CN116270713B - miR-1246在制备促进腱骨愈合药物中的应用 - Google Patents
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Abstract
本发明属于生物医药技术领域，具体涉及miR‑1246在制备促进腱骨愈合药物中的应用。研究证明，所述miR‑1246可以促进腱骨界面接近生理性的愈合，并增加其生物力学强度，同时促进了肌腱细胞的增殖和迁移。miR‑1246通过作用于巨噬细胞，激活巨噬细胞的PI3K/AKT/mTOR通路，使巨噬细胞发生极化，调控促炎因子和抗炎因子的表达，从而减轻损伤修复早期阶段巨噬细胞诱导的炎症反应，有效促进腱骨的修复和再生，避免移植体带来的整合不良、免疫排斥等不良临床效果和较高的复发率，能够解决腱骨愈合不良的临床难题。并且，采用miR‑1246进行治疗时，安全、高效，更有利于该新药的临床转化过程。
Description
技术领域
本发明属于生物医药技术领域。更具体地，涉及miR-1246在制备促进腱骨愈合药物中的应用。
背景技术
肩袖撕裂(Rotator cuff tears，RCT)是最常见的肌肉骨骼损伤类型之一。由外伤、退化变性等因素导致，常引起严重的疼痛，甚至残疾。肩袖撕裂损伤约占肩关节疾病的50％，在我国肩袖撕裂损伤患病率逐年递增，给社会和患者带来沉重的负担。目前，修复肩袖损伤有多种治疗方式，包括肌腱-骨固定技术、自体移植物、同种异体移植物和异种移植物等。如中国专利申请CN107496052A公开了一种肩袖补片，经编工艺编织而成，生物力学强度好，能够为肩袖关节提供足够的力学支撑；中国专利申请CN211534754U公开了一种用于加快肩袖腱骨愈合的自动PRF植入铆钉，所具有的储药槽内储存有能够加快肩袖腱骨连接处愈合的生物材料，通过铆钉主体直接植入到腱骨连接处，减少后期植入生物材料的步骤，利用生物材料在术后初期加快肩袖腱骨连接处的愈合。
但是上述治疗方案大部分是依靠移植材料提供力学支撑，不可避免的存在移植体整合不良、免疫排斥反应等缺陷，没有从根本上解决腱骨愈合不良的问题，后续移植部位撕裂的复发率仍可高达90％。可见，现有技术仍无法提供一种能够真正解决腱骨愈合失败问题的有效方法。
发明内容
本发明要解决的技术问题是克服现有腱骨损伤依靠移植材料治疗过程中存在的移植体整合不良、免疫排斥反应发生、易复发和力学性能差等缺陷和不足，提供一种miR-1246在制备促进腱骨愈合药物中的应用，通过诱导巨噬细胞极化，调控炎症因子释放，促进自身肌腱细胞的增殖和迁移，从而促进腱骨界面接近生理的愈合，增强其生物力学强度，从损伤修复的早期阶段对炎症进行精细调控，即使不依靠移植材料也能达到显著促进腱骨愈合的效果。
本发明上述目的通过以下技术方案实现：
现有技术研究表明，巨噬细胞驱动的炎症是肌腱修复早期阶段的关键特征，而过度炎症则是导致不良临床效果的关键因素。动物实验研究显示，由于促炎型M1巨噬细胞可导致炎症、细胞外基质(ECM)降解和细胞死亡，阻碍了肌腱的愈合。体外证据也支持这一观点，即肌腱愈合不良很大程度上是由于M1巨噬细胞分泌的促炎因子的有害作用。但相反的，抗炎型M2巨噬细胞则倾向于抵抗炎症反应、调节ECM平衡，从而促进组织愈合。现有技术大多数的技术对于改善腱骨修复过程的尝试主要是针对愈合的后期阶段，但是都收效甚微。因此，本发明在损伤修复的早期阶段对炎症进行精细调节以改善腱骨的不良愈合。所采用的miRNA是一类由内源性基因编码的单链小分子RNA，广泛存在生物体中参与转录后基因表达调控，能够调节多种重要的生物学功能。本发明实验研究首次发现，miR-1246可以显著促进腱骨愈合。
因此，本发明要求保护miR-1246在制备促进腱骨愈合药物中的应用，所述miR-1246的序列为：AAUGGAUUUUUGGAGCAGG。
进一步地，所述miR-1246促进腱骨界面愈合，提高生物力学强度。
更进一步地，所述miR-1246促进肌腱细胞的增殖和迁移。
进一步地，所述腱骨愈合为人体的肌腱、韧带在骨止点损伤的修复，包括肩袖撕裂修复、肩锁关节韧带重建术后修复、肘关节侧副韧带修复、前后交叉韧带重建术后修复、踝关节侧副韧带修复。
更进一步地，所述药物还包括药学上可接受的载体。miR-1246通过载体的递送能更高效的进入组织和细胞内，以促进腱骨愈合。
优选地，所述载体选自脂质体、外泌体、聚合物、介孔二氧化硅纳米颗粒、量子点和金属基纳米颗粒中的一种或多种。
优选地，所述药物的剂型为注射剂或外用药剂。更优选地，所述注射剂包括注射液、粉针剂；所述外用药剂包括凝胶、膏剂、粉剂、酊剂、喷剂。
另外的，本发明还提供了miR-1246在制备诱导巨噬细胞极化试剂中的应用，所述miR-1246的序列为：AAUGGAUUUUUGGAGCAGG。
进一步地，所述miR-1246作用于巨噬细胞，抑制促炎因子的表达。
更进一步地，所述促炎因子包括TNFα、IL-1β、IL-6和COX2。
进一步地，所述miR-1246作用于巨噬细胞，促进抗炎因子的表达。
更进一步地，所述抗炎因子包括IL-1RA、IL-4、IL-10。
本发明具有以下有益效果：
本发明首次提供了miR-1246在制备促进腱骨愈合药物中的应用，研究证明，所述miR-1246可以促进腱骨界面接近生理的愈合，并增加其生物力学强度，同时促进了肌腱细胞的增殖和迁移。miR-1246通过作用于巨噬细胞，激活巨噬细胞的PI3K/AKT/mTOR通路，使巨噬细胞发生极化，调控促炎因子和抗炎因子的表达，从而减轻损伤修复早期阶段巨噬细胞诱导的炎症反应，有效促进腱骨界面的修复和再生，避免了移植体带来的整合不良、免疫排斥等不良临床效果和较高的复发率，能够解决腱骨愈合不良的临床难题。并且，采用miR-1246进行治疗时，安全、高效，更有利于该新药的临床转化过程。
附图说明
图1为实施例1中大鼠肩袖撕裂伤模型手术步骤及损伤部位注射药物的图。
图2为实施例1中AgomiR-1246治疗组(A)和AgomiR-NC对照组(B)腱骨结合部愈合情况的大体观察图。
图3为实施例1中腱骨结合部肌腱组织的生物力学测试示意图(A)和各组腱骨界面肌腱的极限失效载荷(B)、刚度(C)比较数据统计图；其中，数据以均数±SEM表示，*P<0.05，**P<0.01，****P<0.0001。
图4为实施例1中各组肩袖腱骨结合部的病理组织学图。
图5为实施例2中由LPS激活的巨噬细胞和肌腱细胞的Transwell共培养体系示意图(A)和经miR-1246mimics处理后肌腱细胞在光学显微镜下20倍(B)和40倍(C)的形态和数量照片。
图6为实施例2中各组miR1246的表达水平数据统计图(A)和各组PI3K、PAKT、PS6相关蛋白表达的蛋白印迹图(B)。
图7为实施例2中各组炎症因子的表达数据统计图(A)和各组间炎症因子的蛋白表达数据统计图(B)。
图8为实施例3中划痕实验检测各组肌腱细胞的迁移能力显微镜下照片(A)、各组肌腱细胞的形态和数量显微镜下照片(B)和各组肌腱细胞的增殖能力数据统计图(C)。
图9为miR 1246促进肩袖撕裂伤后腱骨愈合的作用及机制示意图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
RAW264.7巨噬细胞系购自ATCC细胞库。动物体内注射应用的miR 1246模拟物AgomiR 1246(利用甲基化+胆固醇修饰，由厂家提供，为常规技术手段)，对照AgomiR-NC以及体外细胞模型实验中应用的miR 1246模拟物miR 1246mimics，对照miR 1246mimics NC以及转染试剂盒(为纳米材料载体，由厂家提供，为常规技术手段，其他常用载体也能达到相同效果)均可购自广州锐博生物技术有限公司。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1miR-1246对RCT后腱骨愈合的影响
(1)构建大鼠RCT模型：
大鼠全身麻醉后，上肢肩部备皮并消毒。采用长约3cm的经大结节纵行皮肤切口，钝性分离皮下组织、肌肉，暴露冈上肌，沿肱骨止点锐性离断冈上肌，剥离肱骨大结节残存组织直到骨质。为了消除大结节上残余冈上肌止点组织对组织学观察的影响，术中需彻底切除大结节上的止点组织。取距冈上肌止点约0.3cm、距边缘1/4处两点为进针点，使用两根2-0不可吸收普理灵缝线对肌腱残端行褥式缝合，一侧进针点由内向外，另一侧则有外向内，两根普理灵尾线留取同等长度。将两根PDS线穿入直径1mm的空针针芯，并使用空针针头于肱骨大结节下缘缓慢钻开两组平行骨隧道，出口定位足印区下界，拔出针头将两根PDS线留于骨隧道中。将骨两组隧道中的PDS引线分别与向对应的肌腱残端的两根普理灵缝线拉出骨隧道，将冈上肌腱牵拉锚定缝合在足印区上，建立大鼠单侧冈上肌损伤修复模型，具体如图1中的A～B所示。
(2)药物治疗：
AgomiR 1246治疗组：在肩袖腱骨结合部缓慢注射5nmol AgomiR 1246溶液约200μl，避免药物溢出，每周注射2次。AgomiR-NC对照组：在肩袖腱骨结合部予以注射AgomiR-NC约200μl，注射方法和频次同前。操作过程参见图1中的C。
(3)取材腱骨结合部的组织：
于造模后1周，即修复早期阶段取材腱骨结合部组织。以手术区域冈上肌肌腱止点为中心，完整取下包含完整肩关节的肱骨近端组织及完整冈上肌，剔除皮肤肌肉和软组织，保留完整冈上肌肌腱与肱骨大结节交界区域，即冈上肌-肱骨复合体。
(4)大体观察：
给予药物后1周，观察手术区域切口愈合情况(有无感染等炎症反应)、有无因组织挛缩或粘连而导致的肩关节活动度受限。并取材腱骨结合部组织，观察各组标本手术区有无炎症反应、周围组织粘连情况、肌腱与肱骨之间的间隙及肌腱和腱骨止点外形变化。
实验结果如图2所示，由图可见，给予AgomiR 1246注射后1周，大体观察可见，腱骨界面愈合良好，伤口区域未见感染征及明显炎症反应，腱骨界面周围组织未见因组织挛缩或粘连而导致的肩关节活动度明显受限。而对照组给予AgomiR-NC注射后1周，可见腱骨界面愈合不良，周围组织有粘连，肌腱表面不光滑。
(5)生物力学评估：
根据生物力学检测仪的操作规范，将冈上肌-肱骨复合体固定在AGX-S拉力测试试验机上。设定传感器范围100N，拉伸速度为5mm/min。将肩胛骨与肱骨分别固定在试验机的拉力臂和底座上，调整固定方向，保证拉力的方向与冈上肌纤维附着走行一致。开启拉力测试，精确记录拉力曲线变化，测试最大拉伸载荷冈上肌组织。分为NC：正常组，RCT：未给予任何处理的模型对照组，RCT+AgomiR-NC对照组，RCT+AgomiR 1246治疗组。所有试样均以恒定拉伸速度加载至肌腱拉伸断裂。详细记录其最大断裂载荷(ultimate failure load，N)，载荷-位移曲线的线性斜率为作为刚度(stiffness，N/mm)。
示意图及生物力学性能指标如图3所示。由图可见，和AgomiR-NC对照组相比，AgomiR 1246治疗组肩袖腱骨结合部的极限失效载荷明显提高；AgomiR 1246治疗组表现出刚度性能明显增强。
(6)组织病理学检测：
将腱骨结合部组织在4％多聚甲醛中固定，5％硝酸进行完全脱钙，用石蜡包埋，制成约20μm厚的石蜡切片，并用HE、阿尔新蓝进行染色。
病理结果如图4所示，可见RCT+AgomiR 1246治疗组腱骨界面周围的组织未见明显免疫炎症反应，肌腱和骨质之间呈现出了连续性，肌成纤维细胞向骨质内的生长，导致了更有序排列的肌腱胶原纤维和细胞形成，显示出了组织特征的改善。而在对照组样本中，可见肌腱胶原纤维和细胞失去了其特有的平行方向，肌腱与骨质之间生长不连续性。
实施例2miR-1246对巨噬细胞的影响
(1)肌腱细胞和巨噬细胞系共培养体系的建立
将正常大鼠肌腱组织进行消化，体外分离肌腱细胞，将P5代肌腱细胞接种于transwell的下室内在12孔板中以5×104个/孔的密度扩增培养；在肌腱细胞培养24h后与RAW264.7共培养，制备共培养体系：RAW264.7细胞接种于transwell的上室内，以5×104细胞/室的密度接种，RAW264.7细胞接种6小时后，加入脂多糖(LPS，1μg/mL)刺激8小时，以构建巨噬细胞炎症模型。肌腱细胞和巨噬细胞系共培养体系和经miR-1246处理后肌腱细胞在光学显微镜下的数量和形态照片，具体可参见图5。
(2)转染miR1246 mimics进入巨噬细胞中
根据制造商的说明书，利用转染试剂盒将miR1246 mimics，miRNA mimics NC递送进入巨噬细胞内，在37℃细胞培养箱中孵育24h，24h后更换为新鲜培养基继续培养24h，取细胞获得转染后的细胞样本，分为miR1246 mimics+LPS组；miRNA mimics NC+LPS对照组。
(3)RT-qPCR检测巨噬细胞中miR1246的表达水平
应用TRIzol分别从miR1246 mimics+LPS组，miRNA mimics NC+LPS对照组的巨噬细胞中提取总RNAs，使用Bulge-Loop miRNA特异性逆转录引物(RiboBio)进行逆转录，并使用针对靶向miRNAs合成的特异性引物(RiboBio)进行qPCR，U6作为内源性对照。将表达数据统一标准化，计算巨噬细胞中miR1246的相对表达量的差异。
实验统计结果如图6A所示，由图可见，与miRNA mimics NC组相比，miR1246mimics组的巨噬细胞中miR1246的表达量明显升高。
(4)Westernblot检测miR1246对巨噬细胞中PI3K/AKT/mTOR信号通路的调控作用
PI3K/AKT/mTOR信号通路与巨噬细胞极化密切相关，应用Westernblot方法比较各组PI3K/AKT/mTOR信号通路相关蛋白表达水平的差异。
检测结果如图6B所示，miR1246的过表达使得巨噬细胞中PI3K/AKT/mTOR通路中的PI3K、PAKT、PS6相关蛋白因子的表达显著上调。
(5)RT-qPCR和ELISA检测巨噬细胞相关下游炎症因子
为了进一步探究miR1246的作用机制，根据试剂盒说明书，应用qRT-PCR法和ELISA试剂盒定量共培养体系中细胞上清液中炎症因子的基因和蛋白表达水平，以评估miR1246对M1和M2型巨噬细胞释放的促炎和抗炎因子的影响，包括促炎因子TNFα、IL-1β、IL-6、COX2；抗炎因子IL-1RA、IL-4、IL-10。
实验结果如图7所示，由图可见，miR1246 mimics组中M1型巨噬细胞分泌的促炎因子TNFα、IL-1β、IL-6、COX2的表达量显著下调；M2型巨噬细胞分泌的抗炎因子IL-1RA、IL-4、IL-10的表达量显著上调。
实施例3miR-1246对炎症环境下肌腱细胞的影响
利用划痕实验检测肌腱细胞的迁移能力：在96孔板后均匀划横线，大约每隔0.5～1cm一道，横穿过孔，每孔至少穿过5条线；孔中加入一定数量的细胞，接种原则为过夜后融合率达到100％；用枪头比着直尺，垂直于细胞平面，沿着平板背面的线在细胞层上进行划痕；用PBS洗细胞3次，去除划下的细胞，加入无血清培养基；放入37度5％CO2培养箱培养。按0h、24h，48h取样，拍照。
应用细胞增殖实验(CCK8)检测各组肌腱细胞的增殖能力：消化两组细胞、离心、计数和重悬后制备细胞悬液，按300个/孔的接种密度种于96孔板中，细胞培养箱中培养24h后开始观察1～7天细胞的生长状况。紫外分光光度计上(450nm)测溶液的OD值，以培养时间为横坐标，以细胞光密度值为纵坐标，绘制细胞增殖曲线。
实验结果如图8所示，由图可见，划痕实验显示miR1246表达上调后不仅可以显著促进肌腱细胞的迁移，而且可以促使肌腱细胞的增殖能力明显增强。
综上所述，miR1246通过作用于巨噬细胞，激活巨噬细胞的PI3K/AKT/mTOR通路，促使巨噬细胞发生极化，调控促炎因子(TNFα、IL-1β、IL-6、COX2)和抗炎因子(IL-1RA、IL-4、IL-10)的表达，以减轻损伤修复早期阶段巨噬细胞诱导的炎症反应，从而有效促进了腱骨的修复和再生；同时miR-1246还促进了自身肌腱细胞的增殖和迁移。相关机制说明具体参见图9。在上述共同作用下达到了促使腱骨界面接近生理的愈合，增加了其生物力学强度的治疗效果，避免了移植体带来的整合不良、免疫排斥等不良临床效果和较高的复发率，解决了腱骨愈合不良的临床难题，并且实验过程中未出现任何毒副反应，安全性高。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (6)
1.miR-1246在制备促进腱骨愈合药物中的应用，其特征在于，所述miR-1246的序列为：AAUGGAUUUUUGGAGCAGG；
所述腱骨愈合为人体的肌腱、韧带在骨止点损伤的修复，具体为肩袖撕裂修复。
2.根据权利要求1所述应用，其特征在于，所述miR-1246促进腱骨界面愈合，提高生物力学强度。
3.根据权利要求1所述应用，其特征在于，所述miR-1246促进肌腱细胞的增殖和迁移。
4.根据权利要求1所述应用，其特征在于，所述药物还包括药学上可接受的载体。
5.根据权利要求4所述应用，其特征在于，所述载体选自脂质体、外泌体、聚合物、介孔二氧化硅纳米颗粒、量子点和金属基纳米颗粒中的一种或多种。
6.根据权利要求1所述应用，其特征在于，所述药物的剂型为注射剂或外用药剂。
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