CN117257956B - miR-942-5p在制备治疗光老化的药物中的应用 - Google Patents
miR-942-5p在制备治疗光老化的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域,具体涉及miR‑942‑5p在制备治疗光老化的药物中的应用。具体地,本发明提供了以下任意活性成分在制备抗衰老产品中的应用:1)miR‑942‑5p和/或miR‑942‑5p前体、2)多核苷酸,所述多核苷酸能被转录形成miR‑942‑5p、3)载体,所述载体上含有1)或2)、4)递送载体,所述递送载体中含有以上1)‑3)中至少一种、5)促miR‑942‑5p表达的试剂。
Description
技术领域
本发明属于生物医药领域,具体涉及miR-942-5p在制备治疗光老化的药物中的应用。
背景技术
皮肤是人体的第一道防线,最容易受到外界环境,如紫外线(UV)辐射、空气污染物、寒冷和风的影响而受损和衰老。其中,诱导皮肤老化最重要的因素是紫外线辐射。它也被称为光老化,由于紫外线辐射来自太阳,其特征是表皮角化不良、异常增生、胶原蛋白减少、弹性丧失、皱纹等。紫外线照射可引起真皮层(光损伤部位)中基质金属蛋白酶(MMPs)的表达,从而降解真皮胶原。此外,紫外线可以通过活性氧介导炎症(ROS)并导致衰老。因此,开发缓解光老化的活性分子药物对皮肤护理的发展具有重要意义。
外泌体(exosome)是由真核细胞分泌的细胞外囊泡之一,大小从40nm到160nm不等,近些年已经有很多利用外泌体护理皮肤的产品陆续出现在医美市场,但是大部分产品都局限于用功能性细胞如间充质干细胞等分泌的外泌体直接在皮肤上使用,用于改善皮肤成纤维的功能,但是效果并不理想。然而,由于其特殊的结构,外泌体作为药物载体进行药物运输有独特的优势,主要体现在外泌体的膜结构与细胞膜相同,可以提高药物进入细胞的效率。
miRNA是一段核苷酸组成在20-25bp之间的内源性单链非编码微小RNA分子,以完全配对或不完全配对的方式与mRNA的3’端非编码区相结合。miRNA具有生物活性,既可介导mRNA的降解,也可抑制蛋白质的翻译。此外,miRNA还具有调节基因的能力,能在人体的各个系统和组织器官中发挥重要的调节作用。研究发现,在皮肤衰老进程中miRNA表达异常,并且通过干预特定的miRNA可以调节皮肤衰老过程。因此,miRNA是皮肤衰老预防和治疗中的有效靶点。
发明内容
为提供一种抗成纤维细胞(Fibroblast,FB)衰老的方法从而有效抵抗皮肤衰老,本发明提供了以下技术方案:
第一方面,本发明提供了以下任意活性成分在制备抗衰老产品中的应用:
1)miR-942-5p和/或miR-942-5p前体、
2)多核苷酸,所述多核苷酸能被转录形成miR-942-5p、
3)载体,所述载体上含有1)或2)、
4)递送载体,所述递送载体中含有以上1)-3)中至少一种、
5)促miR-942-5p表达的试剂。
优选地,所述抗衰老包括抗成纤维细胞衰老、抗皮肤衰老。
优选地,所述衰老是光照射所导致的;具体地,所述光包括可见光、紫外线、红外线。
更具体地,所述衰老是紫外线照射所导致的。
优选地,所述抗衰老在细胞的角度表现在β-半乳糖苷酶染色程度降低、细胞活率增加、I型胶原蛋白(Collagen I)分泌量增加、基质金属蛋白酶1(MMP1)分泌量降低或ROS含量降低。
优选地,所述抗衰老在皮肤状态的角度表现在皮肤厚度上升(恢复)、胶原层比例上升(恢复)、皮下胶原损伤恢复。
优选地,所述miR-942-5p如SEQ ID No.4所示。
优选地,所述递送载体包括外泌体、病毒载体、脂质纳米粒(lipid nanoparticle,LNP)、聚合物纳米载体、无机纳米载体或蛋白载体。
优选地,所述递送载体是外泌体。
优选地,所述外泌体包括外源性加载方法或内源性加载方法而制备得到的外泌体。
具体地,所述外源性加载方法是在制备和纯化外泌体后装载miR-942-5p,所述内源性加载是适当的细胞经过基因工程处理或与miR-942-5p共培养以产生工程外泌体。
优选地,所述外泌体的来源包括生物体液、生物组织或细胞培养液。
优选地,所述生物体液包括液、唾液、尿液、滑液、羊水、乳汁。
优选地,所述生物体液、生物组织包括任意生物,所述外泌体可以来源为牛奶等任意动物的乳汁。
优选地,所述细胞培养液可以是任意类型细胞的培养液,具体地,所述细胞包括外周血单个核细胞、间充质干细胞、成纤维细胞、免疫细胞等。
具体地,所述分离方法基于不同原理可以包括超速离心法、密度梯度离心、色谱法、超滤法、微流控技术、磁珠免疫法和多聚物沉淀法等。
优选地,所述外泌体分离自间充质干细胞的细胞培养液。
本发明所述术语“外泌体(Exosomes)”是细胞分泌到胞外的一种囊泡(Extracellular Vesicles,EVs),具有双层膜结构和茶托状形态,含有丰富的内含物(包括核酸、蛋白和脂质等),参与细胞间的分子传递。外泌体广泛存在于细胞培养上清以及各种体液中,包括血液、淋巴液、唾液、尿液、精液、乳汁等,同时也存在于组织样本中,如脑组织、肌肉组织、脂肪组织等。
另一方面,本发明提供了预防和/或修复皮肤、细胞损伤的方法,所述方法包括向待修复对象施用表达miR-942-5p或促进miR-942-5p表达的试剂。
优选地,所述施用对象可以是皮肤,更具体地,作用于人皮肤上的细胞。
优选地,所述细胞是成纤维细胞。
优选地,所述试剂是miR-942-5p的外泌体或其相关产品。
具体地,所述施用的途径可包括例如皮内、透皮、皮下、胃肠外、经鼻、静脉内、肌肉内、鼻内、气管内、腹膜内、肿瘤内、灌注、灌洗、直接注射和口服施用。
更具体地,所述施用的途径是皮下注射。
优选地,所述损伤包括光照射所导致的损伤,具体地,所述光包括可见光、紫外线、红外线。
优选地,所述损伤包括紫外线照射所导致的损伤。
优选地,所述方法是施用对象包括动物,例如灵长目动物、啮齿动物、兔形目动物、牛科动物、羊科动物等,最优选地的使用对象为人。
优选地,所述方法是非治疗目的的。
另一方面,本发明提供了制备预防和/或修复皮肤、细胞损伤的产品的方法,所述方法包括将miR-942-5p导入外泌体,或者,将miR-942-5p导入母细胞并诱导母细胞形成外泌体。
优选地,所述外泌体的来源包括生物体液、生物组织或细胞培养液。
优选地,所述生物体液包括液、唾液、尿液、滑液、羊水、乳汁。
优选地,所述生物体液、生物组织包括任意生物,所述外泌体可以来源为牛奶等任意动物的乳汁。
优选地,所述细胞培养液可以是任意类型细胞的培养液,具体地,所述细胞包括外周血单个核细胞、间充质干细胞、成纤维细胞、免疫细胞等。
优选地,所述母细胞包括外周血单个核细胞、间充质干细胞、成纤维细胞、免疫细胞等。
优选地,所述导入的方法包括共孵育、电转(电穿孔)、超声、冻融等。
优选地,所述导入的方法是电转。
另一方面,本发明提供了一种抗衰老产品,所述抗衰老产品表达miR-942-5p或促进miR-942-5p的表达。
优选地,所述抗衰老包括抗成纤维细胞衰老、抗皮肤衰老。
具体地,所述抗衰老产品的剂型和施用方式没有特别限制。代表性的施用方式包括但并不限于:口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)注射、和局部给药。
优选地,用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂;用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。
优选地,用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液,以及用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。
优选地,用于局部给药的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。由活性成分在无菌条件下与药学上可接受的载体及任何防腐剂、缓冲剂,或必要时可能需要的推进剂一起混合而成。
具体地,所述抗衰老产品可以是化妆品或药物组合物。
优选地,所述抗衰老产品中还可以含有湿润剂,剥落剂,润肤剂。
优选地,所述润肤剂包括植物油、矿物油、牛油树脂、可可脂、凡士林、脂肪酸、甘油三酯、苯甲酸酯、肉豆蔻酸酯、棕榈酸酯、硬脂酸酯、糖脂、磷脂、角鲨烯、甘油、玫瑰果油、酸渣树油、葡萄籽油、鳄梨油、李子油、巴卡斯果油、蓝藻油、杏仁油、阿甘油、荷荷巴油中的一种。
具体地,所述抗衰老产品可以是乳膏、外用贴剂、水凝胶贴剂、透皮贴剂、软膏、凝胶、液体、粉末、洗剂、血清、乳剂、油、粘土、保湿剂、泡沫、面膜、摩丝、气雾剂、喷雾剂、清洁剂、调色剂或洗发剂。
另一方面,本发明提供了miR-942-5p表达量的检测试剂在判断细胞和/或皮肤衰老程度中的应用。
也即,本发明提供了miR-942-5p表达量的检测试剂在制备判断细胞和/或皮肤衰老程度的产品中的应用。
优选地,所述细胞是成纤维细胞。
优选地,所述细胞衰老程度体现于β-半乳糖苷酶染色显色细胞比例高、着色深,所述细胞衰老还体现于细胞活率、I型胶原蛋白(Collagen I)分泌量、基质金属蛋白酶1(MMP1)分泌量或ROS含量。
优选地,所述皮肤衰老程度体现于皮肤厚度、胶原层比例、皮下胶原损伤。
优选地,所述衰老是光照射所导致的;具体地,所述光包括可见光、紫外线、红外线。
更具体地,所述衰老是紫外线照射所导致的。
优选地,所述检测试剂包括以下方法中检测基因表达量时所使用的试剂:核酸扩增方法、Southern杂交方法、Northern杂交方法、点杂交方法、荧光原位杂交方法、DNA微阵列方法、ASO法、高通量测序平台方法。
另一方面,本发明提供了检测皮肤、细胞衰老程度的方法,所述方法包括检测成纤维细胞中miR-942-5p的表达量。
优选地,所述miR-942-5p的表达量的表达量越低则代表衰老程度越高。
优选地,所述表达量的检测方法包括:核酸扩增方法、Southern杂交方法、Northern杂交方法、点杂交方法、荧光原位杂交方法、DNA微阵列方法、ASO法、高通量测序平台方法。
优选地,所述核酸扩增方法包括变温扩增和恒温扩增。
优选地,所述变温扩增主要包括经典的聚合酶链式反应(Polymerase ChainReaction,简称PCR)和连接酶链式反应(Ligase Chain Reaction,简称LCR)。
优选地,所述恒温扩增包括链置换扩增(Strand displacement amplification,简称SDA)、滚环扩增(Rolling Circle amplification,简称RCA)、环介导扩增(LoopMediated Amplification,简称LAMP)、依赖解旋酶恒温扩增(Helicase- dependentIsothermal DNA Amplification,简称HDA)、依赖核酸序列的扩增(Nucleic acidsequence based amplification,简称NASBA)、转录依赖的扩增系统(Transcription-based Amplification System,简称TAS)。
附图说明
图1为本发明实施案例一利用光老化体外诱导的成纤维细胞(Fibroblast,FB)的衰老表征结果图,图A是β-半乳糖苷酶染色示意图,图B是衰老染色阳性细胞的统计图。
图2为本发明实施案例一中利用RT-PCR检测光老化诱导的衰老FB细胞(S)和未光老化诱导的年轻FB细胞(Y)中miR-942-5p的相对表达量。
图3为本发明实施案例二中利用RT-PCR检测电转miR-942-5p后的外泌体孵育FB细胞48h后的miR-942-5p表达量。
图4为本发明实施案例三中外泌体miR-942-5p(ex-miR-942-5p)在FB细胞中的抗衰表征结果图,图A是β-半乳糖苷酶染色示意图,图B是衰老染色阳性细胞的统计图。
图5为本发明实施案例三中利用细胞活力CCK8试剂盒检测FB细胞细胞活率的结果图。
图6为本发明实施案例三中利用ELISA试剂盒检测FB细胞Collagen I分泌量的结果图。
图7为本发明实施案例三中利用ELISA试剂盒检测FB细胞MMP1分泌量的结果图。
图8为本发明实施案例三中利用活性氧试剂盒检测FB细胞中活性氧含量的结果图。
图9为本发明实施案例四中光老化皮肤小鼠模型皮肤组织的HE染色结果图。
图10为本发明实施案例四中光老化皮肤小鼠模型皮肤组织的masson染色结果图。
具体实施方式
下面结合具体实施例对本发明作更进一步的说明,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例一、光老化细胞模型构建以及miR-942-5p和成纤维细胞衰老的相关性验证
利用UVB照射的方式体外诱导成纤维细胞(Fibroblast,FB)衰老,并利用β-半乳糖苷酶染色检测细胞衰老情况,绝大多数正常细胞被认为仅有有限的分裂能力,在不能分裂后就进入衰老(senescence)状态。衰老细胞通常体积变大,并表达在pH6.0时有高酶活性的β-半乳糖苷酶(β-galactosidase)。以X-Gal为底物,在衰老特异性的β-半乳糖苷酶催化下会生成深蓝色产物。从而在光学显微镜下很容易观察到变成蓝色的表达β-半乳糖苷酶的细胞。
1、紫外线照射成纤维细胞构建光老化细胞模型
①培养FB细胞,按照1×106cell/10cm2进行传代;
②将FB细胞按照1×105cell/孔的细胞密度接种至6孔板中;
③细胞接种均匀,且融合程度是否达到70%~80%时进行诱导,去除原有培养液,PBS洗涤3次后,加入能覆盖细胞的新鲜PBS;
④打开塑料盖,使用UVB源紫外灯管(320 nm)以10 J/cm2剂量照射,随后丢弃PBS,补充新鲜培养基,细胞培养至新的处理或者分析。
2、β-半乳糖苷酶染色检测细胞衰老状态
①6孔板中培养的细胞,吸除细胞培养液,用PBS或HBSS洗涤1次,加入1毫升β-半乳糖苷酶染色固定液,室温固定15分钟。对于其它类型的培养板,固定液及后续溶液的用量参照此比例进行操作。
②吸除细胞固定液,用PBS或HBSS洗涤细胞3次,每次3分钟。
③吸除PBS或HBSS,每孔加入1毫升染色工作液。染色工作液的配制方法参考表1。
表1、染色工作液配方
④37℃孵育过夜,可以用parafilm或保鲜膜封住6孔板防止蒸发。注意:37℃孵育不能在二氧化碳培养箱中进行。
⑤普通光学显微镜下观察。利用Image-J进行阳性细胞分析统计。
结果如图1所示,通过图1A发现β-半乳糖苷酶染色在S组中大部分细胞均具有较深的蓝色染料着色,而在Y组仅发现少部分细胞有较浅的蓝色染料着色,图1B统计了β-半乳糖苷酶染色的阳性细胞,在Y组中阳性细胞比率为10.15%,在S组中阳性细胞比率为77.84%,以上结果说明FB衰老细胞模型已经被成功构建。
3、RNA提取和RT-PCR检测
利用RT-PCR检测衰老(S)和未衰老(Y)FB细胞中miR-942-5p的相对表达量,具体操作如下:
(1)RNA提取
①吸去培养液,加入1mL Trizol,室温放置5min后反复吹打细胞;
②将细胞裂解物移入一个1.5mL EP管,加入0.2倍体积的氯仿,振荡15s,室温放置3min后,12000rpm,离心10min;
③吸取上层水相至新的EP管中,然后加入等体积的异丙醇,颠倒混匀;12000rpm,离心5min,弃去收集管中的滤液;
④向离心管中加入1ml百分七十冰浴乙醇,12000rpm离心5min,弃掉收集管中的滤液;残留乙醇并在室温下晾干,加入50μL DEPC水,盖上盖子,室温放置3min溶解RNA沉淀;
⑤用超微量分光光度计测定抽提的RNA浓度和纯度,-80℃保存。
(2)逆转录
使用提取的RNA进行逆转录,逆转录引物序列如SEQ ID No.1所示,反应体系如表2;SEQ ID No .1:CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACATGGC。
表2、逆转录反应体系
(3)RT-PCR检测
逆转录的cDNA继续进行PCR扩增。其中,用于检测miR-942-5p产物的引物序列如SEQ ID No.2-3所示,所示PCR扩增体系如表3:
SEQ ID No.2:AGTGTCGTCAGTCTTCTCTGTTTTG
SEQ ID No.3:CTCAACTGGTGTCGTGGAGTC
表3、PCR反应体系
结果如图2所示,miR-942-5p的表达量在S组FB细胞中相对于Y组FB细胞中有显著的下调,说明miR-942-5p可能是一种潜在的成纤维细胞衰老标志物。
实施例二、外泌体miR-942-5p(ex-miR-942-5p)对成纤维细胞感染效果验证
外泌体miR-942-5p的制备:利用电转的方式将miR-942-5p导入外泌体(间充质干细胞来源)中制备含有miR-942-5p的外泌体ex-miR-942-5p。将1010个外泌体与miR-942-5p在4℃共孵育30min,随后利用电转仪器Neon转染系统(赛默飞世尔中国)进行电转,电转参数为电压1000v,脉宽10ms,脉冲6次,电转后在4℃共孵育30min,随后用于细胞实验。
miR-942-5p序列如SEQ ID No.4所示:UCUUCUCUGUUUUGGCCAUGUG
将空载外泌体(ex)和ex-miR-942-5p分别孵育FB细胞,48h后提取RNA,RT-PCR检测miR-942-5p的表达量,操作方法同实施例一中步骤3。
结果如图3所示,发现外泌体成功将miR-942-5p递送进入FB,并提高表达量554.1倍。
实施例三、在成纤维细胞上验证外泌体miR-942-5p(ex-miR-942-5p)的抗衰效果验证
用空载外泌体分别孵育未衰老FB细胞和衰老FB细胞作为对照组中的未衰老组(Y)和衰老组(S),用ex-miR-942-5p孵育衰老FB细胞作为药物处理组(ex-miR-942-5p),检测ex-miR-942-5p对于衰老FB细胞各项功能如细胞活率、I型胶原蛋白(Collagen I)分泌、基质金属蛋白酶1(MMP1)分泌和活性氧(ROS)含量的影响。
(1)β-半乳糖苷酶染色
染色方法同实施例一中步骤2相同。
结果如图4所示,通过图4A发现β-半乳糖苷酶染色在Y组仅发现少部分细胞有较浅的蓝色染料着色,在S组中大部分细胞均具有较深的蓝色染料着色,而相对于S组,ex-miR-942-5p组蓝色染料着色的细胞数量变少并且着色深度变浅,图4 B统计了β-半乳糖苷酶染色的阳性细胞,在Y组中阳性细胞比率为8.35%,在S组中阳性细胞比率为78.56%,在ex-miR-942-5p组中阳性细胞比率为39.14%,以上结果说明ex-miR-942-5p能显著抑制FB细胞衰老。
(2)CCK8检测细胞活率
通过细胞活力Cell Counting Kit-8检测试剂盒(上海碧云天生物科技有限公司),简称CCK8试剂盒,检测FB细胞的增殖能力,CCK8是一种基于WST-8而广泛应用于细胞增殖和细胞毒性的快速、高灵敏度检测的试剂盒。WST-8是一种类似于MTT的化合物,在电子耦合试剂存在的情况下,可以被线粒体内的一些脱氢酶还原生成橙黄色的formazan。细胞增殖越多越快,则颜色越深;细胞活性越低,则颜色越浅。对于同样的细胞,颜色的深浅和细胞数目呈线性关系。具体步骤如下:
①按实验分为Y、S、ex-miR-942-5p组,每孔加入100微升2000个FB细胞接种于96孔板中,每组6个复孔。
②培养48h后,每孔加入10微升CCK-8溶液。
③在细胞培养箱内继续孵育3小时。
④在450nm测定吸光度。
如图5所示,相较于Y组,S组的衰老FB细胞活率有明显的下降,而相较于S组,ex-miR-942-5p组的FB细胞活率明显上升。
(3)ELISA检测collagen I分泌量
通过人胶原蛋白1(collagen I)酶联免疫试剂盒(北京索莱宝科技有限公司)检测ex-miR-942-5p对FB细胞对于collagen I分泌量的影响,具体操作步骤如下:
①按实验分为Y、S、ex-miR-942-5p组,每孔加入2毫升/5万个FB细胞,每组3个复孔。
②培养72h后,每孔收取500μl培养基上清。
③利用collagen I的ELISA试剂盒进行检测,具体说明书见试剂盒。
④在450nm测定吸光度。
结果如图6所示,相较于Y组,S组的衰老FB细胞collagen I分泌量有明显的下降,而相较于S组,ex-miR-942-5p组的FB细胞的collagen I分泌量大幅度上升,并且其比Y组未衰老FB细胞的collagen I分泌量更高,证明ex-miR-942-5p可以增强衰老FB细胞的collagen I分泌量并超过未衰老FB细胞的collagen I分泌量。
(4)ELISA检测MMP1分泌量
通过人基质金属蛋白酶1(MMP-1)的ELISA检测试剂盒(北京百奥莱博科技有限公司)检测ex-miR-942-5p对FB细胞对于MMP1分泌量的影响,具体操作步骤如下:
①按实验分为Y、S、ex-miR-942-5p组,每孔加入2毫升/5万个FB细胞,每组3个复孔。
②培养72h后,每孔收取500μl培养基上清。
③利用MMP1的ELISA试剂盒进行检测,具体说明书见试剂盒。
④在450nm测定吸光度。
结果如图7所示,相较于Y组,S组的衰老FB细胞MMP1分泌量有明显的上升,而相较于S组,ex-miR-942-5p组的FB细胞的MMP1分泌量大幅度下降,并且通过统计发现其与Y组相比没有统计学差异,证明ex-miR-942-5p可以降低衰老FB细胞的MMP1分泌量并与未衰老FB细胞几乎相同。
(5)活性氧检测试剂盒检测ROS含量
通过活性氧(ROS)检测试剂盒(上海碧云天生物科技有限公司)检测ex-miR-942-5p对FB细胞中ROS含量的影响,具体操作步骤如下:
①按照1:1000用无血清培养液稀释DCFH-DA,使终浓度为10微摩尔/升。
②细胞收集后悬浮于稀释好的DCFH-DA中,细胞浓度为一百万至二千万/毫升,37℃细胞培养箱内孵育20分钟。每隔3-5分钟颠倒混匀一下,使探针和细胞充分接触。
③用无血清细胞培养液洗涤细胞三次,以充分去除未进入细胞内的DCFH-DA
④用流式细胞仪进行检测细胞的平均荧光强度,用flowjo软件进行数据分析。
如图8所示,相较于Y组,S组的衰老FB细胞中ROS含量有明显的上升,而相较于S组,ex-miR-942-5p组的FB细胞中ROS含量大幅度下降,并且通过统计发现其与Y组相比没有统计学差异,证明ex-miR-942-5p可以降低衰老FB细胞中ROS含量并与未衰老FB细胞几乎相同。
实施例四、在光老化皮肤小鼠模型上验证外泌体miR-942-5p(ex-miR-942-5p)的抗衰效果验证
(1)光老化小鼠模型构建及实验分组
选择雌性nude小鼠,年龄约6周,选用320nm的UVB紫外线灯照射8周,每天照射1h。
在正常小鼠和光老化模型小鼠左侧皮下注射空载外泌体作为对照组中的未衰老组(Y)和衰老组(S),在衰老组小鼠左侧皮下注射ex-miR-942-5p作为药物处理组(ex-miR-942-5p)。
(2)石蜡切片HE和Masson染色
通过石蜡切片HE和Masson染色检测ex-miR-942-5p对于光老化皮肤小鼠模型皮肤的影响。
如图9A和图9B所示,通过石蜡切片HE染色发现,相对于Y组小鼠皮肤,S组小鼠皮肤厚度有明显的降低,证明光老化皮肤小鼠模型的成功构建;而相对于S组小鼠皮肤,ex-miR-942-5p组的小鼠皮肤厚度有明显的上升,证明ex-miR-942-5p可以恢复由于光老化造成的小鼠皮肤厚度降低。
如图10所示,通过石蜡切片massson染色发现,相对于Y组小鼠皮肤,S组小鼠蓝色胶原层比例有明显的降低,证明光老化皮肤小鼠模型的皮下胶原受到损伤而减少;而相对于S组小鼠皮肤,ex-miR-942-5p组的小鼠皮肤蓝色胶原层比例有明显的上升,证明ex-miR-942-5p可以恢复由于光老化造成的小鼠皮下胶原损伤。
Claims (6)
1.以下任意一种活性成分在制备治疗光照射所导致的皮肤衰老的药物中的应用:
1)miR-942-5p;
2)递送载体,所述递送载体中含有miR-942-5p;
所述miR-942-5p具有如SEQ ID NO.4所示的序列。
2.如权利要求1所述应用,其特征在于,所述衰老是紫外线照射所导致的。
3.如权利要求1所述应用,其特征在于,所述递送载体包括外泌体、脂质纳米粒、聚合物纳米载体、无机纳米载体或蛋白载体。
4.如权利要求1所述应用,其特征在于,所述递送载体是外泌体。
5.如权利要求4所述应用,其特征在于,所述外泌体分离自间充质干细胞的细胞培养液。
6.检测miR-942-5p表达量的试剂在制备检测皮肤衰老程度的试剂盒中的应用,其特征在于,所述miR-942-5p具有如SEQ ID NO.4所示的序列。
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