-3p基因的表皮干细胞外泌体高效靶向黑色素，抑制细胞的生长和迁移，进而抑制肿瘤生长。
进一步的，所述过表达miR-27b-3p基因的表皮干细胞外泌体能够作用于黑色素细胞的经验证的途径是通过抑制PIK3R3蛋白表达实现的。磷脂酰肌醇-3-激酶(PIK3R3)是信号通路中重要的信号因子，参与细胞增殖、生长、分化、细胞迁移和凋亡等多个生理过程，因此，抑制PIK3R3蛋白能够抑制细胞的增殖及迁移。
本发明提供了一种过表达miR-27b-3p基因的表皮干细胞外泌体，所述过表达miR-27b-3p基因的表皮干细胞外泌体的制备方法包括以下步骤：
1)将miR-27b-3p转染表皮干细胞，经筛选获得miR-27b-3p过表达表皮干细胞；
2)将所述步骤1)得到的转基因干细胞进行培养后收集上清，离心去除漂浮活细胞，得到含外泌体的上清，采用离心法得到外泌体。
本发明还提供了上述技术方案所述过表达miR-27b-3p基因的表皮干细胞外泌体在制备治疗黑色素瘤的药物中的应用。
本发明还提供了上述技术方案所述过表达miR-27b-3p基因的表皮干细胞外泌体在制备美白皮肤的化妆品中的应用。
有益效果
本发明通过miR-27b-3p转染表皮干细胞并收获了所述转基因干细胞来源的外泌体，将所述的外泌体再负载筛选的化合物后，通过实验验证了所述外泌体能够比不负载化合物的转基因外泌体更显著的抑制黑色素细胞中PIK3R3蛋白的表达同时也能够抑制黑色素细胞的增殖与迁移，具有较好的药用和化妆品应用前景。
附图说明
图1干细胞miR-27b-3p表达量图
图2干细胞外泌体粒径分布图
图3细胞增殖检测结果图
图4Western blotting检测结果图
具体实施方式
下面结合具体实施例介绍本发明的技术方案。下述实施例中未特别强调的实验材料均为常规实验材料，无特别要求，都是本领域技术人员容易获得的常规材料。
实施例1人表皮干细胞外泌体的制备
1、miR的制备
miR-27b-3p：5’-uucacaguggcuaaguucugc-3’，miR-27b-3p inhibitor：5’-gcagaacuuagccacugugaa-3’，所述核酸由上海吉玛制药技术有限公司合成。
2、细胞培养：
人表皮干细胞(购自北纳生物，编号BNCC340781)，10％FBS+90％高糖DMEM培养基培养，37℃，5％CO2培养。收集生长状态良好的表皮干细胞，离心计数，以5×103每孔铺于96孔板内，37℃，5％CO2培养24h。
3、转染：
1)转染前一天，在96孔板中用适量不含抗生素的培养基接种培养细胞，使转染时细胞的汇合度达到50％；
2)转染样品按照如下方法准备寡聚物-Lipofecta mineTM2000复合物：
a.用25μl不含血清的Opti-MEMI培养基(Gibco)分别稀释miR-27b-3p和miR-27b-3p inhibitor、阴性对照(无核酸Opti-MEMI培养基)，加入孔内后终浓度为50nM，轻轻混匀，每个转染设3个复孔；
b.使用前轻轻混匀Lipofecta mineTM2000(Invitrogen)，然后取0.25μl稀释到25μl的Opti-MEMI培养基，轻轻混匀后在室温下孵育5min；
c.孵育5min后，稀释的Lipofecta mineTM2000分别与稀释的a步骤的核苷酸及对照混合，轻轻混匀后在室温下孵育20min，以允许复合物的形成；
3)将复合物加入到每一个包含细胞和培养基的孔中，轻轻地前后摇动培养板混合；核苷酸的终浓度为50nM。
4)37℃，5％CO2培养箱继续孵育72小时。
实施例2转基因的表皮干细胞的鉴定
采用RT-qPCR法检测转基因表皮干细胞中miR-27b-3p的表达。将实施例1制备的细胞生长状态良好且数量达到80％时，加入1mL TRIzol和200μL氯仿裂解，然后将样品摇动30s，4℃，14000×g离心15min收集沉淀物后加入60μL DEPC水以溶解沉淀物，通过加入等体积的苯酚、氯仿和异丙醇萃取，将1μL RNA稀释50倍，并在酶标仪上测定吸光度(OD)值。miR-27b-3p正向引物:ctcaactggtgtcgtggagt，反向引物:acactccagctggguucaca，内参U6正向引物:ctcgcttcggcagcaca，反向引物:aacgcttcacgaatttgcgt，采用2-ΔΔCT法分析实验数据，检测miR-27b-3p的表达，挑选了两个阳性转基因细胞以及对照细胞进行检测，结果如图1所示。
从图1可以看出，转基因的表皮干细胞其miR-27b-3p的表达量相对于未转基因的表皮干细胞其表达量提高了接近3倍，得到了显著的提高表达。因此，选择miR-27b-3p的表达量最高的转基因干细胞用于后续外泌体的制备。
实施例3转基因干细胞外泌体的制备
外泌体的收集:取第3代细胞，细胞融合生长至80-90％时，换无血清培养基培养48h，收集细胞上清液。收集的细胞上清液300×g离心10min，去除死细胞和大的细胞碎片，2000×g离心10min，去除死细胞和细胞碎片，10000×g/min离心30min去除细胞碎片较大的囊泡，0.22μm针头滤器过滤，去除微泡及可能存在的凋亡小体。用20mL空针将上清液转移至超速离心管中，106×g离心60min，去除上清液，收集沉淀，得到粗提取的外泌体，106×g离心60min，去除上清液，用100μL PBS溶解沉淀，得到较纯的外泌体。以上操作均在4℃，无菌条件下进行。取5μL外泌体溶液，用BCA法测外泌体的蛋白浓度，BCA蛋白浓度测定结果为1.72g/L；取1滴外泌体滴于铜网上，体积分数1％磷钨酸负染，室温干燥后，用透射电子显微镜观察外泌体外形及检测外泌体直径；取20μL外泌体溶液，用PBS稀释至200μL，用粒度仪检测直径分布。外泌体外形多为椭圆状，有膜结构，直径为(62.358±11.114)nm，粒度仪鉴定其直径集中分布在50-80nm，见图2。取外泌体，采用实施例2的检测方法来检测其中miR--27b-3p的表达量结果发现，其表达量与在转基因干细胞中的表达量基本相似都高表达。
实施例4外泌体负载化合物的制备
将50ug阿霉素、50ug谷胱甘肽分别与等质量的外泌体混合，通过电转的方法制备化合物负载外泌体。电击条件为电压420V，电容150μF，于4mm电转杯进行电转。随后用倒置离心超滤膜过滤，去除未转染进外泌体内的游离化合物，分别制备得到阿霉素-转基因外泌体、谷胱甘肽-转基因外泌体。
实施例5细胞增殖检测
取对数生长期MU89人黑色素细胞，用0.05％胰蛋白酶+0.53mmol/L EDTA溶液消化后，以新鲜培养液配成密度为6×103个/mL的细胞悬液，接种于96孔细胞培养板，每孔180μL，每孔分别加入阿霉素-转基因外泌体、谷胱甘肽-转基因外泌体以及转基因外泌体和未转基因外泌体各50μL，以空白培养基作为空白对照，5个样品设置4个样品质量浓度梯度，每个梯度重复3次。取96孔板，将培养板置于37℃、5％CO2的条件培养箱中分别孵育0、24、48、72、96h，向待测孔加入10μL CCK-8溶液。每组细胞、每个时间点取3个样品检测，将培养板在培养箱内孵育。用酶标仪测定在450nm处的吸光度。结果如图3所示，CCK-8检测显示外泌体均可以抑制细胞的增殖，但是转基因的外泌体本身比未转基因外泌体具有更好的黑色素细胞抑制效果，而阿霉素-转基因外泌体也具有比谷胱甘肽-转基因外泌体更好的细胞抑制效果(图3)。
实施例6采用Western blotting法检测PIK3R3的蛋白表达量
将阿霉素-转基因外泌体以及未转基因的外泌体作用后的黑色素细胞中提取蛋白，采用SDS-PAGE法，应用10％凝胶、80V电压进行电泳90min，并电印迹到PVDF膜上，封闭后加入TBST一抗(山羊抗人PIK3R3多克隆抗体)，放置于4℃冰箱摇床上孵育过夜，洗膜后，加入山羊抗兔IgG/辣根酶标记二抗，孵育2h后暗室曝光，将曝光后的胶片冲洗干净后晾干采集图像。结果如图4所示，在转基因外泌体作用于黑色素细胞后，黑色素细胞的PIK3R3的蛋白表达量被显著的抑制，这表明所述的miR能够作用于PIK3R3的蛋白。
实施例7Transwell侵袭实验检测黑色素瘤细胞迁移能力
将Transwell小室置于24孔板内，将Transwell小室内膜均匀涂抹Matrigel胶50μl(0.2μg/μl)，37孵育15min，使胶凝固；消化、离心、计数细胞后，按照2.5×104/ml用无血清培养基稀释细胞，制成细胞悬液；按照每孔200μl，将细胞悬液加入Transwell上室，同时在Transwell下室加入10％FBS+培养基600μl，放入37℃孵箱培养；甲醛固定，结晶紫染色10min，然后用棉签轻轻擦拭内膜上的细胞。显微镜下技术，计数4个高倍视野(×40)下穿过滤膜的细胞数。所述细胞分别对应于：采用转基因外泌体处理过的MU89人黑色素细胞、采用未转基因外泌体处理过的MU89人黑色素细胞、以及单纯的MU89人黑色素细胞，实验重复3次。细胞侵袭穿过Matrigel基质胶的能力可以反映细胞的侵袭能力。Transwell实验结果如下表1所示。
表1细胞侵袭穿过Matrigel基质胶的能力
| 分组 | 透过Matrigel基质胶的细胞数量 |
| 转基因外泌体 | 32.90±4.25 |
| 阿霉素-转基因外泌体 | 20.52±3.68 |
| 谷胱甘肽-转基因外泌体 | 30.77±4.25 |
| 未转基因外泌体 | 76.14±6.51 |
| 空白对照 | 343.55±25.19 |
从表1可以看出:采用阿霉素-转基因外泌体处理过的MU89人黑色素细胞通过Matrigel基质胶的数量(20.52±3.68)，采用谷胱甘肽-转基因外泌体处理过的MU89人黑色素细胞通过Matrigel基质胶的数量(30.77±4.25)，转基因外泌体处理过的MU89人黑色素细胞通过Matrigel基质胶的数量(32.90±4.25)、采用未转基因外泌体处理过的MU89人黑色素细胞通过Matrigel基质胶的数量(76.14±6.51)、以及单纯的MU89人黑色素细胞通过Matrigel基质胶的数量为(343.55±25.19)，由此可见，采用阿霉素-转基因外泌体处理过的黑色素细胞通过Matrigel基质胶的能力得到了最显著的抑制。
实施例8外泌体对裸鼠黑色素瘤移植瘤的抑制活性
将实验裸鼠于独立通气笼盒SPF条件下适应性驯养1周。1周后在裸鼠腋窝皮下接种0.1mL的MU89人黑色素细胞悬液(含5×106个对数生长期的细胞)。当肿瘤体积生长至100mm3左右(约12d)的时候开始分组腹腔给药。分组如下，每组各8只均腹腔注射给药，连用14d。
对照组：生理盐水+1％DMSO；
实验组1：转基因外泌体组：15mg/(kg·d)；
实验组2：未转基因外泌体组：15mg/(kg·d)；
实验组3：阿霉素-转基因外泌体组：15mg/(kg·d)；
实验组4：谷胱甘肽-转基因外泌体：15mg/(kg·d)；
抑瘤率(％)＝(1-外泌体组肿瘤体积/对照组肿瘤体积)×100％。数据以均数±标准差表示，SPSS13.0统计软件处理数据，采用完全随机设计的单因素方差分析分析组间差异的显着性，以P＜0.05为差异有统计学意义。表3为各组抑瘤率(％)。
表1外泌体治疗后终末移植瘤体积(cm3)
| 终末移植瘤体积(cm<sup>3</sup>) | 抑瘤率 | |
| 对照组 | 1.88±0.21 | / |
| 实验组1 | 0.42±0.11 | 77.66％ |
| 实验组2 | 0.92±0.34 | 51.06％ |
| 实验组3 | 0.31±0.09 | 83.51％ |
| 实验组4 | 0.53±0.27 | 71.81％ |
外泌体干预过程中，各组移植瘤裸鼠没有出现药物引起的死亡，干预前各组移植瘤裸鼠体质量差异无统计学意义，干预后各组移植瘤裸鼠体质量差异亦无统计学意义。与对照组比，转基因外泌体组肿瘤体积显著减小，特别是阿霉素-转基因外泌体组抑制效果最好，体积显著减小(P＜0.05)。
实施例9安全性实验
试验材料和方法
动物：普通级实验动物大白兔。体查动物未见异常。
环境条件：室温：22℃-25℃，相对湿度：60％-70％；
试验方法：
依据《化妆品卫生规范》(2007年版)急性皮肤刺激性试验，试验前24小时，将试验动物背部脊柱两侧毛剪掉，去毛范围3厘米*3厘米。取受试的外泌体0.5ml直接涂抹在右侧皮肤上，以洁净纱布覆盖，用无刺激性胶布固定2小时。另外一侧为阴性对照。除去受试物后的1小时、24小时、48小时72小时观察受试动物接触部位皮肤反应并做记录。
结论：
在本试验条件下，外泌体对大白兔急性皮肤刺激试验按皮肤刺激强度分级为无刺激性，具有较好的安全性。
Claims (9)
1.一种具有抗黑色素瘤作用的外泌体，其特征在于：由过表达miR-27b-3p基因的表皮干细胞来源的外泌体经化合物负载制备而成，所述化合物为用于治疗黑色素瘤的化合物。
2.如权利要求1所述的外泌体，其特征在于所述化合物为阿霉素。
3.如权利要求1所述的的外泌体，所述化合物负载方法为将阿霉素与等质量的外泌体混合，通过电转的方法制备化合物负载外泌体。
4.如权利要求3所述的外泌体，其中电转的方法为：电击条件为电压420V，电容150μF，于4mm电转杯中进行电转。
5.如权利要求3所述的外泌体在用于制备抑制黑色素细胞增殖的药物中的用途。
6.如权利要求3所述的外泌体在用于制备抑制黑色素细胞增殖的美白化妆品中的用途。
7.如权利要求5或6所述的用途，其特征在于：所述过表达miR-27b-3p基因的表皮干细胞外泌体的制备方法包括以下步骤：
1)将miR-27b-3p转染表皮干细胞，经筛选获得miR-27b-3p过表达表皮干细胞；
2)将所述步骤1)得到的转基因干细胞进行培养后收集上清，离心去除漂浮活细胞，得到含外泌体的上清，采用离心法得到外泌体。
8.如权利要求7所述的用途，所述转染为电转化或者脂质体转化。
9.如权利要求7所述的用途，其特征在于所述黑色素细胞为MU89人黑色素细胞。
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