CN113599500A - 一种治疗骨骼代谢性疾病的药物及其应用 - Google Patents
一种治疗骨骼代谢性疾病的药物及其应用 Download PDFInfo
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Abstract
本发明涉及一种治疗骨骼代谢性疾病的药物及其应用。所述药物包括上皮调节蛋白(Epiregulin)。本发明首次发现Epiregulin参与骨髓基质细胞成骨/成脂分化过程，通过提高自噬相关基因ULK‑1、Beclin‑1、Atg5、Atg7的表达，激活自噬信号通路，能够提高骨髓基质干细胞成骨分化标志基因Runx2、Osterix、碱性磷酸酶、骨桥蛋白的表达，并抑制成脂分化标志基因过氧化物酶体增殖物激活受体γ、CCAAT增强子结合蛋白α、脂肪酸结合蛋白4和adipsin的表达，从而促进骨髓基质干细胞成骨分化，抑制成脂分化，并首次发现Epiregulin蛋白可以有效防治去卵巢小鼠的骨丢失。
Description
技术领域
本发明属于生物医药技术领域，涉及一种治疗骨骼代谢性疾病的药物及其应用。
背景技术
骨髓基质干细胞(Bone mesenchymal stem cells，BMSCs)是一类具有多向分化潜能的细胞，可分化为成骨细胞、脂肪细胞、软骨细胞、腱细胞、成肌细胞和内脏中胚层细胞。骨髓基质干细胞作为成骨细胞与脂肪细胞共同的祖细胞，在成脂与成骨的分化过程中存在着此消彼长的竞争关系，在骨的维持中起着至关重要的作用，成骨和成脂分化失衡是许多人类疾病中常见的现象，如骨质疏松症，骨质疏松症一般与年龄有关，是一种骨骼代谢性疾病，其特征是骨密度低、骨量减少、骨骼微结构恶化、脂肪细胞浸润、骨骼脆性增加，导致骨折风险增加。
骨骼是一个动态器官，在机械应力与重力作用下，它通过持续的重塑来维持其矿化平衡及自身的结构完整。在体内微环境中，成骨细胞、骨细胞和破骨细胞合成并分泌旁分泌信号分子，包括生长因子、细胞因子和趋化因子，使骨形成与骨吸收平衡，这一过程也与血管生成耦合，共同维持骨骼的重塑和结构。
表皮生长因子(epidermal growth factor，EGF)家族配体及其受体的信号网络是目前研究最多的信号***之一，它以多种方式调节细胞功能，包括细胞增殖、存活、粘附、迁移和分化。EGF家族配体包括EGF、双向调节蛋白 (ampiregulin，AREG)和转化生长因子α(transforming growth factorα，TGFα)、肝素结合表皮生长因子(Heparin bindinggrowth factor，HB-EGF)、β细胞调节素(β-cellulin，BTC)和上皮调节蛋白(epiregulin，EREG)；表皮生长因子受体(epidermal growth factor receptor，EGFR)一种是位于多种类型细胞表面的 170-kDa糖蛋白，其特征是配体依赖的酪氨酸激酶活性。
上皮调节蛋白(Epiregulin，Ereg)基因位于人4号染色体和小鼠5号染色体上，与其它三个表皮生长因子家族成员，即EGF、Areg和β-cellulin相邻。 Epiregulin是含有46个氨基酸的分泌型蛋白，具有双重生物学活性，表现在可促进成纤维细胞、肝细胞、平滑肌细胞和角质形成细胞的增殖，但又抑制多种肿瘤来源的上皮细胞系的生长，Epiregulin在正常生理状态和病理状态下都具有多种功能。
目前现有技术中尚未有记载Epiregulin具有调控骨稳态和骨量功能的研究报道。
发明内容
针对现有技术的不足和实际需求，本发明提供一种治疗骨骼代谢性疾病的药物及其应用，本发明首次发现上皮调节蛋白能够调控骨髓基质干细胞定向分化，促进骨髓基质干细胞向成骨细胞分化，并抑制骨髓基质干细胞向脂肪细胞分化，从而其能够有效防治骨质疏松。
为达上述目的，本发明采用以下技术方案：
第一方面，本发明提供一种治疗骨骼代谢性疾病的药物，所述治疗骨骼代谢性疾病的药物包括上皮调节蛋白。
骨髓基质干细胞(BMSCs)的分化失衡在骨质疏松症等骨骼疾病的病因学中具有重要意义，在衰老和/或病理条件下，BMSCs向成骨细胞和脂肪细胞分化的平衡状态可能被打破，表现为向脂肪细胞分化偏移，从而引起骨髓内脂肪的堆积而导致骨量减少，威胁骨骼健康。
上皮调节蛋白(Epiregulin，Ereg)是一种表皮生长因子，基因位于人4号染色体和小鼠5号染色体上，其成熟肽是含有46个氨基酸的分泌型蛋白，成熟肽氨基酸序列如SEQ NOID.1或SEQ NO ID.2所示，在正常生理状态和病理状态下均具有多种功能，包括通过调节血管生成和血管重构以及刺激细胞增殖来促进炎症、伤口愈合和组织修复，且与不同的恶性肿瘤的发生有关，包括膀胱癌、胃癌、结肠癌、乳腺癌、肺癌、头颈癌和肝癌等。
SEQ NO ID.1：
VSITKCSSDMNGYCLHGQCIYLVDMSQNYCRCEVGYTGVRCEHFFL。
SEQ NO ID.2：
VQITKCSSDMDGYCLHGQCIYLVDMREKFCRCEVGYTGLRCEHFFL。
根据本发明，所述治疗骨骼代谢性疾病的药物促进骨髓基质干细胞成骨分化。
根据本发明，所述治疗骨骼代谢性疾病的药物抑制骨髓基质干细胞成脂分化。
根据本发明，所述治疗骨骼代谢性疾病的药物激活自噬信号通路。
本发明中，首次发现上皮调节蛋白能够调控骨髓基质干细胞定向分化，可通过激活自噬信号通路促进骨髓基质干细胞向成骨细胞分化，并可抑制骨髓基质干细胞向脂肪细胞分化，从而提高骨密度，增加骨量，防治骨质疏松等骨骼代谢性疾病。
优选地，所述治疗骨骼代谢性疾病的药物还包括药学上可接受的辅料。
优选地，所述辅料包括赋形剂、稀释剂、载体或助溶剂中的任意一种或至少两种的组合。
第二方面，本发明提供上皮调节蛋白在制备促进骨髓基质干细胞成骨分化的药物中的应用。
第三方面，本发明提供上皮调节蛋白在制备抑制骨髓基质干细胞成脂分化的药物中的应用。
第四方面，本发明提供上皮调节蛋白在制备激活自噬信号通路的药物中的应用。
第五方面，本发明提供上皮调节蛋白在制备治疗骨骼代谢性疾病的药物中的应用。
优选地，所述骨代谢性疾病包括骨质疏松等。
与现有技术相比，本发明具有以下有益效果：
本发明首次发现Epiregulin调节骨髓基质细胞成骨/成脂分化过程，通过提高自噬相关基因ULK-1、Beclin-1、Atg5、Atg7的表达，激活自噬信号通路，能够提高骨髓基质干细胞成骨分化标志基因Runx2、Osx、Alp、Opn的表达，并抑制成脂分化标志基因Pparγ、C/ebpα、Fabp4和adipsin的表达，从而促进骨髓基质干细胞成骨分化，抑制成脂分化，并首次发现Epiregulin蛋白可以有效防治去卵巢小鼠的骨丢失。
附图说明
图1A为骨髓基质干细胞上皮调节蛋白基因(Ereg)mRNA表达水平图；
图1B为骨髓基质干细胞成骨分化标志基因Runx2、Osx、Alp、Opn的mRNA 表达水平图；
图1C为骨髓基质干细胞成骨分化标志基因Runx2、Osx、Alp、Opn的蛋白表达Western blotting检测结果图；
图1D为骨髓基质干细胞成骨分化标志基因Runx2、Osx、Alp、Opn的蛋白表达水平图；
图1E为骨髓基质干细胞碱性磷酸酶染色结果图；
图2A为骨髓基质干细胞成脂分化标志基因Pparγ、C/ebpα、Fabp4和adipsin 的mRNA表达水平图；
图2B为骨髓基质干细胞成脂分化标志基因Pparγ、C/ebpα、Fabp4和adipsin 的蛋白表达Western blotting检测结果图；
图2C为骨髓基质干细胞成脂分化标志基因Pparγ、C/ebpα、Fabp4和adipsin 的蛋白表达水平图；
图2D为骨髓基质干细胞油红O染色结果图，标尺长度为100μm；
图2E为骨髓基质干细胞油红O染色后在520nm吸光度(OD520)结果图；
图3A为经Epiregulin处理的骨髓基质干细胞碱性磷酸酶染色结果图；
图3B为经Epiregulin处理的骨髓基质干细胞成骨分化标志基因Runx2、Osx、 Alp、Opn的mRNA表达水平图；
图4A为过表达Epiregulin的间充质细胞系C3H10T1/2自噬相关基因ULK-1、Beclin-1、Atg5、Atg7的mRNA表达水平图；
图4B为过表达Epiregulin的间充质细胞系C3H10T1/2自噬相关蛋白LC3β、 P62、Beclin-1、ATG5、ATG7的Western blotting检测结果图；
图4C为过表达Epiregulin的间充质细胞系C3H10T1/2自噬相关蛋白LC3β、 P62、Beclin-1、ATG5、ATG7表达水平结果图；
图4D为敲减Epiregulin的间充质细胞系C3H10T1/2自噬相关基因ULK-1、Beclin-1、Atg5、Atg7的mRNA表达水平图；
图4E为敲减Epiregulin的间充质细胞系C3H10T1/2自噬相关蛋白LC3β、 P62、Beclin-1、ATG5、ATG7的Western blotting检测结果图；
图4F为敲减Epiregulin的间充质细胞系C3H10T1/2自噬相关蛋白LC3β、 P62、Beclin-1、ATG5、ATG7表达水平结果图；
图5为小鼠胫骨结构图；
图6A为小鼠胫骨骨小梁相对容积(BV/TV)结果图；
图6B为小鼠胫骨单位体积组织的骨矿物含量(BMC/TV)结果图；
图6C为小鼠胫骨骨小梁数量(Tb.Th)结果图；
图6D为小鼠胫骨骨小梁分离度(Tb.Sp)结果图；
图6E为小鼠胫骨骨小梁结构模型指数(SMI)结果图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本发明实施例中涉及的材料与方法如下所述。
(1)细胞培养
从5周龄的C57BL/6J小鼠(斯贝福北京生物技术有限公司)股骨和胫骨中分离得到原代骨髓基质干细胞，用含有10％胎牛血清的α-MEM重悬后进行贴壁培养，培养3天后，去除非粘附的造血细胞，更新培养基，观察贴壁细胞的汇合程度，待其达到85％即可进行第一次消化，传至第三代后可用于后续实验。
待接种的细胞汇合度达到60％即可进行成骨诱导，成骨诱导液组分如下： 50μg/mL抗坏血酸、5mmol/Lβ-甘油磷酸钠，将各组分按比例加入含有10％FBS 和1％双抗的α-MEM，成骨诱导液配制完成后即可进行成骨诱导，72h收集细胞检测成骨相关因子mRNA和蛋白的变化，3d更换一次新的成骨诱导液，诱导 14d后可以固定细胞，进行碱性磷酸酶染色(ALP染色)。
当接种的细胞汇合度达到100％可进行成脂诱导，成脂诱导液组分如下：5 mg/L胰岛素、50μM吲哚美辛、0.25mM 3-异丁基-1-甲基黄嘌呤、0.5μM***，将各组分按比例加入含有10％FBS和1％双抗的α-MEM，成脂诱导液配制完成后即可进行成脂诱导，48h收集细胞检测成脂相关因子mRNA的变化， 72h收集细胞检测成脂相关因子蛋白的变化。72h后更换为只含5mg/L胰岛素的成脂诱导液继续诱导，待脂滴成熟后进行油红O染色。
(2)实时荧光定量PCR(quantitative real-time polymerase chain reaction，qRT-PCR)
诱导3d，利用总RNA提取试剂盒(Omega，Norcross，Georgia)提取细胞总RNA，使用RevertAid H Minus第一链cDNA合成试剂盒(Thermo Fisher Scientific，Waltham，Massachusetts)进行逆转录，利用SYBR Green荧光PCR 试剂盒(生工生物技术，中国上海)进行实时荧光定量PCR，以β-actin为内参，采用2－ΔΔCt计算方法得出各个目的基因的相对表达量，ΔCt＝目的基因Ct值-内参基因Ct值；ΔΔCt＝实验组ΔCt-对照组ΔCt，所有实验均重复3次。
(3)蛋白质印迹法(Western Blotting)
诱导3d利用RIPA裂解液提取细胞总蛋白，配制10％SDS-PAGE凝胶，将提取的总蛋白进行电泳，蛋白分离后，转移到硝酸纤维素膜上，用5％脱脂牛奶进行室温封闭，于4℃过夜孵育一抗，检测成骨/成脂分化相关因子与自噬信号通路相关因子的表达，检测的蛋白包括：成骨细胞分化关键因子锌指结构转录因子Osterix(Osx)、骨桥蛋白(osteopontin，OPN)、碱性磷酸酶(alkaline phosphantase，ALP)、Runt相关转录因子2(runt relatedtranscription factor 2， Runx2)、脂肪细胞分化关键转录因子过氧化物酶体增殖物激活受体γ (peroxisome proliferator-activated receptorγ，PPARγ)、CCATT增强子结合蛋白 (CCATT enhancer binding protein alpha，C/EBPα)、脂肪酸结合蛋白4(fattyacid-binding protein 4，FABP4)、β肌动蛋白(β-actin)、自噬相关基因5(ATG5)、自噬相关基因7(ATG7)、自噬关键调控蛋白Beclin-1、微管相关蛋白轻链3β (LC3β)。将免疫复合物与辣根过氧化物酶标记的二抗进行孵育，使用ECL化学发光试剂盒(proteintech，中国武汉)检测目的蛋白的表达情况。
(4)质粒构建与转染
由PCR扩增Epiregulin cDNA的编码区(SEQ NO ID.3)，然后使用Basic SeamlessCloning and Assembly Kit(Transgene，中国北京)将其克隆到pcDNA3.1 载体BamHI/EcoRI位点上，构建Epiregulin过表达质粒，利用JetPRIME转染试剂(Polyplus，Illkirch，France)将Epiregulin过表达质粒或空载体转染细胞4h，然后更换新的培养基，对于功能缺失性研究，利用lipofectamine RNAi-max试剂 (Invitrogen，Carlsbad，California，USA)将20nmol/L的对照或Epiregulin siRNA 转染细胞18h，然后更换新的培养基。
SEQ NO ID.3：
atggagacgctccctgcctcttgggtcttgacgctgctttgtctaggttcccaccttctacaggcagttatcagcaca accgtgatcccatcatgcatcccaggagaatccgaggataactgtaccgccttagttcagatggaagacgatccccgtgt ggctcaagtgcagattacaaagtgtagttctgacatggacggctactgcttgcatggccagtgcatctacctggtggacat gagagagaaattctgcagatgtgaagtgggctacactggtctgcgatgtgagcacttctttctaactgttcaccaacccttga gcaaagaatacgttgcgttgacagtgattctcattttcctgtttctcatcataaccgctggatgcatatactatttctgcagatgg tacaaaaatcgaaaaagtaaaaaatcgagggaggaatatgagagagtgacctcaggggacccagtgctgccacaggtc tga。
(5)慢病毒包装与感染
将Epiregulin cDNA的编码区克隆到pCDH-CMV-MCS-EF1-copGFP载体上，利用慢病毒包装试剂盒(吉满生物，中国上海)，并通过293T细胞完成慢病毒的包装，得到Epiregulin过表达慢病毒，将空pCDH-CMV-MCS-EF1-copGFP载体进行包装，得到对照病毒，将Epiregulin过表达慢病毒或对照病毒(MOI＝20) 感染原代骨髓基质干细胞并进行诱导分化。
(6)碱性磷酸酶染色
成骨诱导14d后进行碱性磷酸酶染色，用1×PBS缓冲液洗涤细胞，4％多聚甲醛固定10min，用BCIP/NBT染液(碧云天，中国上海)孵育15min。
(7)油红O染色
成脂诱导5d进行油红O染色，用1×PBS缓冲液清洗细胞，用4％多聚甲醛固定10min，用60％异丙醇清洗细胞，加入提前配制好的0.3％油红染液(溶解于60％异丙醇)，室温染色8min，用100％异丙醇提取脂肪细胞中的油红染料，在520nm处测定吸光度。
(8)统计学分析
所有实验均进行三次重复，所得实验数据以均数±标准差表示，两组独立样本比较采用t检验进行组间比较，存在两个独立变量的多组间比较采用Two-way ANOVA，如果差异具有显著性，采用Tukey检验进行两两比较。当P＜0.05时则认为存在显著差异，具有统计学意义。
实施例1
本实施例分析Epiregulin过表达慢病毒对BMSCs成骨分化的影响。
利用Epiregulin过表达慢病毒感染BMSCs(Ereg OE LV组)，以感染对照病毒BMSCs作为对照(Ctrl OE LV组)，48h收集细胞提总RNA，利用qRT-PCR 检测过表达效率。成骨诱导3d时收集细胞提取总RNA，检测成骨相关基因 mRNA的表达，结果如图1A和图1B所示，感染Epiregulin过表达慢病毒的 BMSCs中，上皮调节蛋白基因(Ereg)、Runt相关转录因子2基因(Runx2)、锌指结构转录因子Osterix基因(Osx)、碱性磷酸酶基因(ALP)和骨桥蛋白基因(Opn)的mRNA表达均显著上调，分别为对照组的3.4、2.5、2.5、2.4倍；诱导3d提取细胞总蛋白，检测成骨相关基因蛋白表达，结果如图1C和1D所示，感染Epiregulin过表达慢病毒的BMSCs中，上皮调节蛋白(Ereg)、Runt 相关转录因子2(Runx2)、锌指结构转录因子Osterix(Osx)、碱性磷酸酶(ALP) 和骨桥蛋白(OPN)的蛋白表达均显著上调，分别为对照组的2.9、2.6、2.8、 2.3倍；诱导至14d时进行ALP染色，结果如图1E所示，染色结果显示，与对照相比，ALP染色明显加深。
综上所述，感染Epiregulin过表达慢病毒的BMSCs中，成骨分化标志基因 Runx2、Osx、Alp、Opn的mRNA和蛋白表达水平均上调，说明Epiregulin过表达慢病毒感染BMSCs后，可以促进细胞成骨分化，即上皮调节蛋白能够促进BMSCs成骨分化。
实施例2
本实施例分析Epiregulin过表达慢病毒对BMSCs成脂分化的影响。
利用Epiregulin过表达慢病毒感染BMSCs(Ereg OE LV组)，以感染对照病毒BMSCs作为对照(Ctrl OE LV组)，待细胞密度达到100％时进行成脂诱导，诱导48h收集细胞提取总RNA，检测成脂关键基因mRNA的表达，如图2A 所示，感染Epiregulin过表达慢病毒的BMSCs中，成脂分化标志基因Pparγ、 C/ebpα、Fabp4和adipsin的mRNA表达均下降，分别下降了73％、71％、76％、 68％；诱导3d提取细胞总蛋白，在蛋白水平上检测成脂相关基因编码蛋白的表达，如图2B和图2C所示，感染Epiregulin过表达慢病毒的BMSCs中，PPARγ、 C/EBPα、FABP4的蛋白表达水平均降低，分别下降了48％、56％、41％；诱导至5d时，进行油红O染色，如图2D和图2E所示，染色结果显示，过表达 Epiregulin后脂肪细胞分化明显减少，OD520值下降了62.5％。
综上所述，感染Epiregulin过表达慢病毒的BMSCs中，成脂分化标志基因 Pparγ、C/ebpα、Fabp4和adipsin的mRNA和蛋白表达水平均下降，脂肪细胞分化明显减少，表明Epiregulin过表达慢病毒感染BMSCs后，可以抑制细胞成脂分化。
实施例3
本实施例分析Epiregulin重组蛋白对BMSCs成骨分化的影响。
在BMSCs培养基中加入不同剂量(5ng/mL、10ng/mL和20ng/mL)的 Epiregulin重组蛋白(Prospec，中国上海)，以PBS溶液(vehicle)进行处理作为对照，随后进行成骨诱导，诱导3d时收集细胞提取总RNA，检测成骨相关基因mRNA的表达，诱导至14d时进行ALP染色，染色结果显示(图3A)，与对照组相比，随Epiregulin浓度增加，ALP染色依次加深，qRT-PCR检测结果(图3B)显示成骨分化标志基因Runx2、Osx、Alp、Opn的mRNA表达明显增加，表明Epiregulin重组蛋白处理BMSCs后，可以促进细胞成骨分化。
实施例4
本实施例分析Epiregulin对自噬信号通路的影响。
利用JetPRIME转染试剂(Polyplus，Illkirch，France)将Epiregulin过表达质粒转染间充质细胞系C3H10T1/2(Ereg Construct组)，提高内源性Epiregulin 的表达，以转染空质粒作为对照(Vector组)，利用lipofectamine RNAi-max试剂将20nmol/LEpiregulin siRNA(siRNA-1和siRNA-2，分别针对Epiregulin基因不同靶点，序列分别如SEQ NO ID.4、5和SEQ NO ID.6、7所示)转染间充质细胞系C3H10T1/2，降低内源性Epiregulin的表达，以转染无Epiregulin基因干扰功能的siRNA(SEQ NO ID.8、9)作为对照，转染48h收集细胞提取总RNA，检测自噬相关基因ULK-1、Beclin-1、Atg5和Atg7的表达，72h提取总蛋白，检测LC3β、P62、Beclin-1、ATG5和ATG7相关蛋白的表达，qRT-PCR检测结果(图4A)显示过表达Epiregulin后ULK-1、Beclin-1、Atg5、Atg7的mRNA 表达均上调，如图4B和图4C所示，过表达Epiregulin后LC3Ⅱ/LC3Ⅰ的比值明显增加，P62降低、Beclin-1、ATG5、ATG7均增加，表明自噬信号通路被激活；相反，如图4D所示，降低内源性Ereg表达后，ULK-1、Beclin-1、Atg5、 Atg7的mRNA表达均下降，如图4E和4F所示，LC3Ⅱ/LC3Ⅰ的比值明显下降，P62增加、Beclin-1、ATG5、ATG7均降低，表明自噬信号通路被抑制。
SEQ NO ID.4：(正义RNA)5’-GCAUCUACCUGGUGGACAUTT-3’。
SEQ NO ID.5：(反义RNA)5’-AUGUCCACCAGGUAGAUGCTT-3’。
SEQ NO ID.6：(正义RNA)5’-GCUGGAUGCAUAUACUAUUTT-3’。
SEQ NO ID.7：(反义RNA)5’-AAUAGUAUAUGCAUCCAGCTT-3’。
SEQ NO ID.8：(正义RNA)5’-UUCUCCGAACGUGUCACGUTT-3’。
SEQ NO ID.9：(反义RNA)5’-ACGUCGCACGUUCGGAGAATT-3’。
实施例5
本实施例分析重组人Epiregulin蛋白干预后对去卵巢小鼠骨量的影响。
将10周龄C57BL/6J雌鼠随机分为三组：Sham/Vehicle组、OVX/Vehicle 组和OVX/EREG组，每组7只，Sham/Vehicle组进行假手术，术后三天腹腔注射PBS缓冲液；OVX/Vehicle组和OVX/EREG组进行卵巢去势手术，术后三天分别腹腔注射PBS缓冲液和Epiregulin重组蛋白(Prospec，中国上海)，Epiregulin 每次剂量为50μg/kg，隔日注射一次。干预6周后处死小鼠，进行取样，将胫骨固定后移至70％乙醇内，以备μCT拍摄。将处理好的标本使用Scanco Medical Viva CT 80进行扫描，扫描程序主要参数设置为：分辨率6.72μm，电压55kV，电流145μA。拍摄完毕后使用配套重构和分析软件进行数据分析，分析范围为胫骨干骺端生长板以下100μm处至远端1mm范围内区域，结构图如图5所示，主要测定以下骨形态静力学参数：骨小梁相对容积(Bone Volume/Tissue volume， BV/TV)、单位体积组织的骨矿物含量(Bone Mineral Contents per Tissue Volume， BMC/TV)、骨小梁数量(TrabecularNumber，Tb.N)、骨小梁厚度(Trabecular Thickness，Tb.Th)、骨小梁分离度(TrabecularSeparation/Spacing，Tb.Sp)和骨小梁结构模型指数(Structure Model Index，SMI)。结果如图6A-图6E所示，与Sham/Vehicle组比较，OVX/Vehicle组小鼠骨小梁骨量明显减少，静力学参数胫骨小梁骨容积(BV/TV)、骨密度(BMC/TV)显著降低，骨小梁数量(Tb.N) 显著减少，骨小梁分离度(Tb.Sep)和结构模型指数(SMI)显著增加，表明去卵巢骨质疏松小鼠模型构建成功。给予Epiregulin重组蛋白(EREG)干预后，与OVX/Vehicle组相比，OVX/EREG组小鼠骨小梁骨丢失得到显著改善，胫骨小梁骨容积(BV/TV)、骨密度(BMC/TV)显著增加、骨小梁数量(Tb.N)增加，骨小梁分离度(Tb.Sp)和结构模型指数(SMI)降低，表明Epiregulin能够改善去卵巢小鼠骨质疏松。
综上所述，本发明首次发现Epiregulin参与骨髓基质细胞成骨/成脂分化过程，Epiregulin通过调节自噬信号通路可以促进骨髓基质干细胞成骨分化，此外亦可抑制脂肪细胞分化；首次发现Epiregulin能够有效防治去卵巢小鼠的骨丢失。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 天津医科大学朱宪彝纪念医院（天津医科大学代谢病医院、天津代谢病防治中心）
<120> 一种治疗骨骼代谢性疾病的药物及其应用
<130> 20210723
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<212> PRT
<213> 人工序列
<400> 2
Val Gln Ile Thr Lys Cys Ser Ser Asp Met Asp Gly Tyr Cys Leu His
1 5 10 15
Gly Gln Cys Ile Tyr Leu Val Asp Met Arg Glu Lys Phe Cys Arg Cys
20 25 30
Glu Val Gly Tyr Thr Gly Leu Arg Cys Glu His Phe Phe Leu
35 40 45
<210> 3
<211> 489
<212> DNA
<213> 人工序列
<400> 3
atggagacgc tccctgcctc ttgggtcttg acgctgcttt gtctaggttc ccaccttcta 60
caggcagtta tcagcacaac cgtgatccca tcatgcatcc caggagaatc cgaggataac 120
tgtaccgcct tagttcagat ggaagacgat ccccgtgtgg ctcaagtgca gattacaaag 180
tgtagttctg acatggacgg ctactgcttg catggccagt gcatctacct ggtggacatg 240
agagagaaat tctgcagatg tgaagtgggc tacactggtc tgcgatgtga gcacttcttt 300
ctaactgttc accaaccctt gagcaaagaa tacgttgcgt tgacagtgat tctcattttc 360
ctgtttctca tcataaccgc tggatgcata tactatttct gcagatggta caaaaatcga 420
aaaagtaaaa aatcgaggga ggaatatgag agagtgacct caggggaccc agtgctgcca 480
caggtctga 489
<210> 4
<211> 21
<212> RNA
<213> 人工序列
<400> 4
gcaucuaccu gguggacaut t 21
<210> 5
<211> 21
<212> RNA
<213> 人工序列
<400> 5
auguccacca gguagaugct t 21
<210> 6
<211> 21
<212> RNA
<213> 人工序列
<400> 6
gcuggaugca uauacuauut t 21
<210> 7
<211> 21
<212> RNA
<213> 人工序列
<400> 7
aauaguauau gcauccagct t 21
<210> 8
<211> 21
<212> RNA
<213> 人工序列
<400> 8
uucuccgaac gugucacgut t 21
<210> 9
<211> 21
<212> RNA
<213> 人工序列
<400> 9
acgucgcacg uucggagaat t 21
Claims (10)
1.一种治疗骨骼代谢性疾病的药物，其特征在于，所述治疗骨骼代谢性疾病的药物包括上皮调节蛋白。
2.根据权利要求1所述的治疗骨骼代谢性疾病的药物，其特征在于，所述骨骼代谢性疾病包括骨质疏松症。
3.根据权利要求1或2所述的治疗骨骼代谢性疾病的药物，其特征在于，所述治疗骨骼代谢性疾病的药物还包括药学上可接受的辅料；
优选地，所述辅料包括赋形剂、稀释剂、载体或助溶剂中的任意一种或至少两种的组合。
4.根据权利要求1-3任一项所述的治疗骨骼代谢性疾病的药物，其特征在于，所述治疗骨骼代谢性疾病的药物促进骨髓基质干细胞成骨分化。
5.根据权利要求1-4任一项所述的治疗骨骼代谢性疾病的药物，其特征在于，所述治疗骨骼代谢性疾病的药物抑制骨髓基质干细胞成脂分化。
6.根据权利要求1-5任一项所述的治疗骨骼代谢性疾病的药物，其特征在于，所述治疗骨骼代谢性疾病的药物激活自噬信号通路。
7.上皮调节蛋白在制备促进骨髓基质干细胞成骨分化的药物中的应用。
8.上皮调节蛋白在制备抑制骨髓基质干细胞成脂分化的药物中的应用。
9.上皮调节蛋白在制备激活自噬信号通路的药物中的应用。
10.上皮调节蛋白在制备治疗骨骼代谢性疾病的药物中的应用。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101384707A (zh) * | 2006-02-16 | 2009-03-11 | 布鲁塞尔大学 | 用于骨髓干细胞（bmsc）成骨分化的方法及其用途 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101384707A (zh) * | 2006-02-16 | 2009-03-11 | 布鲁塞尔大学 | 用于骨髓干细胞（bmsc）成骨分化的方法及其用途 |
Non-Patent Citations (2)
| Title |
|---|
| SACHIN THOMAS，BITHIAH GRACE JAGANATHAN: "Signaling network regulating osteogenesis in mesenchymal stem cells", JOURNAL OF CELL COMMUNICATION AND SIGNALING, vol. 16, no. 1, pages 47 - 61, XP037647552, DOI: 10.1007/s12079-021-00635-1 * |
| YANGYANG CAO, RUITANG SHI, HAOQING YANG, JIANPENG ZHANG, LIHUA GE, RUNTAO GAO, ZHIPENG FAN: "Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans-differentiation of adipose-derived mesenchymal stem cells via MAPKs pathway", CELL BIOL INT, vol. 44, no. 4, pages 1046 - 1058 * |
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