CN115137828B - miR-302c-3p在制备治疗椎间盘退变药物中的应用 - Google Patents
miR-302c-3p在制备治疗椎间盘退变药物中的应用 Download PDFInfo
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Abstract
本发明公开了miR‑302c‑3p在制备治疗椎间盘退变药物中的应用。本发明通过转染mimics在NPCs内过表达miR‑302c‑3p,同时运用特异性miRNA inhibitor下调NPCs的miR‑302c‑3p水平,对比miR‑302c‑3p Gain/Loss对NPCs的影响,发现miR‑302c‑3p对退变椎间盘有再生激活的作用;因此上调miR‑302c‑3p表达的试剂可以用于制备治疗椎间盘退变药物。本发明发现小核酸分子miR‑302c‑3p,利用“非细胞形式”的外泌体新技术,既避开ESCs成瘤性和免疫排斥问题又利用其强大的无限增殖能力,本发明将为椎间盘退变性疾病的生物治疗提供新的选择。
Description
技术领域
本发明属于生物医学领域,具体涉及miR-302c-3p在制备治疗椎间盘退变药物中的应用。具体而言,过表达miR-302c-3p能够促进退变椎间盘中髓核细胞增殖能力和细胞外基质合成能力,可以有效维持大鼠椎间盘退变中椎间盘高度,miR-302c-3p激动剂可以作为缓解椎间盘退变的药物。
背景技术
椎间盘退变(intevertebral disc degeneration,IVDD)是一种与年龄相关的退行性疾病。目前,临床保守治疗,即对患者注射抗炎药,如类固醇、肌肉松弛剂等,虽可短期缓解背痛,间接促进自我修复,但不能改变椎间盘退变进程。而细胞疗法所移植细胞在椎间盘内低氧、低营养、低pH值、高渗透压的微环境中难以适应,因而存活率极低,是影响细胞移植治疗的主要原因。
外泌体(exosomes)是近几年干细胞研究的新突破之一。有研究表明,起修复作用的不是植入的干细胞本身,而是干细胞分泌的各种物质,外泌体是这些物质中起主导作用的成分。
最近的研究显示干细胞来源的外泌体对缺血梗死的心肌、肺、肝、肾和脑组织均有较好的修复作用。外泌体用于临床治疗显示了巨大的潜力。然而目前外泌体对退变组织修复作用的研究较少。考虑到椎间盘组织低氧酸性的“恶劣”细胞生存环境,“非细胞形式”的外泌体无疑极具临床应用优势。
目前研究表明,外泌体作为胚胎干细胞旁分泌因子,含有与其无限增殖能力相关的独特miRNAs、蛋白质和特有细胞周期活性,具有模拟干细胞特性,可以实现无细胞治疗。目前尚无胚胎干细胞来源的外泌体在椎间盘退行性病变治疗药物中的应用研究。
外泌体的生物学活性由其来源细胞的生物学特性决定,现有外泌体主要来自于间充质干细胞(Mesenchymal stem cells,MSCs),其所含有的各种分子(包括蛋白质,脂质,DNAs,mRNA和miRNA)与人类胚胎干细胞(embryonic stem cells,ESCs)来源的外泌体存在很大差异。
发明内容
本发明的目的在于提供miR-302c-3p在制备治疗椎间盘退变药物中的应用。
本发明的目的通过下述技术方案实现:
上调miR-302c-3p表达的试剂在制备治疗椎间盘退变药物中的应用;
本发明通过转染mimics在NPCs(nucleus pulposus cells,NPCs)内过表达miR-302c-3p,同时运用特异性miRNA inhibitor下调NPCs的miR-302c-3p水平,对比miR-302c-3p Gain/Loss对NPCs的影响,发现miR-302c-3p对退变椎间盘有再生激活的作用;
所述上调miR-302c-3p表达的试剂包括miR-302c-3p mimics、miR-302c-3pagomir或化合物类促进剂;
miR-302c-3p或其靶基因在制备治疗椎间盘退变药物中的应用;
所述miR-302c-3p的靶基因优选LATS2基因;
靶向降解LATS2(促进YAP信号的活性在髓核细胞核内表达)的试剂在制备治疗椎间盘退变药物中的应用;
所述靶向降解LATS2的试剂优选靶向降解LATS2的mRNA;
本发明通过生物信息学软件预测miR-302c-3p下游靶基因大肿瘤抑制因子(LATS2),进一步证实miR-302c-3p靶向降解LATS2通路对IVDD起到缓解作用。同时本发明建立针刺大鼠尾部致椎间盘退变模型,经X射线检测观察椎间盘高度指数,行HE、番红O固绿组织学染色及细胞外基质蛋白免疫组化进一步在整体水平上评估ESCs-exo内源性miR-302c-3p对大鼠IVDD细胞外基质变化及衰老相关标志的影响。
所述的药物还含有其他活性成分和辅料(载体);
所述的辅料(载体)优选缓释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂等;
所述药物的剂型为气雾剂、片剂、胶囊剂、滴丸、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质剂、透皮剂、口含剂、栓剂或冻干粉针剂等。
本发明相对于现有技术具有如下的优点及效果:
本发明发现ESCs-exo中逆转退变椎间盘髓核组织活性的主要有效成分——小核酸分子miR-302c-3p,利用“非细胞形式”的外泌体新技术,既避开ESCs成瘤性和免疫排斥问题又利用其强大的无限增殖能力,本发明将为椎间盘退变性疾病的生物治疗提供新的选择。此外,相比直接干细胞移植,小核酸分子的“非细胞形式”使其稳定易保存,适合GMP(good manufacturing practice,GMP)标准化生产,临床应用的可操作性更高。
附图说明
图1是miR-302c-3p的表达水平;其中,A为NPCs、ESC、ESCs-exo中miR-302c-3p表达水平;B为NPCs经过不同传代次数后的miR-302c-3p表达水平;C为NPCs经ESCs-exo处理后miR-302c-3p表达水平。
图2是NPCs中miR-302c-3p的表达水平。
图3是NPCs的衰老特异性染色(SA-β-gal染色)实验结果。
图4是EdU细胞增殖实验的荧光显示照片。
图5是EdU细胞增殖实验中EdU阳性细胞百分比。
图6是CCK8检测NPCs经不同处理后的细胞活力。
图7是通过TargetScan、miRBase、TarBase构成韦恩图预测miR-302c-3p潜在下游靶标数目。
图8是miR-302c-3p对LATS2和YAP在转录水平上的影响。
图9是miR-302c-3p对LATS2和YAP在蛋白表达水平上的影响。
图10是miR-302c-3p对LATS2和YAP在蛋白表达水平上的影响。
图11是衰老特异性染色实验(SA-β-gal染色)结果荧光照片(图片标尺为100μm)。
图12是衰老特异性染色实验中NPCs中衰老阳性细胞比例(*与Control相比,#与miR-302c NC相比,***p<0.001,#p<0.05,##p<0.01,###p<0.001)。
图13是免疫荧光检测经不同处理后NPCs中YAP入核情况(图片标尺为10μm)。
图14是Western blot检测NPCs经不同处理后相关蛋白表达水平。
图15是造模及给药后大鼠椎间盘高度变化。
图16是造模及给药后大鼠椎间盘高度指数(DHI)。
图17是大鼠尾部椎间盘HE染色、番红O-固绿染色以及p21、MMP3、YAP免疫组化的结果(图中标尺4×:500μm,40×:100μm)。
以上各图中,*p<0.05,**p<0.01,***p<0.001。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
检测ESCs-exo(人类胚胎干细胞外泌体)中miR-302c-3p的表达水平,包括以下步骤:
(1)人类胚胎干细胞(embryonic stem cells,ESCs)(由中山大学中山医学院国家干细胞重点实验室提供,人胚胎干细胞系H9)在培养过程中,在细胞融合度达到70%左右,且状态良好,无存在过多分化细胞的情况下,将细胞培养液吸出,加入无血清人多潜能性干细胞培养基(北京赛贝生物CA1014500)培养24h后,收集上清液;
(2)外泌体提取(超高速离心法):将步骤(1)收集的上清液于4℃、300×g离心10min,去除死细胞以及细胞碎片;取上清于4℃、2000×g离心10min,去除生物聚合物以及凋亡小体。取上清于4℃、10000×g,离心30min,去除大型囊泡。取上清于100000×g、4℃,离心80min。离心结束后,弃上清液,加入适量PBS轻轻重悬沉淀,集中到一个超速离心管中。确认配平后,100000×g,4℃,离心80min再次离心除去蛋白质杂质,得到纯化的ESCs-exo沉淀。弃上清,用适量PBS重悬将ESCs-exo转移到超净工作台中,用0.22μm针式过滤器过滤除菌并分装到1.5ml离心管,用BCA法对其定量;
(3)分别提取NPCs(退变髓核细胞,由椎间盘髓核组织进行组织培养获得,椎间盘髓核组织标本来自暨南大学附属第一医院接受微创治疗退行性脊椎疾病的椎间盘退变患者,髓核组织样本收集均获得暨南大学附属第一医院医学伦理委员会的批准,并由病人家属签署知情协议书)、ESCs(由中山大学中山医学院国家干细胞重点实验室提供,人胚胎干细胞系H9)和ESCs-exo(步骤2所得的ESCs-exo沉淀)的总RNA,检测A260/A280,比值处于1.8-2.1之间。
以U6为miRNA内参标准,miR-302c-3p及NC前引物由广州锐博生物科技有限公司合成。后续Poly(A)Tailing,RT-PCR步骤参照miDETECT A Track miRNA qRT-PCR StarterKit说明书(广州锐博)。
如图1A所示,miR-302c-3p在ESCS和ESCs-exo中的表达量很高,而在NPCs中表达量很低。
如图1B所示,随着NPCs复制性传代,miR-302c-3p的表达量逐渐降低。
将含有10ug/ml ESCs-exo(步骤2所得的ESCs-exo沉淀)的DMEM/F12培养基用于培养NPCs作为ESCs-exo组,对比Control组(没有添加ESCs-exo的DMEM/F12培养基),ESCs-exo组的NPCs在处理24小时后miR-302c-3p表达量显著增加(图1C),表明ESCs-exo携带的miR-302c-3p可以被NPCs摄取。
实施例2
miR-302c-3p促进NPCs增殖试验,包括以下步骤:
1.功能获得性(gain-of-function)和功能缺失性(loss-of-function)研究(小核酸过表达或敲减技术):选取处于对数生长期的NPCs,生长状态良好。将细胞接种细胞培养板上。当细胞密度达到50%左右时,开始细胞转染。
Hsa-miR-302c-3p mimics、mimic NC、hsa-miR-302c-3p inhibitor及inhibitorNC序列由广州锐博生物科技有限公司合成。
转染分组为:
空白对照组(Control);
ESCs-exo组(用10μg/mL ESCs-exo转染);
ESCs-exo+miR-302c-3p inhibitor对照组(用10μg/mL ESCs-exo+100nMinhibitor NC+转染试剂转染);
ESCs-exo+miR-302c-3p inhibitor(用10μg/mL ESCs-exo+100nM inhibitor+转染试剂转染);
miR-302c-3p mimics对照组(用50nM mimics NC+转染试剂转染);
miR-302c-3p mimics组(用50nM mimics+转染试剂转染)。
按照广州锐博生物公司的miRNA产品使用说明书进行细胞转染,以及miDETECT ATrack miRNA qRT-PCR Starter Kit说明书对转染效率进行验证。结果显示,用miR-302c-3p mimics转染NPCs 48小时后,miR-302c-3p表达水平显著增加(图2)。
2.衰老特异性染色步骤如下,选取处于对数生长期的NPCs,于6孔板中调整细胞密度为1×106/孔,每组设置3个复孔,培养箱中培养24h。吸除培养液,PBS缓冲液洗3次,加入β-半乳糖苷酶染色固定液0.5mL,固定15min。移除固定液,PBS缓冲液洗3次,每孔加入1mlβ-半乳糖苷酶染色液,封口膜四周封闭,烘箱37℃孵育过夜。显微镜下观察显示,经ESCs-exo或miR-302c-3p mimics处理24h后,衰老特异性染色阳性细胞有所减少(图3)。
3.细胞增殖及活性实验具体步骤:
3.1 EdU检测髓核细胞增殖具体步骤:挑选长势较好的NPCs细胞,用胰酶消化制成单细胞悬液,用计数板计数。96孔板按8000个/孔进行种板,根据所种孔数,提前计算好所需细胞悬液的体积和完全培养基的体积,将其充分混匀后,进行种板。种板结束以后,在显微镜下观察细胞是否种匀,于37℃、5%CO2的培养箱中进行培养,并做三个平行重复组。24h待细胞贴壁后,加入新的培养基和作用物,继续培养48h。吸掉剩余的培养基,PBS清洗遍,加入含50μM EdU的50μL培养基,37℃孵育2h。用4%多聚甲醛固定30min,去固定液,PBS洗三遍。加入50μL 2mg/mL甘氨酸溶液,室温静置5min,PBS洗三遍。加入Apollo反应混合物染色,避光孵育1h,PBS洗三遍。加入0.5%Triton X-100进行细胞打孔20min,PBS洗三遍。加入Hoechst 33342染液进行染色。荧光显微镜下进行镜检,拍照。
3.2 CCK8检测髓核细胞活性具体步骤:取长势较好的P3代细胞于超净台上,用胰酶消化成单细胞悬液,用血球计数板进行细胞计数。按8000个/孔接种于96孔板中,每孔加入200μL完全培养基,在显微镜下观察细胞是否种匀,于培养箱中培养24h。吸出原有的培养基,加入新的培养基和作用物,每组做5复孔,做好标记,置于培养箱中分别培养4天,每隔两天换液。分别在0,24,48,72,96h测其CCK8的值,在每个孔中加入10μL CCK8溶液于37℃中孵育4h,将酶标仪预热,用酶标仪在450nm波长下检测其吸光值。
EdU细胞增殖实验显示,ESCs-exo或miR-302c-3p mimics处理24h后,EdU阳性细胞明显增加(图4-图5)。
CCK8实验进一步验证了过表达miR-302c-3p mimics促进衰老NPCs的增殖能力(图6)。
实施例3
miR-302c-3p通过调控LATS2,激活YAP信号通路促进NPCs增殖
1.应用microRNAs生物信息学网站和软件,对miR-302c-3p潜在可能的靶基因进行预测,通过TargetScan、miRBase、TarBase构成韦恩图预测miR-302c-3p潜在下游靶标数目(图7)。在候选靶基因中,LATS2通过TargetScan预测其与miR-302c-3p有两处相结合位点,因为LATS2是Hippo信号通路的关键核心激酶,LATS2激活后导致其下游的转录辅助因子YAP磷酸化水平升高并且滞留在细胞质,已被证明可以调控细胞的增殖和分化,故选择LATS2作为靶基因进一步深入研究。
2.髓核细胞核蛋白及胞浆蛋白提取:按实施例2中的方法,对NPCs转染miR-302c-3p 48h后,按细胞核蛋白与细胞浆蛋白提取试剂盒(上海,碧云天生物)提取核蛋白及胞浆蛋白。
结果显示在转录水平上,过表达miR-302c-3p可明显上调YAP mRNA表达水平,而抑制miR-302c-3p可抑制YAP的mRNA表达水平,从而证实了ESCs-exo来源的miR-302c-3p通过靶向LATS2进而调节YAP的表达(图8)。
在蛋白表达水平上,过表达miR-302c-3p可明显上调核内YAP蛋白表达水平,而经ESCs-exo和miR-302c-3p inhibitor同时作用NPCs后核内YAP表达水平明显下降(图9-图10)。
以上结果进一步证实了miR-302c-3p通过靶向LATS2,调控YAP入核,从而促进NPCs增殖,缓解NPCs衰老。
3.衰老特异性染色:各组髓核细胞转染分组为:
空白对照组(Control)、ESCs-exo组(含10μg/mL ESCs-exo)、ESCs-exo+miR-302c-3p inhibitor对照组(含10μg/mL ESCs-exo+100nM inhibitor NC+转染试剂)、ESCs-exo+miR-302c-3p inhibitor(含10μg/mL ESCs-exo+100nM inhibitor+转染试剂)、miR-302c-3p mimics对照组(含50nM mimics NC+转染试剂)、miR-302c-3p mimics组(含50nM mimics+转染试剂)、ESCs-exo+VP组(含10μg/mL ESCs-exo+0.5μM维替泊芬)、VP组(含0.5μM维替泊芬)。
经特异性衰老细胞染色发现,ESCs-exo处理或过表达miR-302c-3p后,衰老特异性染色阳性细胞明显减少,而同时使用ESCs-exo+miR-302c-3p inhibitor或ESCs-exo+VP组,衰老特异性染色阳性细胞显著增加(图11-图12)。
4.YAP免疫荧光步骤:按上述转染方法处理髓核细胞48h后,弃培养液,PBS洗3遍后用加入100μL 4%多聚甲醛固定髓核细胞15min。PBS洗3遍,每次3min。使用含0.5%TritonX-10的PBS,室温通透细胞20min,PBS洗涤3遍。使用即用型山羊血清室温封闭30min。弃封闭液,按1:100稀释YAP一抗(Novus),在4℃冰箱孵育过夜。次日,用PBS洗涤2遍。加入1:200稀释比例的荧光二抗,室温避光孵育1h。PBS洗涤3遍,加入即用型DAPI染液避光染色10min。PBS洗涤3遍,用防荧光淬灭剂封片,荧光显微镜下镜检采集图像。对经过不同处理的NPCs进行YAP免疫荧光染色,结果可见,经ESCs-exo处理或过表达miR-302c-3p后,YAP入核明显增加,而同时使用ESCs-exo+miR-302c-3p inhibitor或ESCs-exo+VP组,YAP滞留在细胞质中(图13)。
5.蛋白印迹结果显示,对比Control组,经ESCs-exo处理或过表达miR-302c-3p后,NPCs中核YAP蛋白表达水平显著性增加,重点突出的是过表达miR-302c-3p组,衰老相关分泌因子p21,MMP3,ADAMTS4,以及靶基因LATS2蛋白水平都有明显的下降,而使用ESCs-exo+miR-302c-3p inhibitor或ESCs-exo+VP处理NPCs,则作用效果相反(图14)。
以上结果表明,ESCs来源miR-302c-3p靶向LATS2,通过促进YAP入核,进而上调YAP信号活性,促进NPCs增殖,缓解NP衰老。
实施例4
miR-302c-3p在大鼠椎间盘退变模型中的作用
1.大鼠尾部椎间盘退变模型的具体造模方法:选取12周龄SD大鼠(北京华阜康),根据大鼠体重腹腔注射一定量的戊巴比妥钠,待大鼠充分麻醉后,将大鼠俯卧位放置于手术单上,尾巴轻轻拉直,75%酒精消毒后,先通过手触诊定位,即大鼠尾部靠近坐骨尾椎的第一节椎间盘,记号笔标记定位尾椎椎间盘4-5,5-6,6-7,7-8共4个椎间盘,碘伏消毒。用手术刀避开血管划开皮肤,在体视显微镜下,钝性分离筋膜,暴露出椎间盘。选用21G穿刺针,套筒控制针头长度为5mm。垂直穿刺椎间盘,依次穿刺纤维环、髓核,深度5mm,停顿30s,旋转出针。使用4-0手术缝合线缝合尾部皮肤,碘伏消毒,依次对尾部椎间盘进行穿刺。术后,每天观察大鼠活动、饮食情况,观察穿刺针眼处有无红肿、溃疡、化脓等,若出现感染症状则淘汰该大鼠。在术后2周,触诊定位尾椎椎间盘,用34G针头在4-5,5-6,6-7,7-8处分别注射PBS(2μL per disc)、ESCs-exo(2μg in 2μL PBS per disc)、ESCs-exo+miR-302c-3pantagomir(2μg and 5nmol in 2μL PBS per disc)和miR-302c-3p agomir(5nmol in 2μLPBS per disc)。
miR-302c-3p agomir/antagomir(广州锐博)是特殊化学修饰的miRNA激动剂/拮抗剂,分别通过模拟miR-302c-3p的作用和竞争性抑制/特异性降解内源miRNA的作用而进行功能获得性(gain-of-function)和功能缺失性(loss-of-function)研究。在造模当日和注射后2、4、6周进行影像学观察大鼠椎间盘高度变化,并分别对上述不同批次大鼠进行安乐死,然后取材进行组织学分析。
2.X射线观察大鼠椎间盘高度变化具体步骤:在造模当日和注射后2、4、6周分别使用小动物X射线数字成像系统对大鼠尾椎进行拍摄,戊巴比妥钠麻醉大鼠,将大鼠俯卧位垂直投射成像,尾巴自然拉直,拍摄部位包含腰椎和尾椎。大鼠尾椎3-9椎间盘相对高度测量方法:将椎间盘宽度分成三等分,测量中点及其两侧点相邻椎体及椎间盘的高度,计算椎间盘高度指数(Disc height index%,DHI%)=椎间盘高度平均值/椎体高度平均值×100。
结果显示,miR-302c-3p agomir和ESCs-exo组注射后2周,与PBS组相比,椎间盘高度有所回升,但无法恢复到穿刺前水平,具有统计学差异。相反,ESCs-exo+miR-302c-3pantagomir组DHI%虽然有一定程度上升但无统计学差异(图15-图16),表明ESCs-exo和miR-302c-3p agomir能一定程度上缓解大鼠尾部椎间盘退变。以上结果表明ESCs-exo来源miR-302c-3p能缓解大鼠IVD退变,而抑制ESCs-exo中miR-302c-3p的表达则逆转了ESCs-exo的治疗效果。
3.大鼠尾部椎间盘切片制作具体步骤如下:在大鼠安乐死后,骨剪取下大鼠尾椎3-9节椎间盘,剔去皮肤和四周肌肉,于4%多聚甲醛固定液室温固定48h。放入8%盐酸甲醛脱钙液中,每隔1h用针头穿刺椎体,直至针头能够轻易穿过椎体骨头即可终止脱钙,流水过夜。按照髓核组织石蜡包埋方法进行常规乙醇梯度脱水,透明,浸蜡,包埋和切片,其时间适度延长。通过组织学染色HE、番红O-固绿染色及免疫组化,结果显示,Control组椎间盘髓核完好,PBS组和ESCs-exo+miR-302c-3p antagomir组的NP组织萎缩,形状不规则,AF与NP界限模糊,AF纤维片层呈蛇形状,中央NPCs骤减。相反,注射ESCs-exo或miR-302c-3p agomir组可显著减轻椎间盘的形态恶化。番红O着色与阴离子的浓度近似成正比关系,可间接反映基质中蛋白多糖的含量和分布。
对比Control组,PBS组和ESCs-exo+miR-302c-3p antagomir组中,细胞外基质成分发生明显变化,椎间盘中蛋白聚糖明显减少,纤维化加重。而注射ESCs-exo或miR-302c-3p agomir细胞外基质成分未发生较大改变。免疫组织化学染色显示,ESCs-exo组和miR-302c-3p agomir组的p21阳性细胞和MMP3阳性面积明显小于PBS组,相反地,YAP阳性面积明显增加,表明受损椎间盘的自我修复(图17)。这些结果表明,椎间盘退变大鼠模型体内注射miR-302c-3p agomir后,可以有效维持椎间盘高度,同时椎间盘内髓核细胞数量及增殖能力显著增强的同时伴随YAP核信号的上调。
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 (4)
1.上调miR-302c-3p表达的试剂在制备治疗椎间盘退变药物中的应用;
所述上调miR-302c-3p表达的试剂包括miR-302c-3p mimics,其核苷酸序列为:
5-uaagugcuuccauguuucagugg-3;
3-auucacgaagguacaaagucacc-5。
2.根据权利要求1所述的应用,其特征在于:所述的药物还含有其他活性成分和辅料。
3.根据权利要求2所述的应用,其特征在于:所述的辅料为缓释剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂。
4.根据权利要求1所述的应用,其特征在于:所述药物的剂型为气雾剂、片剂、胶囊剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质剂、口含剂、栓剂针剂。
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