CN116942635A - 一种外泌体药物递送系统及其制备方法和应用 - Google Patents
一种外泌体药物递送系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明提出了一种外泌体药物递送系统及其制备方法和应用,属于医药技术领域。包含装载有miR‑204和/或miR‑211的外泌体,具有制备预防和/或治疗骨关节炎药物中的应用,将miR‑204和/或miR‑211靶向递送至软骨细胞,通过下调Runx2表达及功能,影响软骨细胞退变标记物的表达,延缓OA疾病的进展。本发明发现Runx2mRNA的3’UTR中包含miR‑204/211结合位点,而荧光素酶活性试验则进一步证实miR对Runx2转录具有调控作用。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种外泌体药物递送系统及其制备方法和应用。
背景技术
OA的发病与进展受多种因素调控,目前认为,OA易感性与年龄增长具有明确的相关性,随着衰老导致的氧化应激损伤在细胞内不断积累,激活软骨细胞分解代谢,引发软骨细胞退变、钙化、甚至凋亡,最终导致关节滑膜增生、关节液炎性因子激增、软骨钙化、软骨下骨硬化等一系列OA特异性病理表现。在脊椎动物体内,FoxO(Forkhead-box class O)转录因子家族主要参与生物体发育及衰老的生理调控,其中FoxO1主要在骨骼及软骨中表达,能有效对抗软骨细胞内环境稳态失衡,抑制软骨细胞分解代谢。
研究人员对药物载体的选择逐渐转向外泌。外泌体(exosomes)是一种直径在30-150nm之间的茶托型结构的小囊泡,是细胞分泌的具有双层脂膜结构的微囊泡,包含RNA、蛋白质、microRNA、DNA片段等多种成分,由膜脂和膜蛋白组成的生物膜所包围,其结构和组成与细胞膜相似。全部真核细胞和一些原核细胞均可分泌,主要分布在血液、唾液、尿液、羊水和母乳等多种体液中。它是由细胞质膜内陷形成早期的核内体,核内体内陷包裹物质形成多泡体,而后多泡体在与质膜融合后得以释放。正是外泌体的结构(磷脂双分子层和囊泡状结构)和生理特性,使它们具有很好的生物相容性。并且外泌体可以自由的穿过血脑屏障(BBB),低免疫原性和毒性使其可以很容易的被任何器官和组织中的细胞摄取,且不会引起机体的免疫反应,具有无毒、生物相容性好、组织和肿瘤靶向性和循环半衰期长等优点,是新型药物递送系统的理性候选者。
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:(1)使用自源外泌体时,外泌体引起的有害免疫反应极低;(2)外泌体在人血液中的稳定性好;(3)转运效率高;(4)外泌体运载药物具有一定的靶向性;(5)外泌体直径在40-100nm之间,所以能够很好地利用增强渗透滞留(EPR)效应,有选择性地渗入到肿瘤或者炎症组织部位。
外泌体(Exosome)是由活细胞分泌的直径约为30-150nm的小囊泡,具有典型的脂质双分子层结构,广泛存在于细胞培养上清液、血清、血浆以及其它生物体液中。外泌体携带有多种蛋白质、脂类、RNA等重要信息,不仅在细胞与细胞间的物质和信息传递中起重要作用,更有望成为药物靶向递送的高效运载工具。RNA干扰是一种能够关闭特定靶基因表达的基因沉默技术。微小RNA(micor-RNA,miRNA)作为内源性单链非编码RNA,具有效率高、靶向强且安全性较高等特点,正逐步成为基因治疗的潜在有效药物。然而,受到其自身稳定性差,易被降解等特点的限制,miRNA在临床转化过程非常曲折。外泌体作为内源性RNA转运工具,可以作为miRNA治疗的潜在载体,通过外泌体靶向性,从而实现高效安全的miRNA递送的目的。研究显示,间充质干细胞(mesenchymal stem cells,MSCs)对组织损伤修复的病理过程,很大程度依赖于其对细胞微环境和细胞功能的调节,而外泌体与该过程中的信息和物质传递息息相关。MSCs源性外泌体(MSCs derived exsome,MSC-Exos)作为旁分泌的关键作用组件,在抑制软骨细胞退变、促进软骨细胞合成代谢、维持软骨稳态方面,其有效性已被不断证实。当外泌体作为高效运载工具的条件下,其负载的关键基因则成为有效延缓骨关节炎的调控核心。
外泌体作为有效药物递送载体的关键是外泌体对治疗药物的“装载”。如今的装载策略大致分两类:被动和主动装载。无论是被动转载和主动转载策略,外泌体的载药量都受到候选药物亲水性、疏水性、药物分子量的影响,同时外泌体膜的完整性也是检验载药策略好坏的重要因素。被动装载的原理是利用治疗药物的由高浓度向低浓度的扩散,使药物被动穿过磷脂双分子层。此方法对候选药物要求很高,即容易穿过磷脂双分子膜结构又不易降解。主动装载是相对于被动装载提出的概念,是一种以增加载药量和扩大候选药物范围为目的的策略总称。主要包括:超声、挤压、电转和药物交联等技术,且电转策略是运用最为广泛。电转则是利用电场使外泌体膜上产生诸多暂时性的小孔,药物同样是以扩散的方式进入到外泌体中,再利用磷脂膜的流动性使小孔恢复完成药物装载。现阶段,研发一种能够高效递送药物,起到高效预防和治疗骨关节炎的作用的药物靶向递送系统将具有广阔的应用前景。
发明内容
本发明的目的在于提出一种外泌体药物递送系统及其制备方法和应用,从Col2CreER-FoxO1f/f小鼠模型入手,并应用细胞转染技术,构建miR-204/211高表达原代间充质干细胞,提取MSCs-Exos后进行体内注射。确了FoxO1-Runx2信号通路是导致OA疾病进展的关键调控靶点,并制备了外泌体靶向递送系统,能够高效的起到预防和治疗骨关节炎的效果。
本发明的技术方案是这样实现的:
本发明提供一种外泌体药物递送系统,包含装载有miR-204和/或miR-211的人骨髓间充质干细胞MSCs细胞外泌体。
本发明进一步保护上述外泌体药物递送系统在制备预防和/或治疗骨关节炎药物中的应用。
作为本发明的进一步改进,所述装载有miR-204和/或miR-211的外泌体将miR-204和/或miR-211靶向递送至软骨细胞,通过下调Runx2表达及功能,影响软骨细胞退变标记物的表达,延缓OA疾病的进展。
本发明进一步保护如上述外泌体药物递送系统的制备方法,包括以下步骤:
S1.依据miRBase数据库中mmu-miR-204及mmu-miR-211序列,合成寡链核苷酸片段及反义序列,为单链核苷酸,合成双链核苷酸序列,克隆至空白质粒中构建表达载体;
S2.将步骤S1中表达载体转染人骨髓间充质干细胞MSCs细胞,过表达,得到过表达的原代MSCs细胞;
S3.从步骤S2中的原代MSCs细胞中提取MSCs-外泌体,得到外泌体药物递送系统。
作为本发明的进一步改进,所述外泌体的提取方法为超高速离心法结合超滤管法。
优选地,所述超高速离心法结合超滤管法方法如下:细胞上清液3-5℃、500-1000r/min离心10-15min除去细胞,然后3-5℃,3000-4000r/min离心15-20min除去细胞碎片等,超滤管浓缩上清液,3-5℃,12000-15000r/min离心20-30min除去大囊泡和蛋白,上清液用0.22μm滤膜过滤,最后4℃,30000-35000r/min离心60-70min,pH=6.7-7的PBS重悬沉淀,3-5℃,30000-35000r/min再次离心60-70min,得到的沉淀即外泌体,用200μL pH=6.7-7的PBS重悬,按照BCA试剂盒步骤进行蛋白定量,-80℃冰箱冻存存。
本发明进一步保护一种载连翘酯苷A的外泌体药物递送系统,包含装载药物的人骨髓间充质干细胞MSCs细胞外泌体,所述药物包括:
1)miR-204和/或miR-211;
2)连翘酯苷A。
本发明进一步保护上述载连翘酯苷A的外泌体药物递送系统在制备预防和/或治疗骨关节炎药物中的应用。
本发明进一步保护上述载连翘酯苷A的外泌体药物递送系统的制备方法,包括以下步骤:
S1.合成miR-204和/或miR-211前体的双链寡聚核苷酸序列,克隆至空白质粒中构建表达载体;
S2.将步骤S1中表达载体转染人骨髓间充质干细胞MSCs细胞,过表达,得到过表达的原代MSCs细胞;
S3.从步骤S2中的原代MSCs细胞中提取MSCs-外泌体;
S4.将步骤S3制得的MSCs-外泌体和连翘酯苷A混合,采用超声孵育的方法,冰浴超声,温育,离心,重悬,得到载连翘酯苷A的外泌体药物递送系统。
作为本发明的进一步改进,所述外泌体的提取方法为超高速离心法结合超滤管法。
优选地,所述超高速离心法结合超滤管法方法如下:细胞上清液3-5℃、500-1000r/min离心10-15min除去细胞,然后3-5℃,3000-4000r/min离心15-20min除去细胞碎片等,超滤管浓缩上清液,3-5℃,12000-15000r/min离心20-30min除去大囊泡和蛋白,上清液用0.22μm滤膜过滤,最后4℃,30000-35000r/min离心60-70min,pH=6.7-7的PBS重悬沉淀,3-5℃,30000-35000r/min再次离心60-70min,得到的沉淀即外泌体,用200μL pH=6.7-7的PBS重悬,按照BCA试剂盒步骤进行蛋白定量,-80℃冰箱冻存。
作为本发明的进一步改进,所述MSCs-外泌体和连翘酯苷A的质量比为1-2:1;所述超声条件为1000-1200W,超声3-5s,关2-3s,重复10次,间隔2-3min,重复一遍;所述温育条件为36-38℃,孵育1-2h;所述离心条件为30000-35000r/min离心60-70min;所述重悬用的溶液为pH为6.7-7的PBS缓冲液。
优选地,所述超声孵育的方法如下:将MSCs-外泌体和连翘酯苷A按照质量比为1-2:1混合,连翘酯苷A的浓度为300-500mg/L,在冰水浴下超声,超声条件:1000-1200W,超声3-5s,关2-3s,重复10次,间隔2-3min,重复一遍。取出,于36-38℃恒温培养箱中恢复1-2h,然后在3-5℃,30000-35000r/min离心60-70min,去除上清及游离的连翘酯苷A,用pH为6.7-7的PBS缓冲液重悬,再次离心60-70min,适量pH为6.7-7的PBS溶液重悬沉淀,即得到载连翘酯苷A的外泌体药物递送系统。
本发明具有如下有益效果:
miR-204与miR-211是两段序列高度相似,含21bp的单链RNA,分别位于小鼠第15号(GRCh38;GCA_000001405.15)和第19号(GRCm38;GCA_000001635.2)染色体。miR-204/211通过调控多种信号通路,广泛参与细胞病理生理过程。在软骨细胞中,miR-204和miR-211通过抑制FoxC1,有效抑制软骨细胞中炎症因子的表达,此外,miR-204和miR-211的表达也受到软骨细胞机械受体的调控,在GATA4转录因子的调控下,miR-204和miR-211调控蛋白多糖影响关节软骨的稳态。
本发明通过质粒转染构建miR-204/211过表达MSCs细胞系,提取MSCs-Exos将有效抑制Runx2转录因子表达,并抑制软骨细胞分解代谢,延缓OA进展(图1)。
Runx2是软骨细胞退变的重要调控因子,miR-204/211对其具有靶向调控作用。本发明中,FoxO1-/-敲除小鼠出现显著OA表型,且Runx2、Col10α1、MMP9、MMP13、Adamts5等基因表达则出现显著升高(图2)。经TargetScan预测,本发明发现Runx2 mRNA的3’UTR中包含miR-204/211结合位点,而荧光素酶活性试验则进一步证实miR对Runx2转录具有调控作用(图4)。
1、本发明明确了FoxO1-Runx2信号通路是导致OA疾病进展的关键调控靶点。应用①DMM诱导小鼠OA模型和②FoxO1-/-敲除小鼠模型,明确FoxO1-/-可以导致小鼠出现显著的OA疾病进展,而Runx2是该调控机制中的关键靶点,有助于进一步明确Runx2在OA疾病治疗中的潜在应用价值,为软骨细胞退变在OA进展中的研究提供理论基础。
2、本发明在动物学和细胞学水平,明确miR204/211的MSCs-Exos能够靶向递送,直达软骨细胞,显著延缓FoxO-/-敲除引发的OA疾病进展,为OA疾病的基因治疗,提供有力的生物医学证据。
3、本发明深入分析Runx2 3’UTR中miR204/211结合位点(RNA-binding site)的活性,分析其结合位点RNA序列。通过构建包含Runx2 3’UTR片段的luciferase报告基因,应用荧光素酶报告系统,通过比较miR204/211过表达与报告基因作用检测荧光值的变化,判断miR204/211对靶基因的作用。再通过对3’UTR区进行定点突变的方法进一步确定miR204/211与Runx2 3’UTR的作用位点。从而解释了本发明外泌体药物递送系统的作用机理。
发明人进行进一步的研究,发现制得的装载有miR-204和/或miR-211的外泌体进一步采用超声孵育的方法制备载连翘酯苷A的外泌体药物递送系统,能明显解决口服吸收较差、生物利用度较低,影响该药的使用等问题,且miR-204和/或miR-211和连翘酯苷A两者的协同作用下,通过抑制软骨细胞分解代谢,进一步提高改善和治疗骨关节炎的效果,具有协同增效的作用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为本发明的实验示意图;
图2为FoxO1、Runx2在OA小鼠膝关节软骨中的表达图;
图3为FoxO1-/-和FoxO1f/f的对比图,其中,A图为免疫组化对比图,B为各种软骨细胞PCR检测标记物表达图;
图4为miR-204和miR-211的相对荧光强度对比图;
图5为miR-204/211的MSCs-Exos的表征结构;其中,A为免疫荧光图;B为透射电镜扫描图;C为共聚焦显微镜检测图;D为Western blot检测图;
图6为miR-204/211的MSCs-Exos关节腔注射12周后的效果图,其中,A为番红O/H&E染色图,B中从左到右,依次是小鼠软骨ORASI评分,软骨面积计算,滑膜评分,以及两个疼痛敏感试验。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种外泌体药物递送系统的制备方法,包括以下步骤:
S1.依据miRBase数据库中mmu-miR-204及mmu-miR-211序列,合成寡链核苷酸片段及反义序列,以此单链为前体,并在序列前端添加BamHI酶切位点,后端添加polyT转录终止序列及SalI酶切位点。合成miR-204和miR-211的双链核苷酸序列,克隆至空白质粒中构建表达载体。所述空白质粒为pGenesil-1质粒,并购于addgene。miR-204和miR-211的质量比为3:1;重组质粒经扩增、筛选、鉴定与测序后获得含mmu-miR-204及mmu-miR-211的重组过表达质粒。
miR-204的核苷酸序列如SEQ ID NO.1所示;miR-211的核苷酸序列如SEQ ID NO.2所示。
S2.采用脂质体转染法,将步骤S1中表达载体转染人骨髓间充质干细胞MSCs细胞,通过选择性培养与筛选,得到过表达的稳定转染原代MSCs细胞;该步骤具体操作如下:
(1)90%接触抑制的小时MSCs细胞经胰酶消化后,以105/孔浓度接种于6孔板中。待80%生长后更换无血清培养基,培养箱静置30分钟。
(2)取pGenesil-1-miR-204及pGenesil-1-miR-211共5.0μg过表达质粒与10μLLipofectamineTM 2000和250μL基本培养基混匀,室温静置20分钟后加入培养皿中。
(3)6小时后更换为含血清培养基,并于24h后在荧光显微镜下观察转染效率。选取荧光细胞数量≥50%培养孔进行扩增和选择性培养。在10-14天后对形成单克隆的细胞进行扩增培养,并获得稳定转染的细胞株。
S3.采用超高速离心法结合超滤管法从步骤S2中的原代MSCs细胞中提取MSCs-外泌体,得到外泌体药物递送系统,记为miR204/211的MSCs-Exos。以空白质粒转染MSCs位对照。在透射电镜下观察外泌体形态,应用WB检测外泌体特异性蛋白表达,应用PCR检测目的基因含量。如图5,miR204/211的MSCs-Exos具有良好的软骨细胞摄取(A),透射电镜提示外泌体结构(B),细胞对外泌体摄取(C),外泌体特异性蛋白表达鉴定(D)。
所述超高速离心法结合超滤管法方法如下:细胞上清液4℃、700r/min离心15min除去细胞,然后4℃,3500r/min离心17min除去细胞碎片等,超滤管浓缩上清液,4℃,13000r/min离心25min除去大囊泡和蛋白,上清液用0.22μm滤膜过滤,最后4℃,32000r/min离心65min,pH=6.9的PBS重悬沉淀,4℃,32000r/min再次离心65min,得到的沉淀即外泌体,用200μL pH=6.9的PBS重悬,按照BCA试剂盒步骤进行蛋白定量,通过计算,最终获得浓度为80-120μg/ml,外泌体溶液,于-80℃冰箱冻存。
1)DMM诱导小鼠OA模型的构建及组织学分析
以12周龄雄性C57/BL6小鼠为实验对象,经戊巴比妥(3%,1ml/kg体重)腹腔麻醉后打开右侧后肢膝关节腔,离断右后肢内侧半月板韧带(Meniscal-ligamentous,ML),形成右后肢内侧半月板不稳定(Destabilization of Medial Meniscus,DMM)后缝合,作为实验组(DMM组);另选同窝小鼠右后肢做假手术组(Sham组),以DMM组和Sham组小鼠左后肢为空白对照组。造模后12周后行Von Fery疼痛敏感试验,并在LABORAS系统中记录小鼠24小时内平均行走速度、后足站立次数及累计时长。收集双侧膝关节样本,石蜡包埋切片后通过ABH/OG、IHC及IF染色分析Runx2蛋白、FoxO1转录因子、软骨细胞分解代谢标记物表达。
结果如图2,由图可知,DMM诱导小鼠膝关节呈现OA表型,软骨细胞中FoxO1转录因子表达显著低于假手术组,而Runx2蛋白表达则显著高于假手术组。
2)FoxO1-/-敲除小鼠模型构建及组织学分析
通过FoxO1f/f小鼠(购自JaxLab,NO.024756)与Col2-CreERT小鼠(购自JaxLab,NO.006774)杂交及其子代自交后,获得Col2-CreER;FoxO1f/f纯合子小鼠。该小鼠模型,经腹腔注射他莫昔芬(Tamoxifen,TMX)后,会将小鼠体内表达Col2α1的阳性细胞(主要为分布于关节软骨中的软骨细胞)中FoxO1基因敲除,从而实现研究小鼠膝关节软骨中FoxO1基因功能的目的。对12周龄Col2-CreER;FoxO1f/f小鼠进行诱导(方法:腹腔注射TMX 1.5mg/10g体重,每日1次,共5次),获得FoxO1-/-敲除纯合子小鼠,并于造模12周后行动物行为学分析,探讨FoxO1对OA疾病进展的影响,收集双侧膝关节样本,行石蜡切片及组织学分析,重点研究FoxO1敲除对Runx2和软骨细胞分解代谢的影响。
结果如图3,由图可知,FoxO1-/-敲除小鼠膝关节出现明显OA表型,且Runx2蛋白表达显著升高。*与FoxO1f/f组相比P<0.05。图3A中,在FoxO1f/f小鼠关节软骨中,FoxO1阳性表达细胞含量较高(深灰箭头),Runx2阳性表达细胞含量较少(空白箭头),而FoxO1-/-敲除小鼠关节软骨中,FoxO1阳性表达细胞含量较少(空白箭头),Runx2阳性表达细胞含量较高(浅灰箭头);图3B中,FoxO1-/-降低软骨细胞标记物表达,促进成骨相关标记物表达升高。
3)FoxO-/-敲除小鼠膝关节注射miR204/211的MSCs-Exos
向①FoxO1-/-+生理盐水、②FoxO1-/-+负载miR-204/211的MSCs-Exos、③FoxO1-/-+空白质粒MSCs-Exos三种小鼠模型膝关节中注射对应成分的外泌体,浓度为20μg/ml,单次注射量为10μl,每周1次。于12周后行Von Fery疼痛敏感试验。收集双侧膝关节样本,石蜡包埋切片后通过ABH/OG、IHC及IF染色分析Runx2蛋白、FoxO1转录因子、软骨细胞分解代谢标记物表达。具体操作步骤如下:
a.Von Fery疼痛敏感试验:小鼠适应环境半小时待不再四处张望、探究,较安静后,用VonFrey测试探针缓慢地轻柔地刺激小鼠待测肢体足底中部,使探针弯曲,持续2秒钟观察小鼠的缩足反应,若由于刺激而出现快速的缩足反应,则标记为阳性并记录反应阈值,反之则增大刺激阈值。实验结果如图6B所示,负载miR-204/211的MSCs-Exos小鼠,其疼痛敏感程度显著好于非治疗组小鼠。
b.ABH/OG染色:1.烤片,60℃,1hr-过夜(肉眼观察组织表面的蜡融化彻底即可)。2.石蜡水化:二甲苯(xylene)I 5mins→二甲苯II 5mins→二甲苯III 5mins→无水乙醇(ethanol)I 3mins→无水乙醇II 3mins→95%乙醇I 3mins→95%乙醇II 3mins→70%乙醇3mins→ddH2O I 3mins→ddH2O II 3mins。3.放入Acid-alcohol90s,纸巾擦干不冲洗。4.放入Alcian Blue Hematoxylin 40-60mins。5.蒸馏水冲洗至无染液流出,约3次。6.分色,放入Acid-alcohol 3s。7.蒸馏水冲洗3遍。8.0.5%氨水15s。9.蒸馏水冲洗2遍。10.95%乙醇1分钟,不冲洗。11.放入Eosin/Orange G 90s-120s。12.脱水:依次将载玻片放入95%乙醇1min→95%乙醇1min→95%乙醇1min→无水乙醇Ⅰ1min→无水乙醇Ⅱ1min→二甲苯Ⅰ1min→二甲苯Ⅱ1min→二甲苯Ⅲ1min。封片,显微镜下观察拍照。实验结果如图2所示,DMM诱导小鼠膝关节呈现OA表型;图6A所示,小鼠在负载miR-204/211的MSCs-Exos治疗后出现软骨保护作用。
c.IF染色:1.烤片→60℃→1小时—过夜(肉眼见组织表面石蜡融化)2.石蜡水化:二甲苯(xylene)I 5mins→二甲苯II 5mins→二甲苯III 5mins→无水乙醇(ethanol)I3mins→无水乙醇II 3mins→95%乙醇I 3mins→95%乙醇II 3mins→70%乙醇3mins→ddH2O I 3mins→ddH2O II 3mins。3.放入抗原修复液中。4.高压锅预热95℃,煮时长根据片子情况调整(3mins-15mins)。5.TBST清洗2-3次,3-5mins/次。6.3%双氧水(甲醇methanal配制,或ddH2O配制)浸泡15mins-25mins。7.TBST清洗2-3次。8.0.5%Tritom(TBS配制)破膜,10mins-15mins。9.10%goat血清(1%BSA配制)封闭30mins(ColX 60mins)。10.TBST清洗2-3次。11.1%BSA配制一抗,每片用量150μL,孵育4℃过夜、室温2.5hours、37℃1hour。12.TBST清洗3次。13.二抗(1:100-1:500)1%BSA稀释,每片用量150μL,孵育30mins-1hour。14.TBST清洗2次。15.配ABC(提前30mins配制以反应),TBST 1ml滴入A、B各1滴,室温染色30mins。16.TBS清洗2次。17.配DAB(2ml buffer+2滴DAB,现用现配),每片用量150μL,显微镜下观察显色。18.复染,苏木精(hematoxylin)染液30秒,流水冲洗至无蓝色,PBS或氨水反蓝20秒,蒸馏水冲洗。19.封片H2O→70%乙醇3mins→95%乙醇I 3mins→95%乙醇II3mins→无水乙醇I 3mins→无水乙醇II 3mins→二甲苯I 3mins→二甲苯II 3mins→二甲苯III 3mins→封片,显微镜下观察拍照。实验结果如图2所示,软骨细胞中FoxO1转录因子表达显著低于Sham组,而Runx2蛋白表达则显著高于Sham组;图3所示,在FoxO1f/f小鼠关节软骨中,FoxO1阳性表达细胞含量较高,Runx2阳性表达细胞含量较少,而FoxO1-/-敲除小鼠关节软骨中,FoxO1阳性表达细胞含量较少,Runx2阳性表达细胞含量较高。
4)荧光素酶检测系统构建
Targetscan生物信息学软件预测miR-204-5p与miR-211-5p与Runx2存在结合位点,如表1所示。利用PCR扩增Runx2基因3’UTR片段,并将其克隆至pmiR-RB-ReportTM的双荧光素酶报告基因载体中,同时构建Runx2基因3’UTR突变载体。应用脂质体转染法,将miR-204-5p,miR-211-5p及阴性对照分别与野生型Runx2-wt 3'-UTR及突变型Runx2-mut 3'-UTR双荧光素酶报告质粒共转染至293T细胞中,通过比较miR204/211过表达与报告基因作用检测荧光值的变化,判断miR-204/211对靶基因的作用。研究结果如图4所示,miR-204/211可以与Runx2基因3’UTR中AAAGGGAC序列结合,从而抑制荧光素酶活性,而对突变型荧光强度无影响。该结果提示miR-204/211对Runx2活性产生抑制作用。
结果如图6,miR-204/211的MSCs-Exos关节腔注射12周后,小鼠OA疾病进展得到缓解。
实施例2
与实施例1相比,步骤S1中仅合成miR-204前体的双链寡聚核苷酸序列,其他条件均不改变。
实施例3
与实施例1相比,步骤S1中仅合成miR-211前体的双链寡聚核苷酸序列,其他条件均不改变。
测试例1miR-204/211过表达MSCs原代细胞构建及功能鉴定
miR-204、miR-211过表达质粒及空白质粒(购自addgene)经脂质体转染后,构建①miR-204过表达MSCs原代细胞(实施例2)、②miR-211过表达MSCs(实施例3)、③miR-204/211双重过表达MSCs(实施例1)、④空白质粒MSCs。通过细胞成骨诱导培养及成软骨诱导培养,研究miR-204/211对成骨与软骨标记物的调控作用,探索Runx2在miR-204/211调控作用下的表达变化。
TargetScan预测miR-204/211中包含Runx2 3’UTR结合位点,对比wt序列和突变序列(GGGTTTGG)荧光素强度,miR-204/211对Runx2转录后调控产生抑制作用(表1,图4)。
表1
实施例4
一种外泌体药物递送系统的制备方法,包括以下步骤:
S1.合成miR-204和miR-211前体的双链寡聚核苷酸序列,克隆至空白质粒中构建表达载体;所述空白质粒购于addgene。
S2.将步骤S1中表达载体转染人骨髓间充质干细胞MSCs细胞,过表达,得到过表达的原代MSCs细胞;
S3.采用超高速离心法结合超滤管法从步骤S2中的原代MSCs细胞中提取MSCs-外泌体,得到外泌体药物递送系统,记为miR204/211的MSCs-Exos。以空白质粒转染MSCs位对照。在透射电镜下观察外泌体形态,应用WB检测外泌体特异性蛋白表达,应用PCR检测目的基因含量。如图5,miR204/211的MSCs-Exos具有良好的软骨细胞摄取(A),透射电镜提示外泌体结构(B),细胞对外泌体摄取(C),外泌体特异性蛋白表达鉴定(D)。
所述超高速离心法结合超滤管法方法如下:细胞上清液4℃、700r/min离心15min除去细胞,然后4℃,3500r/min离心17min除去细胞碎片等,超滤管浓缩上清液,4℃,13000r/min离心25min除去大囊泡和蛋白,上清液用0.22μm滤膜过滤,最后4℃,32000r/min离心65min,pH=6.9的PBS重悬沉淀,4℃,32000r/min再次离心65min,得到的沉淀即MSCs-外泌体,用200μL pH=6.9的PBS重悬,按照BCA试剂盒步骤进行蛋白定量,-80℃冰箱冻存。
S4.将步骤S制得的MSCs-外泌体和连翘酯苷A混合,采用超声孵育的方法,冰浴超声,温育,离心,重悬,得到载连翘酯苷A的外泌体药物递送系统。
所述超声孵育的方法如下:将MSCs-外泌体和连翘酯苷A按照质量比为1:1混合,连翘酯苷A的浓度为400mg/L,在冰水浴下超声,超声条件:1100W,超声3s,关3s,重复10次,间隔3min,重复一遍。取出,于37℃恒温培养箱中恢复2h,然后在4℃,32000r/min离心65min,去除上清及游离的连翘酯苷A,用pH为6.9的PBS缓冲液重悬,再次离心65min,用pH为6.9的PBS溶液重悬沉淀,使得固含量为40%,即得到载连翘酯苷A的外泌体药物递送系统。
实施例5
与实施例4相比,MSCs-外泌体和连翘酯苷A的质量比为2:1,其他条件均不改变。
实施例6
与实施例4相比,MSCs-外泌体和连翘酯苷A的质量比为1.5:1,其他条件均不改变。
测试例2
膝骨关节炎患者70名,分为7组。实施例1-6组的患者在超声引导下缓慢注射1mL实施例1-3制得的外泌体药物递送系统或者实施例4-6制得的载连翘酯苷A的外泌体药物递送系统到患骨关节炎的关节腔,每周1次,连续注射1个月;空白组患者在超声引导下,将等体积的透明质酸缓慢注射到患骨关节炎的关节。
纳入标准:①符合膝骨关节炎诊断标准;②年龄45-75岁;③6个月内膝骨关节炎的Kellgren-Lawrence(K-L)放射学分级为Ⅰ-Ⅲ级;④自愿参加本次试验并签署知情同意书。
排除标准:①备孕、妊娠期或哺乳期妇女;②其他疾病引起的膝部疼痛(如自身免疫疾病、腰骶椎疾病等);③膝关节结构已严重变形或畸形者;④近3个月内接受过针灸治疗者;⑤合并严重内科疾病或精神类疾病者;⑥近3个月内参加其他临床研究者。
1、6个月后疼痛缓解评分和症状缓解平均评分见表2(其中,评分越高疼痛或症状缓解效果越佳,受试者根据实际感觉进行评分)。
表2
结果显示,实施例4-6中载连翘酯苷A的外泌体药物递送系统的效果明显优于实施例1-3中外泌体药物递送系统的效果,且实施例6的效果最佳,可见MSCs-外泌体和连翘酯苷A的质量比为1.5:1时效果最佳。
2.超声测定软骨厚度(mm,n=10)
采用Philips iU22和日立HI VISION Avius彩色多普勒超声诊断仪进行检测,结果见表3。
表3
结果显示,实施例4-6中载连翘酯苷A的外泌体药物递送系统的软骨恢复效果明显优于实施例1-3中外泌体药物递送系统的效果,且实施例6的效果最佳,可见MSCs-外泌体和连翘酯苷A的质量比为1.5:1时效果最佳。
由此可知,装载有miR-204和/或miR-211的外泌体进一步采用超声孵育的方法制备载连翘酯苷A的外泌体药物递送系统,且miR-204和/或miR-211和连翘酯苷A两者的协同作用下,通过抑制软骨细胞分解代谢,促进软骨恢复,进一步提高改善和治疗骨关节炎的效果,具有协同增效的作用。
FoxO1作为软骨细胞退变的重要调控因子,其表达量的降低与软骨细胞退变具有显著的相关性。在FoxO1低表达小鼠模型中,4周龄小鼠即出现关节软骨的异常增厚,其下游Runx2及软骨细胞肥大化标记物显著增高,至5月龄时(相当于人类约40岁)关节软骨出现全层退变。该模型有效证明FoxO1通过调控Runx2广泛参与软骨细胞代谢、增殖、分化及退变的生理调控。此外,手术诱导和过量运动诱导OA模型中也都发现,随着膝关节软骨退变的加剧,FoxO1表达也出现显著的降低。此外,FoxO1低表达促进Runx2激活,加速软骨下骨形成及骨重塑,推动软骨下骨硬化的发生。目前有研究显示,FoxO1主要通过下调Runx2表达,抑制软骨细胞肥大化,从而延缓软骨细胞退变。
Runx2作为参与调控软骨细胞分化,维持细胞功能稳态的关键转录因子,与OA疾病的进展具有显著的相关性。大量研究显示,Runx2在OA患者的软骨细胞中显著高表达,而敲除Runx2的小鼠模型中,衰老引发的关节软骨退变及OA进展得到了显著的缓解。在OA进展过程中,软骨细胞发生“软骨细胞分解代谢激活→软骨细胞肥大化→软骨基质退变→软骨细胞凋亡”等一系列病理变化。在OA早期,AMPK蛋白磷酸化水平逐渐降低,进而导致细胞代谢稳态失衡,而关节软骨细胞中FoxO1水平会反馈性增高,有效抑制Runx2的表达激活,从而有效抑制MMP9、MMP13及Adamts5、Adamts7等细胞外基质蛋白酶的表达,延缓Col2α1、Acan等多重螺旋胶原的代谢分解,抑制软骨细胞退变。然而,随着OA疾病的进展,氧化应激损伤不断积累,软骨细胞中Runx2表达不断增高,进而激活Col10α1、MMP9及MMP13等软骨细胞肥大化标记物和基质蛋白酶升高,进一步破坏软骨基质及软骨细胞微环境,最终引发caspase3,caspase9激活,启动细胞凋亡程序。此外,Runx2进一步抑制软骨细胞弹性纤维表达,软布机制对机械应力耐受力下降也会加剧软骨细胞中MMP9及Adamts5的表达激活,进一步加速软骨细胞及软骨外基质退变。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种外泌体药物递送系统,其特征在于,包含装载有miR-204和/或miR-211的人骨髓间充质干细胞MSCs细胞外泌体。
2.一种如权利要求1所述外泌体药物递送系统在制备预防和/或治疗骨关节炎药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述装载有miR-204和/或miR-211的外泌体将miR-204和/或miR-211靶向递送至软骨细胞,通过下调Runx2表达及功能,影响软骨细胞退变标记物的表达,延缓OA疾病的进展。
4.一种如权利要求1所述外泌体药物递送系统的制备方法,其特征在于,包括以下步骤:
S1.依据miRBase数据库中mmu-miR-204及mmu-miR-211序列,合成寡链核苷酸片段及反义序列,为单链核苷酸,合成双链核苷酸序列,克隆至空白质粒中构建表达载体;
S2.将步骤S1中表达载体转染人骨髓间充质干细胞MSCs细胞,过表达,得到过表达的原代MSCs细胞;
S3.从步骤S2中的原代MSCs细胞中提取MSCs-外泌体,得到外泌体药物递送系统。
5.根据权利要求4所述的制备方法,其特征在于,所述外泌体的提取方法为超高速离心法结合超滤管法。
6.一种载连翘酯苷A的外泌体药物递送系统,其特征在于,包含装载药物的人骨髓间充质干细胞MSCs细胞外泌体,所述药物包括:
1)miR-204和/或miR-211;
2)连翘酯苷A。
7.一种如权利要求6所述载连翘酯苷A的外泌体药物递送系统在制备预防和/或治疗骨关节炎药物中的应用。
8.一种如权利要求6所述载连翘酯苷A的外泌体药物递送系统的制备方法,其特征在于,包括以下步骤:
S1.合成miR-204和/或miR-211前体的双链寡聚核苷酸序列,克隆至空白质粒中构建表达载体;
S2.将步骤S1中表达载体转染人骨髓间充质干细胞MSCs细胞,过表达,得到过表达的原代MSCs细胞;
S3.从步骤S2中的原代MSCs细胞中提取MSCs-外泌体;
S4.将步骤S3制得的MSCs-外泌体和连翘酯苷A混合,冰浴超声,温育,离心,重悬,得到载连翘酯苷A的外泌体药物递送系统。
9.根据权利要求8所述的制备方法,其特征在于,所述外泌体的提取方法为超高速离心法结合超滤管法。
10.根据权利要求8所述的制备方法,其特征在于,所述MSCs-外泌体和连翘酯苷A的质量比为1-2:1;所述超声条件为1000-1200W,超声3-5s,关2-3s,重复10次,间隔2-3min,重复一遍;所述温育条件为36-38℃,孵育1-2h;所述离心条件为30000-35000r/min离心60-70min;所述重悬用的溶液为pH为6.7-7的PBS缓冲液。
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