CN116785434A - 一种cd52+巨噬细胞作为骨关节炎治疗靶点的应用 - Google Patents
一种cd52+巨噬细胞作为骨关节炎治疗靶点的应用 Download PDFInfo
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Abstract
本发明公开了CD52+巨噬细胞作为治疗靶点在筛选或制备治疗骨关节炎的药物中的应用。本发明通过单细胞转录组测序显示，在骨关节炎情况下，炎症性CD52+巨噬细胞数量显著增加，并且特异性高表达CD52，在体内水平，通过关节腔注射CD52特异性抗体可以减轻OA滑膜组织的炎症并有效延缓OA的进程，证实CD52+巨噬细胞可以作为骨关节炎的治疗靶点。因此，CD52+巨噬细胞能够作为骨关节炎的一个潜在的有前途的治疗靶点，靶向CD52+巨噬细胞的药物对骨关节炎的治疗具有积极的作用。本发明有助于推动相关靶向巨噬细胞药物的制备，提高相关抗炎药物的疗效，并为新药物研发提供线索。
Description
技术领域
本发明涉及生物医药领域，尤其涉及一种CD52+巨噬细胞作为骨关节炎治疗靶点的应用。
背景技术
膝骨关节炎（Osteoarthritis，OA）是导致下肢功能障碍的主要骨科疾病之一，不仅严重影响患者的生活质量，也极大地增加医疗成本，给家庭和社会带来沉重的经济负担。随着我国老龄化社会的到来，OA的发病人数呈增加趋势，然而目前仍然没有针对OA的特效药物，当病情进展到晚期时，只能接受手术治疗。患者一般在出现膝关节周围不适，包括关节周围红、肿、热、痛，才前往门诊就诊，而上述症状是OA滑膜组织炎症的典型表现，滑膜组织中炎性物质的释放使局部的血管扩张，皮温升高，释放痛性物质造成局部疼痛、发红，并且滑膜液的释放大于吸收，导致关节的肿胀。
滑膜主要由内膜（衬里）和内膜下（衬里下）层组成。衬里层包含巨噬细胞和滑膜成纤维细胞；衬里下层由几层滑膜细胞组成，覆盖在疏松的结缔组织上，富含成纤维细胞、胶原蛋白和其他细胞外基质蛋白。滑膜的一个主要功能是分泌滑液至关节间隙，润滑关节促进关节运动；但是在病理情况下，如滑膜炎，滑膜分泌的促炎因子及软骨基质降解相关的金属基质蛋白酶（MMPs）同样会随滑液运动到关节内各个组成部分，包括关节软骨。滑膜炎与OA的进展密切相关，从机制上分析，关节软骨的降解产物释放到关节腔后可被巨噬细胞摄取成为激活状态，分泌促炎因子，包括IL-1β，TNFα，IL-6 以及软骨细胞外基质降解相关的蛋白酶MMPs（MMP1/3/9/13），ADAMTS4/5，破坏关节软骨，促使更多的关节软骨降解产物释放到关节腔中，进一步刺激巨噬细胞，形成恶性循环。因此，靶向OA早期滑膜巨噬细胞，是延缓OA进展的有效途径。
发明内容
本发明的目的是提出一种CD52+巨噬细胞作为骨关节炎治疗靶点的应用，以解决现有技术中的早中期膝骨关节炎缺乏特异性抗炎药物的问题。
本发明的目的将通过以下技术方案得以实现：
本发明一方面提供了CD52+巨噬细胞作为治疗靶点在筛选或制备治疗骨关节炎的药物中的应用。
本发明另一方面提供了CD52+巨噬细胞的抑制剂在制备治疗骨关节炎的药物中的应用。
进一步的，所述CD52+巨噬细胞的抑制剂为沉默或敲除所述CD52+巨噬细胞的试剂。
进一步的，所述CD52+巨噬细胞的抑制剂为抗体anti-CD52，siCD52#1或siCD52#2中的任意一种。
进一步的，所述药物包括其药学上可接受的载体。
本发明的突出效果为：本发明通过单细胞转录组测序显示，在骨关节炎情况下，炎症性CD52+巨噬细胞数量显著增加，并且特异性高表达CD52（CD52为细胞表面蛋白，主要表达于免疫细胞表面，如淋巴细胞，T细胞，B细胞以及单核细胞，与机体的免疫反应、炎症反应、骨科疾病等相关），在体内水平，通过关节腔注射CD52特异性抗体可以减轻OA滑膜组织的炎症并有效延缓OA的进程，证实CD52+巨噬细胞可以作为骨关节炎的治疗靶点。因此，CD52+巨噬细胞能够作为骨关节炎的一个潜在的有前途的治疗靶点，靶向CD52+巨噬细胞的药物对骨关节炎的治疗具有积极的作用。本发明有助于推动相关靶向巨噬细胞药物的制备，提高相关抗炎药物的疗效，并为新药物研发提供线索。
以下便结合实施例，对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
附图说明
图1A为本发明实施例1中小鼠正常及OA滑膜组织单细胞测序的合并分析UMAP图；
图1B为本发明实施例1中各群巨噬细胞的标志基因表达的UMAP图；
图1C为本发明实施例1中正常及OA组细胞的重叠分析UMAP图；
图1D为本发明实施例1中测序结构分开分析UMAP图；
图1E为本发明实施例1中CD52在各群细胞中表达的UMAP图；
图1F为本发明实施例1中CD52在各群细胞中表达的violin plot图；
图2为本发明实施例1中CD52在对照及OA小鼠滑膜组织中的免疫荧光染色图；
图3为本发明实施例1中CD52在正常及OA患者滑膜组织中的免疫荧光染色图；
图4为本发明实施例1中流式分析图；
图5为本发明实施例2中CD52+巨噬细胞表达炎症相关基因的UMAP分析图；
图6为本发明实施例2中CD52+巨噬细胞相关的生物活性GO分析点图；
图7为本发明实施例2中流式分析图；
图8本发明实施例2中iNOS与CD52免疫荧光共染图；
图9本发明实施例3中CD52的表达结果图；
图10本发明实施例3中Il1的表达结果图；
图11本发明实施例3中Il6的表达结果图；
图12本发明实施例3中Il8的表达结果图；
图13本发明实施例3中iNOS免疫荧光图；
图14为本发明实施例3中膝关节HE染色，Synovitis score分值越高，滑膜组织炎症越严重；
图15为本发明实施例3中软骨组织番红O/快绿染色，Mankin score分值越高，软骨损伤越严重。
实施方式
为使本发明的目的、技术方案及效果更加清楚、明确，以下参照实施例对本发明作进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限本发明。
本发明上下文中所使用的试剂，均通过市售获得。本发明所使用的实验方法均为本领域的常规方法和技术。本发明所有动物实验及相关工作都得到了暨南大学实验动物伦理委员会的批准。
实施例1：单细胞转录组测序显示，OA滑膜组织中CD52+巨噬细胞数量大量增加
通过内侧半月板失稳手术（Destabilization of the Medial Meniscus，DMM）手术构建小鼠创伤性骨关节炎模型。选取3个月大的雄性C57BL/6小鼠，在其右膝进行DMM手术，isoflurane麻醉小鼠，称重，皮下注射5 mg/kg 的Meloxicam，剃除右膝关节附近的毛发，对右膝关节进行消毒。在手术台上铺一条无菌垫，将小鼠固定在无菌垫上，充分暴露右膝关节，固定好右膝关节，在手术时避免晃动。找到髌韧带上方区域，髌韧带为一白色结构，可透过小鼠皮肤观察到，约在膝关节正上方，使用无菌剪刀在髌韧带上方打开一条约5 mm长切口，钝性分离皮肤与皮下组织，用眼科剪沿着髌韧带内侧缘打开膝关节，钝性分离髌下脂肪组织，如出血，可用棉签压迫止血。找到内侧半月板横韧带，利用显微外科专用剪刀完全剪断内侧半月板横韧带，可观察到游离的半月板断端。用7-0的缝合线缝合关节囊，缝一针即可，利用4-0的缝合线缝合皮肤，消毒手术切口，待小鼠苏醒后将小鼠送回动物中心，并进行术后观察。
收集正常以及DMM术后小鼠整个膝关节，保留股四头肌，将股四头肌从股骨近端翻起直至完全暴露膝关节腔，用无菌的镊子取下滑膜组织并放入无菌PBS 中清洗，之后用0.25%含EDTA 的胰酶(Invitrogen) 消化滑膜组织30 分钟，再用300 U/mL 的I 型胶原酶(Worthington Biochemical) 消化滑膜组织45 分钟，将所有的消化液通过直径为70 μm直径的过滤器(Fisher Scientific)过滤，滤液在300 g 离心5 分钟后，去除上清，用含0.04% BSA 的PBS溶液重悬细胞，充分吹打均匀后进行细胞计数，将细胞密度调整到800-1200 细胞/μl，进行10x Genomics 单细胞转录组测序分析，主要包括以下流程：1）标记cDNA 片段，2）测序，3）对原始数据进行预处理，4）利用Seurat进行下游分析，包括差异表达基因GO term、KEGG pathway、GSEA 富集分析以及转录调控网络分析。
选取巨噬细胞群合并分析，可将巨噬细胞分为3群（图1A和1B）。将正常（Normal）及骨关节炎组（OA）分开分析，发现相比于正常组，OA组第一群巨噬细胞数量明显增加（图1C和1D, cluster 1），并且特异性高表达CD52，而正常小鼠滑膜巨噬细胞中基本不表达CD52（图1E和1F），因此将第一群巨噬细胞命名为CD52+巨噬细胞。
对正常及OA小鼠膝关节组织冰冻切片进行免疫荧光染色，用免疫组化笔圈出染色区域，用3%的BSA稀释一抗CD52，iNOS在4℃孵育一抗过夜，PBST清洗3次后，用3%的BSA稀释二抗，室温孵育2小时，PBST清洗3次后，用DAPI Fluoromount-G封片液(Southern Biotech)将玻片封在载玻片上，在共聚焦显微镜(Zeiss LSM 710)下观察并拍照。结果如图2所示，相比于正常组，OA组小鼠滑膜组织中F4/80+CD52+的巨噬细胞明显增加。
同样的方法，对正常及OA患者的滑膜组织冰冻切片进行免疫荧光染色，免疫荧光结果如图3所示，相比于正常组，OA患者的滑膜组织中F4/80+CD52+的巨噬细胞也明显增加。
利用LPS（50 ng/ml）刺激小鼠Raw 264.7巨噬细胞系24 h后，细胞流式分析结果如图4所示，F4/80+CD52+的巨噬细胞同样显著增加。
实施例2：CD52+巨噬细胞为炎症型巨噬细胞
对CD52+巨噬细胞进行基因表达分析，结果如图5所示，该群巨噬细胞高表达炎症相关基因，如Cd86, Irf7, Cytip, Il1b, Il1r2, Il7r。GO分析如图6所示，CD52+巨噬细胞与炎症反应相关的生物学行为有关，如白细胞粘附（Leukocyte cell-cell adhesion）,白细胞增殖（Leukocyte proliferation），淋巴细胞增殖（Lymphocyte proliferation），单核细胞增殖（mononuclear cell proliferation）。将20万个Raw 264.7巨噬细胞种于12孔板，并利用LPS（50 ng/ml）刺激24 h后，PBS清洗3次，冰上孵育CD52以及CD80流式抗体45分钟，进行流式分析，结果如图7所示，CD52+巨噬细胞高表达验证相关基因CD80。免疫荧光染色（方法同上），结果如图8所示，而相比于对照组，OA小鼠滑膜组织中CD52+细胞同样高表达另一种炎症相关蛋白iNOS。综上可知，CD52+巨噬细胞为炎症型巨噬细胞。
实施例3：在体内及体外水平验证沉默CD52+巨噬细胞对滑膜组织炎症以及OA进程的影响
利用CD52的特异性siRNA沉默CD52的表达。将Raw 264.7巨噬细胞按照2*105个/孔的细胞密度种于六孔板中，24 h后，配制瞬转的试剂（配制前，先把所用试剂震荡离心，然后把lipo2000 5 μL，所溶解RNA干扰序列5 μL放入到无血清培养基100 μL中）。3）配好后，离心震荡，室温孵育15 min。4）15 min后，向已经配好转染复合体中加900 μL无血清培养基。5）混匀，把1 mL转染液体加入6孔板中，孵育4 h后，换成2 mL完全培养基。直至72 h后，提取RNA进行qPCR实验，检测炎症基因IL1，IL6，IL8，iNOS的表达。所述siRNA序列为：
CD52-sense#1 5- GAUACAAACAGGAUCCUUGTT -3；
CD52-anti-sense#1 5-CAAGGAUCCUGUUUGUAUCTT-3；
CD52-sense#2 5- CCCACGGGAAGGGUUGAUATT -3；
CD52-anti-sense#2 5- UAUCAACCCUUCCCGUGGGTT -3。
结果如图9-12所示，在巨噬细胞中能有效抑制CD52的表达，并且敲低CD52的表达能有效降低炎症因子IL1，6，8的表达水平。
为了进一步炎症沉默CD52对膝关节内炎症反应的影响，我们采取了另一种策略，即在通过DMM手术后，立即开始关节腔注射PBS或CD52的特异性中和抗体anti-CD52（体积：10 μl），使其局部作用于小鼠关节腔内，每3周重复一次，共持续12周，之后进行组织学分析。免疫荧光染色（方法同上），结果如图13所示，anti-CD52处理组滑膜组织中，炎症因子iNOS的表达量明显少于PBS组。对膝关节组织样品进行HE染色，经过脱蜡水化后，将切片放到苏木精染色液中染色10分钟，之后将切片放到流水中清洗5分钟，随后将切片放置于5%的乙酸中酸化1分钟，酸化后将切片放在流水中清洗5分钟，然后将切片放在伊红染色液中染色1分钟，根据染色情况，可以适当延长或缩短染色时间，梯度酒精脱水后，二甲苯透明并封片，在室温风干切片，显微镜下观察，结果如图14所示，发现anti-CD52处理组的滑膜炎评分明显低于PBS组，证明抑制CD52+巨噬细胞可以减轻DMM术后的膝关节炎症。对膝关节组织样品进行番红O染色，经过脱蜡水化后，将切片放置于0.05%快绿染色液中染色3分钟，在吸水纸上去除多余的快绿染色液，将切片放置在1%的乙酸溶液中酸化10-15秒。酸化后，将切片放在1%的番红O染色液中染色10分钟，梯度酒精脱水后，二甲苯透明并封片，在室温风干切片，显微镜下观察发现，结果如图15所示，PBS组呈现出明显的软骨组织损伤及缺失，而anti-CD52组的软骨组织较完整，只存在轻微的软骨损伤，OA相关的Mankin Score数值更低，表明抑制CD52+巨噬细胞能够有效延缓小鼠OA的进程。
综上所述，这些结果表明，CD52+巨噬细胞可以作为治疗靶点用于筛选或制备治疗骨关节炎的药物。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (5)
1. CD52+巨噬细胞作为治疗靶点在筛选或制备治疗骨关节炎的药物中的应用。
2. CD52+巨噬细胞的抑制剂在制备治疗骨关节炎的药物中的应用。
3.根据权利要求2所述的应用，其特征在于：所述CD52+巨噬细胞的抑制剂为沉默或敲除所述CD52+巨噬细胞的试剂。
4.根据权利要求3所述的应用，其特征在于：所述CD52+巨噬细胞的抑制剂为抗体anti-CD52，siCD52#1或siCD52#2中的任意一种。
5.根据权利要求1或2所述的应用，其特征在于：所述药物包括其药学上可接受的载体。
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| CN117180443B (zh) * | 2023-10-23 | 2024-03-22 | 暨南大学附属第一医院（广州华侨医院） | 滑膜肌成纤维细胞的细胞膜在制备骨关节炎药物的用途 |
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