CN114632153A - Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途 - Google Patents
Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途 Download PDFInfo
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Abstract
本发明涉及异位骨化治疗产品领域,特别是涉及Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途,所述Hedgehog信号通路抑制剂包括SMO抑制剂、Hh蛋白抑制剂、Gli抑制剂,所述Gli抑制剂包括Gli1抑制剂、Gli2抑制剂和Gli3抑制剂。本发明的Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途为临床上异位骨化的治疗提供潜在靶标,Hedgehog信号通路抑制剂例如JQ1可能作为治疗异位骨化的潜在药物,尤其是治疗损伤引起的跟腱异位骨化的药物。
Description
技术领域
本发明涉及异位骨化治疗产品领域,特别是涉及Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途。
背景技术
异位骨化(Heterotopic ossification,HO)是一种在肌肉、肌腱等软组织形成异位骨的病理过程。异位骨化常发生于损伤(如骨折,脊髓损伤)和一些遗传性疾病(如进行性肌肉骨化症等)过程中。异位骨化分为获得性异位骨化和遗传性异位骨化。获得性异位骨化主要发生于深部烧伤、骨折、全髋关节置换术等引起的创伤以及脊髓损伤。遗传性异位骨化是一些罕见遗传病的典型症状,例如进行性肌肉骨化症(FOP)和进行性骨异型增生症(POH)。研究发现,14%的肱骨骨折会形成明显的异位骨化,20%-30%的脊髓损伤会发生异位骨化。异位骨化的形成可导致肌肉、肌腱韧带的功能丧失,给患者带来疼痛并破坏运动系统功能,进而大大降低患者的运动能力和生活质量。异位骨化的机制尚不清楚,临床上也没有有效药物治疗,一些患者主要以手术去除为主,但存在易复发的问题。
肌腱和韧带会发生异位骨化主要是由于包含祖细胞。至今,各种类型的祖细胞已被发现参与异位骨化的形成,包括由肌腱来源祖细胞(Tendon-derived progenitorcells,TDPCs)、循环来源的祖细胞和外周神经的祖细胞(6-8)。肌腱祖细胞的体外研究较多。Bi Yanming等鉴定发现肌腱祖细胞表达Sca1,CD90.2和CD44等表面标志物。此外,以Tie2-/CD45-/Thy1-/6C3-AlphaV+/CD105+为特征的软骨基质祖细胞(BCSP)被发现参与异位骨化的形成。先前的研究表明,Scleraxis(Scx)标记的肌腱祖细胞参与了异位骨化过程中软骨细胞和成骨细胞的产生。然而,肌腱祖细胞在体内的研究依旧有限,异位骨化发生的细胞起源和机制也并不清楚。
刺猬(Hedgehog,Hh)信号在软骨和骨骼发育中起着至关重要的作用,Hh信号的失调导致许多骨疾病,例如骨肿瘤、骨关节炎等。Suppressor of Fused(Sufu)是Hh信号的主要负调节子,其在细胞质中隔离全长Gli蛋白以限制其活性,而Sufu的失活导致严重的异位Hh 途径活化(图1)。进行性骨异型增生症(POH)主要是由于过度激活的Hh信号足以诱导软组织通过膜内骨化异位成骨。但Hh的激活对于肌腱异位骨化中细胞的命运调控仍不清楚。
寻找治疗异位骨化的药物迫在眉睫。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途,用于解决现有技术中的问题。
为实现上述目的及其他相关目的,本发明提供Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途。
如上所述,本发明的Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途,具有以下有益效果:Hedgehog信号通路抑制剂例如JQ1可能作为治疗异位骨化的潜在药物,尤其是治疗损伤引起的跟腱异位骨化,本发明为临床上异位骨化的治疗提供潜在靶标。
附图说明
图1显示为经典Hedgehog信号通路的过程示意图;左图为Hh信号通路处于未激活状态;右图为激活路径。
图2显示为验证Ctsk+Scx+细胞是肌腱祖细胞的数据,各图说明如下:
A.Ctsk-Cre;Rosa26-Ai9;ScxGFP小鼠跟腱的荧光图像,红色是Ctsk标记的细胞,绿色是Scx标记的细胞。
B.流式细胞术反映四种细胞亚群Ctsk-Scx-,Ctsk-Scx+,Ctsk+Scx-和Ctsk+Scx+的比例。
C.流式细胞术反映四种细胞亚群Ctsk-Scx-,Ctsk-Scx+,Ctsk+Scx-和Ctsk+Scx+表达祖细胞标志物的比例。
D.四种细胞亚群Ctsk-Scx-,Ctsk-Scx+,Ctsk+Scx-和Ctsk+Scx+的集落形成能力测定。
E.四种细胞亚群Ctsk-Scx-,Ctsk-Scx+,Ctsk+Scx-和Ctsk+Scx+的集落形成能力测定的定量。
F.Ctsk+Scx+细胞在有/无TGFb3培养条件下的荧光图片。
G.Ctsk+Scx+细胞显示比Ctsk+Scx-细胞更强的三向分化能力。
图3显示为Ctsk-Cre表达细胞中条件性敲除Sufu会诱导自发性的韧带,肌腱和关节异位骨化,各图说明如下:
A.Ctsk-Cre;Sufufl/fl(Ctsk-CKO)小鼠的构建策略。
B.Ctsk-Cre;Sufufl/+(Ctsk-Ctrl)小鼠的CT图像。
C.4周龄,9周龄和20周龄Ctsk-Ctrl小鼠和Ctsk-CKO小鼠后肢腿骨的X-ray和CT图像。
D.4周龄,9周龄和20周龄Ctsk-Ctrl小鼠和Ctsk-CKO小鼠后肢跟骨的X-ray和CT图像。
E.40周龄Ctsk-Ctrl小鼠和Ctsk-CKO小鼠肱骨的CT图像。
F.20周龄后肢腿骨的番红O/固绿染色。
G.20周龄后肢腿骨的苏木精&伊红,番红O/固绿,二型胶原(COLII)和骨桥蛋白(OPN) 的免疫荧光染色。
图4显示为肌腱和韧带中的Ctsk-Cre表达细胞参与异位骨化的形成,各图说明如下:
A.6周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱的COLII和OPN的免疫荧光图片。
B.流式分选6周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞,并用Qrt-PCR检测Hedgehog通路标志物的表达情况。
C.流式分选6周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞,并用Qrt-PCR检测软骨分化标志物的表达情况。
D.流式分选6周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞,并用Qrt-PCR检测成骨分化标志物的表达情况。
E.流式分选6周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞,并用Qrt-PCR检测肌腱分化标志物的表达情况。
F.4,5,6周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱的COLII和OPN 的免疫荧光图片。
G.6周龄Ctsk-Ctrl;Rosa26-Ai9;ScxGFP和Ctsk-CKO;Rosa26-Ai9;ScxGFP小鼠跟腱的 COLII的免疫荧光图片。
H.6周龄Ctsk-Ctrl;Rosa26-Ai9;ScxGFP和Ctsk-CKO;Rosa26-Ai9;ScxGFP小鼠跟腱的 Aggrecan的免疫荧光图片。
I.20周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱的H型血管的免疫荧光图片。
J.20周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱的H型血管的定量统计。
图5显示为Sufu缺失的肌腱来源的细胞呈现增强的软骨和成骨分化能力,各图说明如下:
A.流式分选4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞并进行成软骨分化,结果用阿尔新蓝染色显示分化程度。
B.流式分选4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞并进行成软骨分化,并用RT-qPCR检测软骨分化标志物的表达情况。
C.流式分选4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞并进行成骨分化,分别进行ALP和茜素红染色显示分化程度
D.流式分选4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞并进行成骨分化后上清ALP的活性定量。
E.流式分选4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞并进行成骨分化,并用RT-qPCR检测Hh通路标志物的表达情况。
F.流式分选4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞并进行成骨分化,并用RT-qPCR检测成骨分化标志物的表达情况
G.4周龄Ctsk-Ctrl;Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠跟腱TUNEL染色。
图6显示为Gli1/Gli2的敲除抑制Ctsk-CKO小鼠的异位骨化的发生,各图说明如下:
A.蛋白质印记实验检测Gli1和Gli2的在Ctsk-CKO;Gli1lacz/lacz和Ctsk-CKO;Gli2fl/fl双敲除小鼠跟腱的敲除效率。
B.20周龄Ctsk-Ctrl小鼠,Gli1lacz/lacz小鼠,Ctsk-Cre;Gli2fl/fl小鼠,Ctsk-CKO小鼠,Ctsk-CKO; Gli1lacz/lacz小鼠,and Ctsk-CKO;Gli2fl/fl小鼠的后腿的X-ray和CT图像。
C.20周龄Ctsk-Ctrl小鼠,Gli1lacz/lacz小鼠,Ctsk-Cre;Gli2fl/fl小鼠,Ctsk-CKO小鼠,Ctsk-CKO;Gli1lacz/lacz小鼠,and Ctsk-CKO;Gli2fl/fl小鼠的跟腱的X-ray和CT图像。
D.20周龄Ctsk-Ctrl小鼠,Gli1lacz/lacz小鼠,Ctsk-Cre;Gli2fl/fl小鼠,Ctsk-CKO小鼠, Ctsk-CKO;Gli1lacz/lacz小鼠,and Ctsk-CKO;Gli2fl/fl小鼠的后腿切片的番红O/固绿染色。
E.20周龄Ctsk-Ctrl小鼠,Gli1lacz/lacz小鼠,Ctsk-Cre;Gli2fl/fl小鼠,Ctsk-CKO小鼠,Ctsk-CKO; Gli1lacz/lacz小鼠,and Ctsk-CKO;Gli2fl/fl小鼠的跟腱切片的番红O/固绿染色和OCN染色。
图7显示为小分子抑制剂JQ1可通过抑制Hh信号来改善Ctsk-CKO小鼠的异位骨化进程,各图说明如下:
A.阿尔新蓝染色显示JQ1抑制分选的4周龄Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞的成软骨分化。
B.RT-qPCR结果显示JQ1抑制分选的4周龄Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞的成软骨分化。
C.ALP染色和茜素红染色显示JQ1抑制分选的4周龄Ctsk-CKO;Rosa26-Ai9小鼠跟腱 Ctsk+细胞的成骨分化。
D.RT-qPCR结果显示JQ1抑制分选的4周龄Ctsk-CKO;Rosa26-Ai9小鼠跟腱Ctsk+细胞的成骨分化。
E.JQ1与对照试剂处理4周龄Ctsk-Ctrl和Ctsk-CKO小鼠3周后的CT图片。
F.JQ1与对照试剂处理4周龄Ctsk-Ctrl和Ctsk-CKO小鼠3周后的异位骨体积的定量。
G.JQ1与对照试剂处理4周龄Ctsk-Ctrl和Ctsk-CKO小鼠3周后的后腿切片的番红O/固绿染色。
H.JQ1与对照试剂处理4周龄Ctsk-Ctrl和Ctsk-CKO小鼠3周后的跟腱切片的番红O/ 固绿染色。
图8显示为Hedgehog信号通路抑制剂JQ1可以有效缓解损伤导致的异位骨化,各图说明如下:
A.JQ1与安慰剂处理肌腱损伤后的野生型小鼠的CT图片
B.JQ1与安慰剂处理肌腱损伤后的野生型小鼠的肌腱异位骨化的骨体积定量结果。
C.JQ1与安慰剂处理肌腱损伤后的野生型小鼠跟腱的OPN,OCN,Aggrecan的免疫荧光染色。
具体实施方式
本发明首先提供Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途。
如图1所示,左图为在没有配体的情况下,Hedgehog(简写为Hh)信号通路处于未激活状态,跨膜蛋白受体Patched(Ptch)会抑制七次跨膜蛋白Smoothened(Smo)。转录因子Gli与融合蛋白和融合抑制蛋白(Sufu)的相互作用而被阻止进入细胞核。Hh靶标基因的转录激活被抑制。右图的激活路径通过结合三种哺乳动物配体而启动。配体结合Ptch导致Smo 的抑制,从而激活级联反应,从而导致转录因子Gli与Sufu解离并进入细胞核。核Gli激活靶基因表达,包括Ptch和Gli本身,以及Hip。
所述Hh信号通路抑制剂包括SMO抑制剂、Hh蛋白抑制剂、Gli抑制剂。所述Gli抑制剂包括Gli1抑制剂、Gli2抑制剂和Gli3抑制剂。在一种实施方式中,所述Gli抑制剂选自JQ1,化学式为C23H25ClN4O2S。JQ1是靶向BRD4的小分子抑制剂,可抑制Gli1和Gli2的转录。
具体的,所述Hh信号通路抑制剂选自vismodegib、环杷明(cyclopamine)、sonidegib (LDE225)、BMS-833923、PF04449913、LEQ506、TAK-441、LY2940680、Robotnikinin、 GANT58、HIP-1、HIP-2、HIP-3、HIP-4、三氧化二砷中的一种或几种。
Gli蛋白是分子量较大的多功能转录因子(1000个氨基酸以上),它的家族成员只有在维持全长时才具有转录激活子的功能,启动下游靶基因的转录;当羧基端被蛋白酶水解后就形成了转录抑制子,抑制下游靶基因的转录。
异位骨化(Heterotopic ossification,HO)是一种在肌肉、肌腱等软组织形成异位骨的病理过程。异位骨化包括获得性异位骨化和遗传性异位骨化。
所述异位骨化包括肌肉异位骨化、肌腱异位骨化、韧带异位骨化、关节囊异位骨化、关节软骨异位骨化。所述肌腱异位骨化包括跟腱异位骨化。
所述异位骨化产品包括肌肉异位骨化产品、肌腱异位骨化产品、韧带异位骨化产品、关节囊异位骨化产品、关节软骨异位骨化产品。所述肌腱异位骨化产品包括跟腱异位骨化产品。
在一种实施方式中,所述治疗异位骨化产品为治疗损伤引起的异位骨化产品。具体的,为治疗跟腱损伤引起的异位骨化产品。
所述异位骨化产品通过抑制Ctsk-Cre标记的肌腱祖细胞中的Hh信号通路治疗异位骨化。
所述异位骨化产品通过抑制Ctsk和Scx双阳细胞(Ctsk+Scx+细胞)中的Hh信号通路治疗异位骨化。
所述产品必然包括Hh信号通路抑制剂,并以Hh信号通路抑制剂作为前述功效的有效成分。
所述产品中,发挥前述功用的有效成分可仅为Hh信号通路抑制剂,亦可包含其他可起到前述功用的分子。
亦即,Hh信号通路抑制剂为所述产品的唯一有效成分或有效成分之一。
所述产品可以为单成分物质,亦可为多成分物质。
所述产品的形式无特殊限制,可以为固体、液体、凝胶、半流质、气雾等各种物质形式。
所述产品主要针对的对象为哺乳动物。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。
所述产品包括但不限于药物、保健品、食品等。
所述Hh信号通路抑制剂可以为核酸分子、抗体、小分子化合物。其中,所述核酸分子可以是双链RNA或shRNA。
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围;在本发明说明书和权利要求书中,除非文中另外明确指出,单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实验材料和方法
1.实验动物
所有分析的小鼠是129/Sv背景。在所有分析中均使用了性别匹配的同窝小鼠对照。所有小鼠饲养并维持在无特定病原体(SPF)的条件下。
2.影像学评估。
用二氧化碳对所有小鼠进行安乐死,并将后肢或整个身体保持在70%的乙醇中。使用Faxitron MX-20对骨骼进行X射线图像分析。对于microCT分析,将同年龄和性别分离的后肢或整个身体固定在70%的乙醇中,并使用SkyScan1272(Bruker,Kartuizersweg,比利时)以18μm 的分辨率进行扫描。然后以相同的阈值执行3D重建。
3.组织学分析。
石蜡切片:将股骨,胫骨和跟腱在4℃的预冷4%PFA中固定48小时,并在10%EDTA(pH 7.5)中脱钙。PBS洗涤后,将样品分别用70%,95%和100%的乙醇脱水,在二甲苯浸没后用石蜡包埋,切成7μm厚的切片。切片按照标准染色步骤用苏木精和伊红(H&E)和番红O /固绿(SOFG)进行染色。
冷冻切片:将股骨,胫骨和跟腱在4℃的预冷4%PFA中固定48小时。在10%EDTA中完全脱钙后,将其在30%蔗糖中脱水过夜,然后用冰冻切片包埋剂OCT进行包埋,并使用Leica CM3050 S冷冻切片机以14μm的厚度切片。
4.免疫荧光。
将冷冻的切片风干并用PBS再水化,在室温下用含10%马血清和0.2%Triton X-100的PBS 封闭并透化1小时。然后用如下一抗:山羊抗OPN(R&D,AF808,1:400),山羊抗CD31(R&D,AF3628,1:200),小鼠抗COL2(Abcam,ab185430,1:200),兔抗TRAP(Abcam, ab185716,1:200),兔抗骨钙蛋白OCN(Abcam,ab93876,1:200),兔抗Col10a1(Abclonal, a6889,1:200),兔抗聚集蛋白聚糖Aggrecan(Millipore,ab1031x,1:100),大鼠抗EMCN (SantaCruz,sc-65495,1:200)在4℃下孵育过夜。在用PBS洗涤后,将荧光标记的二抗 (1:1000)在室温避光下孵育1小时。细胞核用DAPI(Sigma,D9542)染色。使用荧光封片剂(Dako,S3023)进行封片。用Olympus FV1200共聚焦显微镜进行成像。
5.细胞分选和培养。
从4周龄小鼠的跟腱分离出原代肌腱细胞。剥离腱鞘后,将肌腱切成小段,并在37℃下用胶原酶II(1.5mg/ml,Sigma)和分散酶II(2mg/ml,Roche)消化30分钟。收集消化液,并以1200rpm离心5分钟。将分离的细胞在含有10%FBS的α-MEM(Corning)的24孔板中培养。然后通过Aria SORP对荧光标记的肌腱来源的细胞进行分选。
6.荧光辅助细胞分选(FACS)。
消化获取Ctsk-Cre;Rosa26-Ai9;ScxGFP小鼠的跟腱细胞,用RBC裂解缓冲液(Beyotime, C3702)去除获得的肌腱来源细胞中的红细胞之后,将获得的肌腱来源细胞用APC抗小鼠 CD24(Biolegend,138505),APC抗小鼠Nestin(Biolegend,655108)染色),APC抗CD44 (Biolegend,559250),APC抗小鼠CD200(Biolegend,123809),PE/Cy7抗小鼠CD105(Biolegend,120409)和APC抗Sca1(eBioscience,17-5981-81)进行冰上孵育30min。PBS 洗涤后,使用流式细胞仪Beckman CytoFlex FCM进行分析。
7.集落形成和体外多能分化实验。
为了进行集落形成实验,从Ctsk-Cre;Rosa26-Ai9;ScxGFP小鼠的跟腱中分选Ctsk-Scx-, Ctsk-Scx+,Ctsk+Scx-和Ctsk+Scx+四群细胞并培养7天。之后用4%PFA固定后,用结晶紫进行染色后统计。
对于成骨分化,简要地说,每孔将大约2x104细胞铺在96孔板,培养液使用的是添加了10% FBS和1%青霉素/链霉素(Thermo Fisher Scientific)的α-MEM(Corning)培养液。12小时后,将培养基换为成骨分化培养基,其包括50μg/ml的抗坏血酸(Sigma-Aldrich)和1mg/ml 的甘油磷酸酯(Sigma-Aldrich)。每2天更换一次培养基。诱导21天后,通过茜素红S染色确定成骨分化。
对于脂肪分化,分化培养基包含溶液A和溶液B。溶液A包含具有10%FBS的α-MEM,50mM 地塞米松(Sigma-Aldrich),100nM罗格列酮(Sigma-Aldrich),500nM 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma-Aldrich),10mg/ml胰岛素(Sigma-Aldrich)和1%青霉素/链霉素(Sigma-Aldrich)。溶液B包含具有10%FBS的α-MEM,10mg/ml胰岛素(Sigma-Aldrich) 和1%青霉素/链霉素(Sigma-Aldrich)。AB液分别进行诱导,一天一换,7天后,通过0.5%油红O染色(Sigma-Aldrich)测定成脂分化能力。
对于软骨分化,收集细胞并将其重悬于含有10%FBS和1%青霉素/链霉素的α-MEM中。将含有2.5x105细胞的液滴(15μl)小心地放置在24孔板每个孔的中间。细胞在37℃粘附2-4 小时后,加入500ml软骨形成培养基,包括1%胰岛素-转铁蛋白-硒溶液(ITS,Sigma-Aldrich), 10ng/mlTGF-β3(Peprotech),100nM地塞米松(添加Sigma-Aldrich),40μg/ml脯氨酸 (Sigma-Aldrich),50μg/ml 1-抗坏血酸2-磷酸酯(Sigma-Aldrich)和1mM丙酮酸钠(Thermo Fisher Scientific)。每2天更换一次培养基。在第10天,分化后细胞进行AlcianBlue染色。
8.ALP活性定量。
为了定量分析相对ALP活性,将7天后培养的细胞与alamarBlue(Thermo FisherScientific) 在37℃下孵育4小时,并使用EnVision(PerkinElmer)在580nm下测定细胞性。然后除去上清液,将细胞与含有6.5mM Na2CO3、18.5mM NaHCO3、2mM MgCl2和1mg/ml磷酸酶底物(Sigma-Aldrich)的ALP底物溶液温育30分钟,并在405nm用EnVision测定ALP活性。
9.TUNEL。
如Promega所述进行用于凋亡测试的TUNEL测定。
10.实时RT-PCR分析。
使用TRIzol(Sigma)制备总RNA,并用PrimeScriptTMRT试剂盒(TaKaRa)反转录为cDNA。使用Bio-Rad CFX96系统进行实时定量PCR。
11.蛋白质印迹。
在含有蛋白酶抑制剂混合物(MCE,HY-K0010)的冷裂解缓冲液中对小鼠跟腱进行匀浆。通过SDS-PAGE分离蛋白质并转移至PVDF膜。用1x TBST缓冲液配制的5%脱脂奶粉(Sangon Biotech,A600669)封闭后,将膜与如下一抗体4℃过夜孵育:Gli1(1:1000,CST,2534S), Gli2(Abcam,ab26056)。然后,将膜与偶联HRP的二抗-抗兔IgG(1:5000,Dako,P0217) 在室温下孵育1小时。使用ECL试剂(Millipore,345818)检测信号。
12.JQ1处理。
对于体外治疗,将JQ1(MCE,HY-13030)溶解在DMSO中,并使用梯度浓度处理细胞。对于Ctsk-CKO小鼠的体内治疗,对四周大的Ctsk-CKO小鼠和同窝对照,或每天腹膜内注射50mg/kg JQ1和对照试剂。3周后,我们对这些后肢进行了放射学评估和组织学分析。对于跟腱损伤的小鼠的给药,首先对10周龄小鼠的跟腱进行跟腱切断手术,然后缝合皮肤,2周后开始进行腹膜内注射50mg/kg,持续6周,然后收样。
实施例1 Ctsk+Scx+细胞是肌腱祖细胞
使用Ai9报告基因小鼠进行了谱系追踪研究,构建了Ctsk-Cre;Rosa26-Ai9小鼠,以红色荧光标记所有Ctsk谱系细胞。肌腱细胞表达Scleraxis(Scx),将Ctsk-Cre;Rosa26-Ai9 小鼠与ScxGFP小鼠(表达Scx的细胞带绿色荧光)杂交获得Ctsk-Cre;Rosa26-Ai9;ScxGFP 小鼠,发现部分Ctsk-Cre阳性细胞表达Scx(图2,A)。然后,通过FACS分析确定四个不同的细胞亚群:Ctsk-Scx-(84.9%±14.3%),Ctsk-Scx+(2.1%±1%),Ctsk+Scx-(10.2%±1.6%)和Ctsk+Scx+(2.9%±1.9%)(图2,B)。此外,Ctsk+Scx+细胞显示富含肌腱祖细胞标志物CD44,CD105,Nestin和Sca1,以及其它已知的干/祖细胞表面标志物,如CD24 和CD200(图2,C)。
体外形成集落被认为是祖细胞特征之一。为了分析四个细胞亚群的集落形成能力,分离并培养Ctsk-Scx-,Ctsk+Scx-,Ctsk-Scx+和Ctsk+Scx+四群细胞。到培养第7天时,Ctsk+Scx+ 细胞显示出比其它细胞高得多的集落形成效率(图2,D和E)。结果发现TGFβ配体3(TGF β3)可以在体外培养Ctsk+Scx+细胞中维持Scx的表达(图2,F)。接下来,比较四个细胞亚群在成骨,成脂肪和成软骨方面的多能分化潜能。Ctsk-Scx-或Ctsk-Scx+细胞均未在体外显示多能分化潜能。Ctsk+Scx+肌腱来源的细胞显示出比Ctsk+Scx-肌腱来源的细胞更高的分化为所有三个谱系的能力(图2,G)。综上所述,这些数据表明Ctsk+Scx+细胞是肌腱祖细胞。
实施例2 Ctsk-Cre表达细胞中Sufu缺失会诱导自发性的韧带,肌腱和关节异位骨化
为了探究Hh的激活是否能导致异位骨化,将Sufufl/fl小鼠与在内源性Ctsk启动子控制下表达Cre的小鼠杂交(图3,A)。Ctsk-Cre;Sufufl/+小鼠(此后称为Ctsk-Ctrl)显示出正常的骨骼特征(图3,B)。从4周开始,Ctsk-Cre;Sufufl/fl(此后称为Ctsk-CKO)小鼠开始出现行动不便的问题。4周,9周和20周龄Ctsk-CKO小鼠后肢的X射线/μ-CT射线照片显示了自发性和进行性关节周围韧带和肌腱骨化(图3,C)。通过μ-CT分析在20周龄Ctsk-CKO小鼠的后肢跟腱中检测到异位骨化(图3,D)。此外,在40周大的Ctsk-CKO小鼠的肱骨周发现了异位骨(图3,E)。在组织学上,番红O/固绿(SOFG)染色显示关节周异位骨化(图 3,F)。SOFG染色和II型胶原蛋白(COLII)的免疫荧光染色显示,来自20周龄Ctsk-CKO 小鼠的跟腱中部有软骨细胞(图3,G)。H&E染色和骨桥蛋白(OPN)染色显示跟腱处有成骨细胞(图3,G)。这些结果表明,表达Ctsk-Cre的细胞中的Sufu缺失引起自发性关节周围,韧带和肌腱骨化。
实施例3肌腱和韧带中的Ctsk-Cre表达细胞参与异位骨化的形成
为了确定Ctsk-CKO小鼠中异位骨化的细胞是来自Ctsk-Cre阳性细胞,交配产生Ctsk-Ctrl; Rosa26-Ai9和Ctsk-CKO;Rosa26-Ai9小鼠。与Ctsk-Ctrl;Rosa26-Ai9小鼠相比,发现来自6 周龄Ctsk-CKO;Rosa26-Ai9小鼠的跟腱的Ctsk+(Ai9+)细胞中的软骨形成标志物-II型胶原蛋白(COLII)和成骨标志物-骨桥蛋白(OPN)的表达水平增加(图4,A)。通过荧光激活细胞分选术(FACS)在Ctsk-CKO;Rosa26-Ai9跟腱中分离了Ctsk+细胞,发现其分选的Ctsk+ 细胞中Sufu的表达明显低于对照组Ctsk-Ctrl;Rosa26 Ai9小鼠(图4,B)。并且Ctsk-CKO中 Hh靶基因Gli1和Ptch1的表达水平显著上调(图4,B)。Ctsk-CKO;Rosa26-Ai9小鼠中的 Ctsk+细胞显示出上调的软骨生成标志物(Sox9,Col2a1,Aggrecan)和成骨标志物(Alp, Ocn,Opn)(图4,C和D)。此外,Ctsk-CKO;Rosa26-Ai9跟腱的Ctsk+细胞显示与肌腱相关的基因Scx,Mkx和Tnmd的表达下调(图4,E)。重要的是,对4、5和6周龄Ctsk-CKO 的COLII和OPN免疫染色表明:HO起始于肌腱中部(图4,F)。Ctsk-CKO中COLII+细胞呈现典型的连续的肌腱细胞的排列,这一事实表明肌腱细胞发生了细胞命运的内在变化(图 4,A)。为了进一步证实,交配Ctsk-CKO;Rosa26-Ai9小鼠与ScxGFP小鼠。表达肌腱标记 ScxGFP和软骨标记-COLII/Aggrecan的细胞出现在跟腱中段(图4,G和H)。由于 CD31highEmcnhigh(H型)血管的形成与新骨的形成相关,在20周大的Ctsk-CKO小鼠跟腱中段H型血管显著增加(图4,I和J)。综上所述,这些数据表明表达Ctsk-Cre的肌腱祖细胞参与了肌腱异位骨化的形成。
实施例4 Sufu缺失的肌腱来源的细胞呈现增强的软骨和成骨分化能力
检测Sufu是否对肌腱祖细胞的分化至关重要。从4周龄Ctsk-Ctrl;Rosa26-Ai9小鼠和 Ctsk-CKO;Rosa-Ai9小鼠的跟腱中分离并培养了Ctsk+细胞。Ctsk-CKO;Rosa-Ai9小鼠跟腱的 Ctsk+细胞显示出增强的软骨分化能力(图5,A)。RT-qPCR显示,在Sufu缺失的肌腱细胞中,软骨生成基因(Sox9,Col2a1,Aggrecan)的表达高于对照组细胞(图5,B)。来自4 周龄Ctsk-CKO;Rosa-Ai9小鼠的肌腱来源细胞在培养7天后比对照组细胞显示出增强的ALP染色和ALP活性(图5,C和D)。培养14天后,Sufu缺失导致表示矿化水平的茜素红染色增加(图5,C)。RT-qPCR显示在Sufu缺陷肌腱来源细胞中Hh通路标志物(Gli1,Gli2, Patch1)明显高于对照细胞,并且成骨基因(Alp,Bsp,Ocn,Runx2,Osx)的表达高于对照细胞(图5,E和F)。进行末端脱氧核苷酸转移酶dUTP缺口末端标记(TUNEL),以确定 Sufu缺失是否会影响肌腱祖细胞的凋亡。然而,4周龄Ctsk-Ctrl;Rosa26-Ai9小鼠和Ctsk-CKO; Rosa-Ai9小鼠的跟腱显示极少的TUNEL阳性细胞(图5,G)。这些数据表明,Sufu缺失可导致肌腱祖细胞的软骨和成骨分化的增强,但不会导致肌腱祖细胞的凋亡。
实施例5 Gli1/Gli2的敲除抑制Ctsk-CKO小鼠的异位骨化的发生
在经典Hh信号中,SUFU缺乏会导致SMO失调,从而无法调节锌指转录因子GLI2的移动。GLI2作用是从纤毛移位到细胞核并激活GLI1启动子。GLI1和GLI2可激活Hh目标基因的转录。为了确定活化的Hh信号对于异位骨化的形成的相关性,我们通过交配Gli1lacz/lacz敲入小鼠或Gli2fl/fl小鼠来缺失Ctsk-CKO小鼠中的Gli1或Gli2,并通过Western blot验证了敲除效率(图6,A)。X射线和μ-CT射线照相印证,20周大的Ctsk-CKO;Gli1lacz/lacz和 Ctsk-CKO;Gli2fl/fl双敲除小鼠显示关节周围和跟腱中的异位骨化情况显著改善(图6,B)。踝关节和跟腱周围的肌腱的异位骨体积明显减少(图6,C)。SOFG染色显示在双敲除小鼠的关节周围区域没有异位骨(图6,D)。双敲除小鼠的跟腱中的番红O阳性软骨细胞和OCN 阳性成骨细胞显著减少(图6,E)。这些结果表明,Hh信号对于调控肌腱祖细胞的细胞命运至关重要。
实施例6 Hedgehog信号通路抑制剂JQ1可通过抑制Hh信号来改善Ctsk-CKO小鼠的异位骨化进程。
JQ1可以阻断Gli1的转录并已被用于治疗小鼠中各种类型的Hh信号过度激活的肿瘤,结果如图7A-D显示JQ1抑制了Ctsk-CKO;Rosa-Ai9小鼠Ctsk+肌腱来源细胞的向软骨和成骨分化。然后从4周开始,每天向Ctsk-CKO小鼠腹膜内注射50mg/kg JQ1,持续3周。经治疗的Ctsk-CKO小鼠的活动能力显着改善。通过μ-CT分析和番红O/固绿染色确定,JQ1 明显改善了关节周围和跟腱的异位骨化(图7,E-G)。此外,此剂量的JQ1对处理的对照小鼠的长骨生长没有明显的有害作用(图7,E和G)。通过番红O/固绿染色评估,跟腱的番红 O阳性细胞明显减少(图7,H)。
综上,本发明利用Cathepsin K-Cre;Rosa26-ai9报告小鼠鉴定出一群由组织蛋白酶K (Cathepsin K,Ctsk)标记的具有自我更新和体外三向分化能力的肌腱干细胞。并且发现 Ctsk阳性的肌腱干细胞的Hedgehog signaling(Hh)上调可引起渐进性肌腱异位骨化,构建了Ctsk-Cre标记的细胞条件性敲除Sufu的自发性异位骨化小鼠模型,该小鼠会产生渐进性、自发性的肌腱和韧带的异位骨化。通过体内体外实验验证Bet domain inhibitor-JQ1可缓解该小鼠模型因Hh上调引起的异位骨化。同时也发现了JQ1可以抑制损伤引起的跟腱异位骨化,为临床上异位骨化的治疗提供潜在靶标。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
Claims (10)
1.Hedgehog信号通路抑制剂在制备治疗异位骨化产品中的用途。
2.根据权利要求1所述的用途,其特征在于,所述Hedgehog信号通路抑制剂选自SMO抑制剂、Hh蛋白抑制剂和Gli抑制剂。
3.根据权利要求2所述的用途,其特征在于,所述Gli抑制剂选自Gli1抑制剂、Gli2抑制剂和Gli3抑制剂。
4.根据权利要求1所述的用途,其特征在于,所述Hedgehog信号通路抑制剂选自JQ1、vismodegib、环杷明、sonidegib、BMS-833923、PF04449913、LEQ506、TAK-441、LY2940680、Robotnikinin、GANT58、HIP-1、HIP-2、HIP-3、HIP-4、三氧化二砷中的一种或几种。
5.根据权利要求1所述的用途,其特征在于,所述异位骨化选自肌腱异位骨化、韧带异位骨化、关节囊异位骨化、关节软骨异位骨化。
6.根据权利要求5所述的用途,其特征在于,所述肌腱异位骨化包括跟腱异位骨化。
7.根据权利要求1所述的用途,其特征在于,所述异位骨化产品通过抑制Ctsk-Cre标记的肌腱祖细胞中的Hedgehog信号通路治疗异位骨化。
8.根据权利要求1所述的用途,其特征在于,所述异位骨化产品通过抑制Ctsk和Scx双阳细胞中Hedgehog信号通路治疗异位骨化。
9.根据权利要求1所述的用途,其特征在于,Hedgehog信号通路抑制剂在制备治疗损伤导致的异位骨化产品中的用途。
10.根据权利要求1所述的用途,其特征在于,所述治疗异位骨化产品选自药物、保健品或食品。
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---|---|---|---|---|
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Non-Patent Citations (1)
Title |
---|
HENG FENG等: "Tendon-derived cathepsin K–expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
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