CN113755580B - 一种治疗和/或缓解淋巴水肿的药物干预靶点及其应用 - Google Patents
一种治疗和/或缓解淋巴水肿的药物干预靶点及其应用 Download PDFInfo
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Abstract
本发明提供了一种药物干预靶点在制备治疗和/或缓解淋巴水肿的药物中的应用，该靶点为TREM1基因及其编码蛋白。本发明还提供了一种药物干预靶点在制备治疗组织纤维化和/或脂肪沉积药物中的应用及一种药物干预靶点在制备抑制有害炎性因子表达或降低促炎巨噬细胞数量药物中的应用，所述靶点均为TREM1基因及其编码蛋白。所述药物为一种能干扰TREM1与其配体结合的多肽药物，在此基础上，包括多肽在内的针对该靶点设计的药物均可用来治疗和缓解淋巴水肿。本发明为淋巴水肿的治疗和/或缓解提供了新的药物靶点和新的药物选择。
Description
技术领域
本发明属于生物医药领域，具体涉及一种治疗和/或缓解淋巴水肿的药物干预靶点及其应用。
背景技术
淋巴水肿是由于淋巴液回流障碍而滞留在组织中而引起的水肿，根据世界卫生组织统计，淋巴水肿在常见慢性病中列第11位，致残类疾病中列第2位，在全球范围内淋巴水肿患者约达1.7亿，而我国淋巴水肿患者的发病率仍在逐年攀升。
淋巴水肿一般表现为肢体皮下脂肪组织的进行性肿胀、脂肪组织的慢性炎症反应，脂肪组织病理性纤维化和过多的脂肪沉积等病理过程。淋巴水肿往往给肿瘤患者带来沉重的生理和心理负担，严重影响患者的生活质量。然而，淋巴水肿这种疾病是世界医学难题，目前仍旧缺乏有效的药物疗法对其进行干预和预防。
本发明人前期通过单细胞转录组对淋巴水肿患者和健康人的腿部皮下脂肪组织进行分析，发现淋巴水肿患者皮下脂肪组织中抗炎巨噬细胞和促炎巨噬细胞的比例为0.76，而健康人皮下脂肪组织中抗炎巨噬细胞和促炎巨噬细胞的比例为2.03。这一改变反映了淋巴水肿患者皮下脂肪组织中呈现出明显的促炎免疫微环境。改变这种促炎的免疫微环境，有可能会降低脂肪组织的纤维化和脂肪沉积，进而改善患肢的水肿。同时，我们观察到促炎细胞特异性地表达TREM1受体，已知该受体激活后会放大炎症反应并调控髓系免疫细胞向炎症部位聚集。多肽LR12是由12个氨基酸(LQEEDAGEYGCM)组成的多肽药物，LR12是一种特殊的TREM-1抑制剂，作为诱饵受体，干扰TREM1与其配体的结合。
专利WO2021105137A1公开了TREM1作为抑制血管闭合的靶点，LR12可以作用于该靶点来治疗镰刀状细胞疾病的血管闭塞和组织损伤。AmirBoufenzer等研究发现，LR12作为TREM1抑制肽，对心肌梗塞引起的炎症细胞的募集和激活具有明显的抑制作用。Ruili Shi等研究发现，LR12可以减轻脂多糖诱导的小鼠急性肺损伤。A Boufenzer等研究发现LR12可通过调节TREM-1提高实验性脓毒症小鼠的存活率，可预防败血症引起的心血管功能障碍。
但是，迄今为止并没有以TREM1为靶点治疗淋巴水肿相关疾病的研究报道。
发明内容
本发明的目的在于提供一种治疗和/或缓解淋巴水肿的药物干预靶点。
为了实现上述目的，本发明采用了如下技术方案：
本发明提供了一种药物干预靶点在制备治疗和/或缓解淋巴水肿的药物中的应用，所述药物干预靶点为TREM1基因及其编码蛋白。其中，所述TREM1基因编码一种免疫受体，该免疫受体表达定位于皮下脂肪组织的促炎巨噬细胞表面。本发明人前期研究发现淋巴水肿患者皮下脂肪组织中促炎巨噬细胞的比例明显升高，促炎巨噬细胞特异性地表达TREM1受体。
优选地，所述淋巴水肿为继发性淋巴水肿；更优选地，所述继发性淋巴水肿为肿瘤次生淋巴水肿。
本发明还提供了一种药物干预靶点在制备治疗脂肪组织纤维化和/或脂肪沉积的药物中的应用，所述药物干预靶点为TREM1基因及其编码蛋白。脂肪组织纤维化是在应对损伤和炎症过程中出现的细胞外基质(ECM)的过量沉积。ECM主要成分包括胶原蛋白及其修饰蛋白、基质金属蛋白、纤连蛋白等。淋巴液淤滞会导致ECM发生不可逆的纤维增生，主要是胶原纤维以及弹力纤维。淋巴水肿引发组织纤维化，组织纤维化反过来加重淋巴水肿，但是目前尚不清楚淋巴水肿导致组织纤维化的机制。脂肪水肿会随着淋巴患者病期延长而逐渐加重，但淋巴水肿导致脂肪沉积的机制目前尚不清楚。本发明提供证据表明以TREM1基因及其编码蛋白为靶点对淋巴水肿小鼠进行药物干预，能够明显抑制淋巴水肿小鼠的组织纤维化和脂肪沉积，表明组织纤维化与脂肪沉积的发病机制与TREM1基因或其编码蛋白相关。
本发明还另外提供了一种药物干预靶点在制备抑制有害炎性因子表达或降低促炎巨噬细胞数量的药物中的应用，所述药物干预靶点为TREM1基因及其编码蛋白。优选地，所述有害炎性因子选自：Il1b或Tnf。本发明提供证据表明用所述药物对淋巴水肿小鼠进行干预能够抑制有害炎性因子Il1b和Tnf的基因表达，进而使免疫微环境从慢性炎症状态恢复到稳态，抑制炎症反应；所述药物还能够抑制巨噬细胞标记基因Cd68的表达，降低促炎巨噬细胞的数量。
在本发明的一种实施方式中，所述药物包括基因敲低技术对所述靶点基因TREM1进行阻断干预或蛋白受体阻断技术对所述靶点基因TREM1编码蛋白进行阻断干预的药物。
优选地，所述基因敲低技术选自shRNA，siRNA或CRISPR/Cas系统的一种或多种。shRNA和siRNA都是在RNA水平上降低蛋白的表达，CRISPR/Cas是在DNA水平上降低蛋白的表达。
优选地，所述蛋白受体阻断技术选自小分子化合物或多肽的一种或多种。更优选地，所述蛋白受体阻断技术为多肽，所述多肽的氨基酸序列如SEQ ID NO：1所示。
本发明中针对TREM1基因及其编码蛋白产物而设计的siRNA药物、小分子抑制剂，抗体类和其他多肽类用于治疗淋巴水肿的药物都包含在本发明的保护范围之内。
本发明的治疗和/或缓解淋巴水肿的药物还包含药物学上可以接受的载体，所述载体包括但不限于：脂质体、稀释剂、缓冲剂、混悬剂、乳剂、颗粒剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着色剂、矫味剂或吸附载体。
本发明所述药物可以制成包括但不限于显微注射剂、适于转染的剂型、注射液、片剂、粉剂、粒剂、胶囊剂。上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明所述药物可以单独施用，或者与其他能够抑制淋巴水肿的药物进行组合施用。
有益效果
本发明公开了一个淋巴水肿的药物靶点，该靶点是TREM1基因及其编码蛋白。本发明中，使用多肽LR12对该靶点进行阻断干预，能够显著地降低组织炎症细胞的浸润及炎症反应、皮下脂肪组织的厚度和纤维化，进而缓解淋巴组织水肿。本发明为淋巴水肿的治疗提供了新的治疗靶点和新的药物选择。
附图说明
图1：A.经过mLR12(n＝5)或Scramble多肽(随机打乱序列做阴性对照；n＝5)治疗处理后，鼠尾直径的变化曲线；B.治疗4周(建模手术6周)后的鼠尾；C.H&E染色结果；D.鼠尾皮下脂肪组织厚度的统计分析；E.天狼星红染色结果；F.免疫荧光染色结果(箭头所指即Cd11b+Trem1+细胞)；G.Cd11b+Trem1+细胞在所有Cd11b+细胞中的比例；H.qRT-PCR Cd68的表达结果；I.qRT-PCR Il1b的表达结果；J.qRT-PCR Tnf的表达结果。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
下述实施例所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所有的材料、试剂等，如无特殊说明，均可从商业途径获得。
实施例1
本实施例证明mLR12(LR12对应的鼠类多肽)可以显著缓解淋巴水肿鼠尾模型的水肿症状。
1、小鼠鼠尾淋巴水肿模型及LR12给药
淋巴水肿鼠尾模型建立方法：使用成年雄性(4-6周龄，C57)小鼠在腹腔内注射10％水合氯醛0.1mL麻醉后，将鼠尾根部用75％乙醇消毒，在距鼠尾根部1cm处切开皮肤并破坏尾部深层淋巴管结构。更为具体的建模方法可参考文献《Zampell JC,Yan A,ElhadadS,Avraham T,Weitman E,Mehrara BJ.CD4+Cells Regulate Fibrosis andLymphangiogenesis in Response to Lymphatic Fluid Stasis.PLoS One.2012；7:e49940》。
分组及给药方法：待小鼠自然恢复2周后，测量小鼠鼠尾切口1cm处的鼠尾直径，然后将其随机分配到对照(n＝5)或治疗(n＝5)组。为了抑制TREM1受体，使用化学合成的鼠类LR12(LQEEDTGEYGCV，SEQ ID NO：1；mLR12)。要求内毒素低于0.5EU/mg。手术2周后，每天通过腹腔注射mLR12(5mg/kg)对小鼠进行治疗，持续6周。对照组小鼠每天腹膜内注射氨基酸顺序随机打乱的mLR12(5mg/kg；mLR12-scramble)。在6周内每3天测量一次小鼠鼠尾切口1cm处的鼠尾直径。手术6周后收集尾部组织样本用于后续实验分析。
如图1A所示，与阴性对照(scramble；n＝5)相比，经过mLR12(n＝5)多肽药物(n＝5)治疗后，鼠尾直径显著减小，恢复到正常直径的速度明显加快。图1B显示治疗4周(建模手术6周)后鼠尾直径几乎已恢复到手术前的水平。
2、H&E染色
对距离小鼠鼠尾手术切口远端1cm处的横截面切片(n＝3)进行常规H&E染色，H&E染色程序在自动染色机上进行。同时对每张切片上测量3处不同的皮下组织(表皮基底层到深筋膜层)厚度进行统计分析。
H&E染色图像显示经过mLR12治疗后鼠尾皮下脂肪组织厚度与阴性对照组相比明显降低(图1C和1D)，反映了mLR12能有效降低淋巴水肿皮下脂肪的沉积。
3、天狼星红染色
切片使用苏木精溶液染色5分钟，后使用0.1％天狼星红溶液染色1小时，染色结束后使用无水酒精脱水，二甲苯透明，最后使用中性树胶进行封片。
天狼星红染色图像显示阴性对照的胶原纤维呈现纤维化的典型特征即不规则排列，而mLR12治疗组的鼠尾胶原纤维排列整齐(图1E)，反映了mLR12在抑制淋巴水肿组织纤维化中的作用。
4、免疫荧光染色
切片脱蜡后，在EDTA(pH＝9.0)溶液中进行抗原修复，使用PBS洗涤3次，每次5分钟，后用山羊血清进行封闭1小时。在4℃环境中过夜孵育一抗CD11b(ab8878,Abcam)和Trem1(bs-4886R,Bioss)。在室温下进行复温30分钟，后使用PBS洗涤3次，每次5分钟。使用Alexa 488(A-11008，Thermo Fisher Scientific)、Alexa 594(ab150116，Abcam)荧光二抗染色40分钟，最后用含有DAPI的封片剂进行封片。
免疫荧光染色显示，与阴性对照组相比，mLR12治疗组的鼠尾组织中Cd11b+Trem1+促炎巨噬细胞数量明显减少,反映了mLR12通过靶向Trem1,能够有效降低组织中促炎巨噬细胞的数量(图1F、1G)
5、RNA提取
使用手术刀片切下小鼠鼠尾手术切口至远端1cm处的组织样本，使用匀浆器进行匀浆，加入1ml TRIzol试剂充分混合，室温放置5分钟，加入200μl氯仿，剧烈振荡15s，室温放置3分钟后，4℃环境中，12000g离心15分钟。将离心后得到的水相转移到新管中，加入500μl异丙醇，上下颠倒，室温放置10分钟后，4℃环境中，10000g离心10分钟。将离心得到的白色胶状RNA沉淀，用80％的乙醇在4℃环境中，7500g离心5分钟清洗2次，后将上清完全弃去，晾干RNA直至透明不可见，加入20μl RNase-free水使其溶解，在56℃环境的金属浴中，充分溶解RNA 10分钟，后测量RNA浓度。
6、逆转录
使用100ng-2ug的RNA模板，与1μl的Oligo dT primer(50μm)、1μl的dNTP Mixture(10mM)充分混合，最后用RNase free H2O补齐体系至10μl，放入PCR仪器中，65℃加热5分钟，使RNA变性，随后放入冰上。在上述反应体系中加入4ul的5×PrimeScript Buffer、0.5ul的RNase Inhibitor(40units)、1μl的PrimeScript Reverse Transcriptase(100-200units)和4.5μl的RNase-free水，充分混匀，总共20ul体系。在PCR仪器中，设定程序为42℃60min，70℃15min进行逆转录，结束后放入冰上冷却，得到cDNA产物。
7、RT-qPCR检测
配制以下反应体系：2X PowerUp SYBR Green Master Mix 5μl、正向引物(10μM/L)0.3ul、反向引物(10μM/L)0.3ul、cDNA模板(5ng/μl)1μl和ddH2O 3.4μl，总共10μl体系，每个样本设置3个平行复孔。扩增程序为：95℃10min,(95℃15s，60℃60s)40个循环。以SYBRGreen作为荧光标记物，在荧光实时定量PCR仪上进行PCR反应，ΔΔCT法进行相对定量。
qRT-PCR结果表明(图1H、1I、1J)，mLR12治疗后鼠尾组织中巨噬细胞标记基因Cd68的表达量显著降低，反映了mLR12能够有效抑制组织中巨噬细胞及炎症反应；mLR12治疗后鼠尾组织中有害炎症因子基因Il1b和Tnf的表达量显著降低，反映了mLR12治疗后组织的免疫微环境发生了从慢性炎症到稳态的转变。
综上，mLR12能够显著地降低组织炎症细胞的浸润及炎症反应、皮下脂肪组织的厚度和纤维化，能够显著缓解淋巴水肿症状。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国医学科学院阜外医院
<120> 一种治疗和/或缓解淋巴水肿的药物干预靶点及其应用
<130> P210229
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> mLR12
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Leu Gln Glu Glu Asp Thr Gly Glu Tyr Gly Cys Val
1 5 10
Claims (4)
1.一种多肽LR12在制备治疗和/或缓解淋巴水肿的药物中的应用，所述LR12通过靶向TERM1、降低促炎巨噬细胞的数量、降低炎症因子基因Il1b和Tnf的表达量来缓解淋巴水肿症状，所述LR12的氨基酸序列如SEQ ID NO：1所示。
2.如权利要求1所述的应用，其特征在于，所述LR12能够降低淋巴水肿皮下脂肪的沉积、抑制淋巴水肿组织纤维化。
3.如权利要求1或2所述的应用，其特征在于，所述药物还包含药物学上可以接受的载体，所述载体包括：脂质体、稀释剂、缓冲剂、混悬剂、乳剂、颗粒剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着色剂、矫味剂或吸附载体。
4.如权利要求3所述的应用，其特征在于，所述药物单独施用，或者与其他能够抑制淋巴水肿的药物进行组合施用。
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