CN114712380A - microRNA在制备治疗或预防骨质疏松的药物中的应用 - Google Patents
microRNA在制备治疗或预防骨质疏松的药物中的应用 Download PDFInfo
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- CN114712380A CN114712380A CN202210551636.XA CN202210551636A CN114712380A CN 114712380 A CN114712380 A CN 114712380A CN 202210551636 A CN202210551636 A CN 202210551636A CN 114712380 A CN114712380 A CN 114712380A
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Abstract
本发明公开了一种miR‑335‑3p在制备治疗或预防骨质疏松的药物中的应用，并提供一种miRNA‑335‑3p抑制剂优化的工程化人脂肪间充质干细胞外泌体。miR‑335‑3p可通过负向调控机制，促进骨质疏松小鼠来源骨髓间充质干细胞的成骨向分化，通过高效和简便的方式将miRNA‑335‑3p抑制剂加载到外泌体中，能够提高外泌体对骨质疏松的治疗效果。另外，经成骨诱导的人脂肪间充质干细胞来源外泌体，相较于普通增殖培养基培养获得的外泌体也具有更好的治疗和预防骨质疏松的效果。
Description
技术领域
本申请涉及生物医药技术领域，具体而言，涉及一种microRNA在制备治疗或预防骨质疏松的药物中的应用。
背景技术
随着老龄化社会的到来，骨质疏松症患者日渐增多。我国现有约九千万骨质疏松症患者，骨质疏松引起骨骼疼痛、骨折等全身问题，同时口腔颌面部骨丢失亦与骨质疏松密切相关，如剩余牙槽嵴重度吸收等。这些问题均严重影响着患者的生活质量，给社会经济带来巨大负担。因此，骨质疏松的合理预防与有效治疗是目前临床上亟待解决的难题。目前骨质疏松的治疗方法主要依赖于药物，包括双磷酸盐类、雌激素类、甲状旁腺素类等，而这些传统治疗方法均存在明显的副作用，比如颌骨坏死、血栓、增加骨肿瘤的发病风险。因此，寻找一种新的、安全、高效且副作用少的骨质疏松症治疗和预防手段显得十分必要和迫切。
目前，对骨质疏松动物模型的干细胞治疗已被证明能取得良好的治疗效果。然而，干细胞治疗存在的问题，包括植入体内后存活率低、免疫排斥、致瘤风险等，限制了其临床推广应用。外泌体是直径为30-150nm的细胞外泌囊泡，具有双层脂质膜结构，包裹蛋白、脂质和核酸等生物活性成分，在细胞间交流和调控细胞生物学功能中发挥重要作用。与干细胞相比，外泌体具有免疫原性低、安全、稳定、容易储存、便于递送等优点。CN110403959A公开一种用于治疗或预防骨质疏松症间充质干细胞外泌体制剂，由骨髓间充质干细胞来源的外泌体、药学上可接受的载体和乙酰水杨酸组成，体内注射200ul含有200ug外泌体+40ug乙酰水杨酸的溶液具有较好的骨质疏松治疗效果。然而，骨髓充质干细胞难于获得，并且会给患者带来巨大痛苦，甚至骨组织缺损，限制了其临床推广应用。
脂肪间充质干细胞可从自身获取，通过吸脂手术获得或来源于废弃的脂肪组织，具有来源广泛、获取便捷、供体痛苦小的优势，因此具备巨大的临床转化潜能。CN114246882A公开了一种间充质干细胞外泌体在制备防治骨质疏松症药物中的应用，包括人脂肪间充质干细胞来源的外泌体和载体制剂。但是该外泌体来源普通培养基培养的脂肪间充质干细胞，其对骨质疏松症治疗或预防效果有限。通过工程化的方式将具有特定功能的生物活性分子，如miRNA或其抑制剂加载到外泌体中，可增强外泌体的治疗效果。CN112190592A公开了一种miR-155-5p高表达的滑膜间充质干细胞来源的外泌体，外泌体中miR-155-5p的表达优选增加67倍，所述外泌体能够减轻骨关节炎损伤并促进软骨再生。CN113373117A公布了一种通过电穿孔技术制备的高表达miR-13474的人脐带间充质干细胞源外泌体用于糖尿病足创面修复。目前，尚无关于miRNA抑制剂修饰的脂肪间充质干细胞来源外泌体用于骨质疏松治疗的报道。
发明内容
本发明提供了一种microRNA在制备治疗或预防骨质疏松的药物中的应用，所述microRNA为miR-335-3p。
本发明还提供一种治疗或预防骨质疏松的间充质干细胞外泌体，所述外泌体中miR-335-3p的表达量降低。
具体的，通过在所述外泌体中转染miR-335-3p抑制剂使外泌体中miR-335-3p的表达量降低。
更具体的，所述外泌体中转染miR-335-3p抑制剂的方法为通过Exo-Fect外泌体转染试剂或电穿孔将miR-335-3p抑制剂转染至间充质干细胞外泌体中；或通过慢病毒将miR-335-3p抑制剂转染至间充质干细胞中。
通过Exo-Fect外泌体转染试剂转染的方法包括：
(1)以200pmol miRNA：300μg外泌体的比例，将miR-335-3p抑制剂与外泌体混合，再加入10μL Exo-Fect溶液，补充适量PBS使得混合体系总体积为150μL；
(2)轻轻混匀体系后，在37℃恒温振荡器中震荡孵育10min，并立即置于冰上冷却；
(3)向冷却后的体系中加入30μL ExoQuick-TC试剂并轻轻混匀以终止反应；
(4)冰上静置30min后，在4℃，14000rpm条件下离心3min，沉淀即为转染miR-335-3p抑制剂的外泌体。
其中，所述miR-335-3p抑制剂为序列如SEQ ID NO.2所示的核苷酸片段；
所述外泌体来源于人脂肪间充质干细胞；优选的，所述外泌体来源于经无外泌体的成骨诱导培养基培养的人脂肪间充质干细胞。
所述无外泌体的成骨诱导培养基的配方如下：Dulbecco改良Eagle培养基(DMEM)，10％(v/v)去除外泌体的胎牛血清，100U/mL青霉素G，100mg/mL链霉素，10nM地塞米松，10mMβ-甘油磷酸和0.2mM抗坏血酸。
本发明的有益效果包括：
(1)本发明基于人脂肪间充质干细胞具有来源广泛、获取便捷、供体痛苦小等优势，提取经成骨诱导的人脂肪间充质干细胞来源外泌体，相较于普通增殖培养基培养获得的外泌体具有更好的治疗和预防骨质疏松的效果。外泌体作为生物活性分子或药物的纳米载体具有先天优势，外泌体的组成成分均来源于细胞，避免了人工合成的脂质体、高分子等材料带来的细胞毒性等问题。相比传统药物治疗，生物安全性更高。与干细胞治疗相比，避免了干细胞的存活率低、免疫排斥、致瘤风险等缺陷。同时，外泌体更易于储存和运输，因此人脂肪间充质干细胞外泌体非常适宜临床转化应用。经成骨诱导的人脂肪间充质干细胞外泌体亦可用于口腔颌面部骨丢失与骨缺损患者的治疗，为颌面部骨再生提供新策略。未来有望用于骨质疏松与骨再生治疗的外泌体库的构建，实现临床推广，提升广大骨质疏松患者生活质量，减轻社会经济负担。
(2)本发明研究发现miR-335-3p可通过负向调控机制，促进骨质疏松小鼠来源骨髓间充质干细胞的成骨向分化；并发现miR-335-3p可能通过下调Aplnr基因发挥抑制成骨向分化的作用。为今后基于miR-335-3p的骨质疏松治疗和促进骨再生策略提供了研究基础。通过高效和简便的方式将miRNA-335-3p抑制剂加载到外泌体中，构建miRNA-335-3p抑制剂优化的工程化人脂肪间充质干细胞外泌体，提高外泌体对骨质疏松的治疗效果。
附图说明
图1为普通光学显微镜下培养的人脂肪间充质干细胞的形态图；
图2为透射电镜下人脂肪间充质干细胞外泌体的形态图；
图3为Western blot鉴定人脂肪间充质干细胞外泌体的标志物；
图4为NTA检测外泌体的粒径分布；
图5为人脂肪间充质干细胞外泌体对小鼠骨髓间充质干细胞碱性磷酸酶活性的定量分析结果；
图6为人脂肪间充质干细胞外泌体对小鼠骨髓间充质干细胞成骨分化相关基因表达的影响；
图7为micro CT显示人脂肪间充质干细胞外泌体对骨质疏松小鼠中骨小梁数量的影响；
图8为micro CT显示人脂肪间充质干细胞外泌体预防骨质疏松的效果；
图9为miRNA-335-3p抑制剂优化的工程化人脂肪间充质干细胞外泌体对小鼠骨髓间充质干细胞碱性磷酸酶活性的定量分析结果；
图10为miRNA-335-3p抑制剂优化的工程化人脂肪间充质干细胞外泌体对小鼠骨髓间充质干细胞成骨分化相关基因表达的影响；
图11为micro CT显示miRNA-335-3p抑制剂优化的工程化人脂肪间充质干细胞外泌体增加了骨质疏松小鼠中骨小梁的数量。
具体实施方式
下面结合实施例对本发明进行进一步说明和描述，但所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明和实施例中，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发明和实施例，都属于本发明保护的范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
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1、人脂肪间充质干细胞(hASCs)外泌体的提取、鉴定和生物安全性评价
(1)hASCs外泌体的提取和鉴定
将hASCs增殖培养基(PM，由Dulbecco改良Eagle培养基(DMEM，Gibco,NY,USA)，10％(v/v)胎牛血清(ScienCell，USA)，100U/mL青霉素G和100mg/mL链霉素(Gibco,NY,USA)组成)和成骨诱导培养基(OM，由DMEM，10％(v/v)胎牛血清，100U/mL青霉素G，100mg/mL链霉素，10nM地塞米松(Sigma-Aldrich,MO,USA)，10mMβ-甘油磷酸(Sigma-Aldrich,MO,USA)和0.2mM抗坏血酸(Sigma-Aldrich,MO,USA)组成)中的FBS组分替换为无外泌体FBS(将FBS在4℃，110000g条件下超高速离心16h，弃去上清液。用0.22μm的无菌滤器将FBS过滤除菌后，保存于4℃冰箱中)，其余组分不变，配制无外泌体的hASCs增殖培养基(Exofree-PM)和成骨诱导培养基(Exofree-OM)。待hASCs(ScienCell，USA)生长至80％汇合度以后，吸去原培养基，用磷酸缓冲盐溶液(PBS)冲洗3次后，加入新鲜的无外泌体的增殖培养基或成骨诱导培养基。将细胞置于37℃，5％二氧化碳浓度的细胞孵箱中培养。3天后收集细胞培养液，用PBS冲洗细胞后加入新鲜的无外泌体的增殖培养基或成骨诱导培养基继续培养。随后每3天收集一次细胞培养液，并加入新鲜无外泌体培养基继续培养。将收集的培养液在4℃，2000g条件下离心20min，收集上清液，弃去沉淀。将上清液在4℃，10000g条件下离心40min，收集上清液，弃去沉淀。用0.22μm的滤器将上清液过滤并收集滤液。将滤液在4℃，110000g的条件下超高速离心70min，弃去上清液，保留沉淀。用大量PBS重悬沉淀，并将重悬液在4℃，110000g的条件下超高速离心70min后，弃去上清液，保留外泌体沉淀。用适量PBS重悬外泌体沉淀，保存于4℃或-80℃，用于后续实验，其中来源于Exofree-PM的外泌体被称为P-Exos。来源于Exofree-OM的外泌体称为O-Exos。
对于提取的外泌体的鉴定结果如图1-3所示。通过透射电子显微镜观察，两种提取物中均可见大量凹面杯状囊泡，高倍镜视野下可见囊泡直径为100nm左右，符合外泌体的形态与大小特征。Western blot结果显示，P-Exos和O-Exos样本中外泌体标志蛋白CD9、CD81和TSG101呈阳性，而两者中外泌体阴性蛋白β-tubulin均无明显表达。NTA结果(如图4所示)显示P-Exos样本中粒子直径主要分布在77-180nm之间，峰值为128nm，粒子平均直径为132.2±54.3nm；O-Exos样本中粒子主要分布在81-192nm之间，峰值为124.4nm，粒子平均直径为136.4±56.8nm。综上可知，提取的P-Exos和O-Exos中含有大量纯度较高的外泌体。
(2)hASCs外泌体的生物安全性评价
以200μg外泌体蛋白：5μLDiR荧光染料(凯基生物，江苏，中国)(溶于200ug/mL乙醇)的比例将外泌体与DiR混合，震荡混匀后于室温下静置孵育30min。孵育完成后，向外泌体/DiR混合体系中加入10mL PBS后混匀，在4℃，110000g条件下超高速离心70min，弃去上清液，获得DiR标记的外泌体沉淀。将DiR标记的P-Exos或O-Exos经尾静脉注射入小鼠体内，注射剂量为每只小鼠200μL，对照组注射200μL PBS。在注射外泌体12h、24h和48h后处死小鼠并取出小鼠心、肺、脾、肝、肾和股骨，拍摄各器官内荧光信号的分布情况，并对股骨内荧光信号强度做定量分析。结果显示DiR的荧光信号(红色)主要集中于肝脏，其次为脾脏，股骨中也有荧光信号出现。荧光信号强度定量分析结果显示，在12h、24h和48h三个时间点，P-Exos组和O-Exos组之间股骨内的DiR荧光信号强度无显著差异，但均显著大于PBS组。
取小鼠的心、肝、脾、肺、肾制成切片并做HE染色，结果显示，无论注射P-Exos还是O-Exos，小鼠的心脏、肺、脾、肝和肾均未观察到明显的病理性改变。结果表明hASCs来源的外泌体具有良好的生物安全性。
实施例2、hASCs外泌体体外对骨质疏松小鼠来源骨髓间充质干细胞(OVXmBMSCs)成骨向分化的影响
(1)hASCs外泌体体外被摄取情况
以100μg实施例1制备的P-Exos或O-Exos外泌体：50μL外泌体红色荧光标记染料PKH26(宇玫博，上海，中国)的比例将外泌体与工作液混合，震荡混匀后于室温下静置孵育10min。孵育完成后，向外泌体-工作液混合体系中加入10mL PBS后混匀，在4℃，110000g条件下超高速离心70min，弃去上清液，获得PKH26标记的外泌体沉淀。用适量PBS重悬外泌体沉淀。将PKH26标记的P-Exos或O-Exos加入到无外泌体血清配制的Exofree-PM中，制成外泌体浓度为50μg/mL的P-Exos增殖培养基或O-Exos增殖培养基。待mBMSCs(武汉普诺赛生命科技有限公司，武汉，中国)生长至90％汇合度后，更换为P-Exos增殖培养基或O-Exos增殖培养基。继续培养细胞12h、24h、48h和72h后，弃去培养基，激光扫描共聚焦显微镜观察细胞摄取外泌体的情况。随着时间推移，P-Exos和O-Exos(红色荧光信号)被mBMSCs缓慢摄取入细胞内。荧光信号定量分析显示，与外泌体共培养24h后细胞内的荧光信号开始明显增强。共培养72h后，细胞内已聚集大量外泌体。
(2)P-Exos和O-Exos在体外对OVXmBMSCs成骨向分化的促进效果
首先，分别从Sham(假手术，切开腹部皮肤找到双侧卵巢后，不进行结扎和切除，直接复位卵巢和子宫，对位缝合腹膜和皮肤)和OVX小鼠的股骨内提取了原代mBMSCs，分别定义为ShammBMSCs和OVXmBMSCs。将50μg/μL的P-Exos或O-Exos与OVXmBMSCs共培养，对照组为采用不含外泌体的培养基处理的OVX mBMSCs(OVX组)和ShammBMSCs(Sham组)。3天和7天后，P-Exos对OVX mBMSCs的ALP染色无明显影响，添加O-Exos后，OVX mBMSCs的染色明显加深。ALP(碱性磷酸酶)活性定量分析结果(如图5所示)与ALP染色的实验结果一致，P-Exos对OVXmBMSCs(OVX+P-Exos组)的ALP活性无明显影响，添加O-Exos后OVX mBMSCs(OVX+O-Exos组)的ALP活性明显增强。通过qTR-PCR实验进一步检测各组细胞内成骨相关基因Runx2、Alp和Ocn的表达情况。结果如图6显示，其中OVX+P-Exos组细胞内成骨相关基因的表达量与OVX组之间并无显著差异，且两者均显著低于Sham组的细胞。OVX+O-Exos组细胞的成骨相关基因表达量显著高于OVX组和OVX+P-Exos组。用Western Blot检测细胞内成骨相关蛋白Runx2的表达情况。结果显示，OVX组细胞合成的Runx2明显少于Sham组，且与OVX+P-Exos组无显著差异。而添加O-Exos可以使OVX mBMSCs中Runx2的表达量明显升高。以上结果表明，P-Exos对OVX mBMSCs的成骨分化无明显促进作用，而O-Exos能够挽救OVX mBMSCs受损的成骨分化能力，促进OVX mBMSCs成骨分化。
实施例3、体内注射hASCs来源外泌体对骨质疏松小鼠的治疗和预防效果
(1)治疗效果
将P-Exos或O-Exos稀释于PBS中，配制成每200μL溶液中含有100μg外泌体蛋白的P-Exos或O-Exos注射液，以200μL PBS溶液作为对照。将Sham或OVX术后三个月的小鼠随机分为四组：Sham+PBS组，OVX+PBS组，OVX+P-Exos组和OVX+O-Exos组。经尾静脉将PBS、P-Exos注射液或O-Exos注射液分别注射入相应组小鼠体内，每只小鼠的注射剂量为200μL。每3天注射一次。经过8次注射后，处死小鼠，取小鼠的股骨拍摄micro-CT。如图7所示，注射P-Exos(OVX+P-Exos组)或O-Exos(OVX+O-Exos组)后，小鼠骨小梁较OVX+PBS组的OVX小鼠更为致密。骨密度和骨形态学分析结果显示，OVX+P-Exos组和OVX+O-Exos组小鼠的骨密度显著大于OVX+PBS组的小鼠。尽管OVX+O-Exos组小鼠的骨密度大于OVX+P-Exos组，但两组之间无统计学差异。注射P-Exos或O-Exos后，小鼠股骨ROI的骨体积分数(BV/TV)、骨小梁数量(TbTh)和骨小梁厚度(TbN)明显高于注射PBS的OVX小鼠(OVX+PBS组)，而骨表面积体积比(BS/BV)和骨小梁间隙(TbSp)则显著低于注射PBS的OVX小鼠(OVX+PBS组)。与骨密度分析结果类似的是，尽管OVX+O-Exos组小鼠的BV/TV、TbTh和TbN大于OVX+P-Exos组，BS/BV和TbSp小于OVX+P-Exos组，但两组之间无统计学差异。组织切片分析结果与micro-CT结果相同。HE和Masson染色显示，OVX小鼠股骨内骨小梁组织较Sham+PBS组明显减少，骨髓组织占据空间增大；注射P-Exos或O-Exos后，OVX小鼠股骨内骨小梁组织增多，骨髓组织占据空间减小。抗酒石酸酸性磷酸酶(TRAP)染色与破骨细胞计数结果显示，OVX小鼠股骨内破骨细胞数量较Sham+PBS组增多，注射P-Exo或O-Exos后，小鼠股骨内破骨细胞数量较OVX组减少。免疫组化染色与定量以及成骨细胞计数结果显示，OVX小鼠股骨内Ocn分泌功能较Sham+PBS组减弱，注射P-Exo或O-Exos后，小鼠股骨内出现大量功能活跃的成骨细胞，股骨内成骨趋势增强。
综上所述，静脉注射P-Exos或O-Exos可有效地缓解OVX小鼠的骨质疏松症状。
(2)预防效果
向hSACs外泌体中加入适量PBS，稀释成外泌体浓度100μg/200μL的外泌体注射液。在小鼠OVX手术或sham(假手术)2周后，按以下分组经尾静脉注射hASCs外泌体或PBS：Sham+PBS，OVX+PBS，OVX+O-Exos。注射剂量为200μL，注射频率为每3天注射一次。经过8次注射后处死小鼠，并取小鼠股骨拍摄microCT，评估骨质情况。如图8显示，OVX+PBS组小鼠股骨内骨小梁较Sham+PBS组更为稀疏，OVX手术使小鼠股骨出现骨质丢失，而OVX+O-Exos组小鼠股骨内骨小梁较OVX+PBS组小鼠更为致密，与Sham+PBS组接近。HE和Masson染色显示，OVX+PBS组小鼠股骨内骨小梁组织较Sham+PBS组明显减少，骨髓组织占据空间增大；OVX+O-Exos组小鼠股骨内骨小梁较OVX+PBS组增多。骨形态学定量分析结果显示，OVX+PBS组小鼠股骨ROI区域BMD较Sham+PBS组明显下降，BV/TV、TbTh和TbN明显低于Sham+PBS组，而BS/BV和TbSp明显高于Sham+PBS组；OVX+O-Exos组小鼠股骨ROI区域BMD则显著高于OVX+PBS组，BV/TV、TbTh和TbN显著高于OVX+PBS组，而BS/BV和TbSp显著低于OVX+PBS组。由以上结果可以得出，OVX手术使小鼠出现骨质疏松，而术后注射O-Exos可以有效预防OVX手术引起的骨质疏松。
实施例4、hASCs来源外泌体促进干细胞成骨向分化的潜在机制
(1)筛选外泌体促成骨作用的关键miRNA
使用exoRNeasy Maxi Kit试剂盒(QIAGEN，Germany)分别提取下列三种外泌体上的RNA：hASCs在Exofree-PM条件下分泌的外泌体(P-Exos)、hASCs在Exofree-OM中向成骨分化第7天分泌的外泌体(O-Exos-7d)以及hASCs在Exofree-OM中向成骨分化第14天分泌的外泌体(O-Exos-14d)。对P-Exos、O-Exos-7d和O-Exos-14d上共计2684个miRNA的表达情况进行检测分析，三种外泌体之间差异表达的基因数量用Venn图展示。对O-Exos-7d vs P-Exos以及O-Exos-14d vs P-Exos之间差异表达的miRNA进行对比。与P-Exos相比，O-Exos-7d上携带的miRNA中有16个miRNA表达上调，21个miRNA表达下调，而O-Exos-14d上携带的miRNA中有41个miRNA表达上调，31个miRNA表达下调。对O-Exos-7d和O-Exos-14d中表达上调的miRNA取交集，发现没有miRNA在O-Exos-7d和O-Exos-14d上同时表达上调。对O-Exos-7d和O-Exos-14d中表达下调的miRNA取交集，发现仅有4个miRNA在O-Exos-7d和O-Exos-14d上同时表达下调：miR-93-3p、miR-501-3p、miR-490-3p和miR-335-3p。因此，将miR-93-3p、miR-501-3p、miR-490-3p和miR-335-3p作为研究O-Exos促进mBMSCs成骨分化机制的候选关键基因。通过qRT-PCR进一步验证了hASCs和外泌体在成骨分化过程中这四个miRNA的表达情况。
使用Poly A加尾法PCR试剂盒检测hASCs成骨分化过程中miR-93-3p、miR-501-3p、miR-490-3p和miR-335-3p的表达情况。在成骨分化第7天和第14天，hASCs中miR-93-3p、miR-501-3p、miR-490-3p和miR-335-3p的表达量均显著下降，其中miR-335-3p的下降幅度最大。接下来，使用茎环法PCR试剂盒对成骨分化的hASCs及其分泌的外泌体上miR-335-3p的表达情况进行进一步验证。在成骨分化过程中hASCs及其分泌的外泌体上miR-335-3p的表达量均显著下降。因此，最终采用miR-335-3p作为研究O-Exos促进成骨分化机制的关键miRNA。
(2)明确miR-335-3p及其靶基因Aplnr在体外促成骨中的作用
通过慢病毒(上海吉玛制药技术有限公司，上海，中国)将miR-335-3p模拟物(miR-335-3p mimics，5’-TTTTTCATTATTGCTCCTGACC-3’，对应于序列表中SEQ ID NO.1)或miR-335-3p抑制剂(miR-335-3p inhibitors，5’-GGTCAGGAGCAATAATGAAAAA-3’，对应于序列表中SEQ ID NO.2，上海吉玛制药技术有限公司，上海，中国)转染进mBMSCs内，构建miR-335-3p敲低/过表达的mBMSCs细胞系，以miR-NC(5’-TTCTCCGAACGTGTCACGT-3’，对应于序列表中SEQ ID NO.3)作为对照。qRT-PCR检测miR-335-3p敲低/过表达效果：转染miR-335-3pmimic后，mBMSCs中miR-335-3p的表达量较转染miR-NC的细胞显著升高；转染miR-335-3pinhibitor后，mBMSCs中miR-335-3p的表达量较转染miR-NC的细胞显著降低。qRT-PCR检测miR-335-3p敲低/过表达细胞系中Aplnr的表达情况，结果显示，miR-335-3p过表达的mBMSCs中，Aplnr的表达量显著下降，而miR-335-3p敲低的mBMSCs中，Aplnr的表达量显著升高。双荧光素酶报告基因实验显示，miR-335-3p mimic降低了Aplnr WT的荧光素酶活性，但对Aplnr Mut的荧光素酶活性无显著影响。由此可以得出结论：Aplnr是miR-335-3p的靶基因，miR-335-3p通过与Aplnr的3’-UTR结合，直接抑制Aplnr的表达。对转染后的mBMSCs进行成骨诱导，并在成骨诱导第3天和第7天检测各组细胞的成骨分化情况。ALP染色结果显示，miR-335-3p mimic组细胞染色较miR-NC对照组明显较浅，而miR-335-3p inhibitor组的染色较miR-NC对照组更深。miR-335-3p mimic组的ALP活性显著低于miR-NC对照组，而miR-335-3p inhibitor组的ALP活性则显著升高。qRT-PCR检测成骨相关基因表达情况，结果显示：在PM培养基中miR-335-3p mimic组和miR-335-3p inhibitor组细胞中Runx2、Alp和Ocn的表达量均与miR-NC对照组无显著差异；在OM培养基中miR-335-3p mimic组细胞中Runx2、Alp和Ocn的表达量显著低于miR-NC对照组，而miR-335-3p inhibitor组细胞中Runx2、Alp和Ocn的表达量显著高于miR-NC对照组。用western blot检测细胞内成骨相关蛋白Runx2的合成情况，结果显示：OM培养基中miR-335-3p mimic组细胞内Runx2表达量较miR-NC对照组下降，而miR-335-3p inhibitor组细胞内Runx2的表达量升高。以上结果表明，miR-335-3p抑制mBMSCs成骨分化，是mBMSCs成骨分化的负向调节因子。
实施例5、加载miR-335-3p抑制剂的工程化hASCs来源外泌体的构建及其对骨质疏松小鼠来源干细胞成骨向分化的影响
使用Exo-FectTM外泌体转染试剂盒(System Biosciences,CA,USA)将miR-335-3pinhibitor装载至O-Exos，构建功能优化的外泌体(O-Exos+anti-miR-335)。将O-Exos作为阴性对照(O-Exos)。转染方法为：使用Pierce BCA蛋白定量试剂盒测定O-Exos浓度。以200pmol miRNA：300μg外泌体的比例将miR-335-3p inhibitor或miR-NC与O-Exos混合，再加入10μL Exo-Fect溶液，补充适量PBS使得混合体系总体积为150μL。轻轻混匀体系后，在37℃恒温振荡器中震荡孵育10min，并立即置于冰上冷却。向冷却后的体系中加入30μLExoQuick-TC(System Biosciences,CA,USA)试剂并轻轻混匀以终止反应。冰上静置30min后，在4℃，14000rpm条件下离心3min。弃去上清液，保留沉淀，得到装载miR-335-3pinhibitor或miR-NC的O-Exos。加入少量PBS重悬外泌体沉淀。
将50μg/mL的O-Exos或O-Exos+anti-miR-335与OVX mBMSCs共培养，以采用不含外泌体的培养基处理的ShammBMSCs(Sham组)和OVX mBMSCs(OVX组)作为对照，在第3天和第7天对细胞的成骨分化能力进行检测。ALP染色显示，OVX+O-Exos+anti-miR-335组的染色比OVX+O-Exos组更深，且二者均深于OVX组染色。如图9所示，ALP活性定量与ALP染色的实验结果一致。添加O-Exos+anti-miR-335后，OVX mBMSCs的ALP活性强于O-Exos组，且二者均强于未添加外泌体的OVX mBMSCs。提取细胞RNA，通过qTR-PCR实验检测各组细胞内成骨相关基因Runx2、Alp和Ocn的表达情况。结果如图10所示，OVX+O-Exos+anti-miR-335组细胞的成骨相关基因表达量显著高于OVX+O-Exos组，且二者均高于OVX组。用western blot检测细胞内成骨相关蛋白Runx2的合成情况。结果显示，添加O-Exos可以使OVX mBMSCs中Runx2的表达量升高，而添加O-Exos+anti-miR-335可以使OVX mBMSCs中Runx2的表达量较添加O-Exos进一步升高。以上结果表明，O-Exos+anti-miR-335具有比O-Exos更强的促OVX mBMSCs成骨分化的作用。
实施例6、加载miR-335-3p抑制剂的工程化hASCs来源外泌体体内注射对骨质疏松小鼠的治疗效果
将O-Exos或O-Exos+anti-miR-335稀释于适量PBS中，配制成每200μL溶液中含有100μg外泌体蛋白的O-Exos注射液或O-Exos+anti-miR-335外泌体注射液，以200μL PBS溶液作为对照。将Sham或OVX术后三个月的小鼠随机分为四组：Sham+PBS组、OVX+PBS组、OVX+O-Exos组和OVX+O-Exos+anti-miR-335组。经尾静脉将PBS、O-Exos注射液或O-Exos+anti-miR-335注射液分别注射入相应小鼠体内，每只小鼠的注射剂量为200μL。每3天注射一次。经过8次注射后，处死小鼠，取小鼠的股骨拍摄micro-CT。结果如图11所示，OVX+O-Exos+anti-miR-335组小鼠骨小梁较OVX+O-Exos组更为致密。骨密度和骨形态学分析结果显示，OVX+O-Exos+anti-miR-335组小鼠的骨密度显著大于OVX+O-Exos组的小鼠，且二者均大于OVX+PBS组。注射O-Exos+anti-miR-335后，小鼠股骨ROI区域的BV/TV、TbTh和TbN显著高于注射O-Exos的OVX小鼠，而BS/BV和TbSp则显著低于注射O-Exos的OVX小鼠。组织切片分析结果与micro-CT结果相同。HE和Masson染色显示，OVX小鼠股骨内骨小梁组织较Sham明显减少，骨髓组织占据空间增大；注射O-Exos和O-Exos+anti-miR-335以后，OVX小鼠股骨内骨小梁组织增多，且OVX+O-Exos+anti-miR-335组骨小梁明显多于OVX+O-Exos组。TRAP染色与破骨细胞计数结果显示，OVX+O-Exos+anti-miR-335组小鼠股骨内破骨细胞数量较OVX+O-Exos减少。免疫组化染色与定量以及成骨细胞计数结果显示，OVX+O-Exos+anti-miR-335组小鼠股骨内Ocn分泌功能较OVX+O-Exos组增强，股骨内成骨趋势强于OVX+O-Exos组。取小鼠的心、肝、脾、肺、肾制成切片并做HE染色，结果显示，无论注射O-Exos或O-Exos+anti-miR-335，小鼠的心脏、肺、脾、肝和肾均未观察到明显的病理性改变。结果表明hASCs来源的外泌体具有良好的生物安全性。综上所述，O-Exos+anti-miR-335具有比O-Exos更强的治疗骨质疏松的效果，并且具有良好的生物安全性。
序列表
<110> 北京大学口腔医学院
<120> microRNA 在制备治疗或预防骨质疏松的药物中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tttttcatta ttgctcctga cc 22
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtcaggagc aataatgaaa aa 22
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttctccgaac gtgtcacgt 19
Claims (9)
1.microRNA在制备治疗或预防骨质疏松的药物中的应用，其特征在于，所述microRNA为miR-335-3p。
2.一种治疗或预防骨质疏松的间充质干细胞外泌体，其特征在于，所述外泌体中miR-335-3p的表达量降低。
3.根据权利要求2所述的外泌体，其特征在于，通过在所述外泌体中转染miR-335-3p抑制剂使外泌体中miR-335-3p的表达量降低。
4.根据权利要求3所述的外泌体，其特征在于，所述外泌体中转染miR-335-3p抑制剂的方法为通过Exo-Fect外泌体转染试剂或电穿孔将miR-335-3p抑制剂转染至间充质干细胞外泌体中；或通过慢病毒将miR-335-3p抑制剂转染至间充质干细胞中。
5.根据权利要求4所述的外泌体，其特征在于，所述通过Exo-Fect外泌体转染试剂转染的方法包括：
(1)以200pmol miRNA：300μg外泌体的比例，将miR-335-3p抑制剂与外泌体混合，再加入10μL Exo-Fect溶液，补充适量PBS使得混合体系总体积为150μL；
(2)轻轻混匀体系后，在37℃恒温振荡器中震荡孵育10min，并立即置于冰上冷却；
(3)向冷却后的体系中加入30μL ExoQuick-TC试剂并轻轻混匀以终止反应；
(4)冰上静置30min后，在4℃，14000rpm条件下离心3min，沉淀即为转染miR-335-3p抑制剂的外泌体。
6.根据权利要求3-5任一项所述的外泌体，其特征在于，所述miR-335-3p抑制剂为序列如SEQ ID NO.2所示的核苷酸片段。
7.根据权利要求2-5任一项所述的外泌体，其特征在于，所述外泌体来源于人脂肪间充质干细胞。
8.根据权利要求7所述的外泌体，其特征在于，所述外泌体来源于经无外泌体的成骨诱导培养基培养的人脂肪间充质干细胞。
9.根据权利要求8所述的外泌体，其特征在于，所述无外泌体的成骨诱导培养基的配方如下：DMEM，10％(v/v)去除外泌体的胎牛血清，100U/mL青霉素G，100mg/mL链霉素，10nM地塞米松，10mMβ-甘油磷酸和0.2mM抗坏血酸。
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