CN116286996A - 一种靶向cxcl16+巨噬细胞的药物载体及其制备方法和应用 - Google Patents
一种靶向cxcl16+巨噬细胞的药物载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向CXCL16+巨噬细胞的药物载体及其制备方法和应用。本发明首先构建了过表达CXCR6的质粒并将其转染至293T细胞中，使293T细胞膜表面过表达CXCR6，最后提取293T细胞膜作为药物载体，从而构建通过CXCR6靶向CXCL16+巨噬细胞的药物载体（MembraneCXCR6）。MembraneCXCR6可进一步负载抗炎药物。本发明有助于推动相关靶向抗炎药物的制备，提高骨性关节炎相关药物的疗效，并为新药物研发提供线索。
Description
技术领域
本发明涉及生物医用领域，尤其涉及一种靶向CXCL16+巨噬细胞的药物载体及其制备方法和应用。
背景技术
膝骨关节炎（Osteoarthritis，OA）是老年人群中常见的关节退行性疾病，导致下肢功能障碍，极大地影响患者的生活质量。目前正逐步进入老龄化社会，65岁及以上人群膝OA的患病率约为50.0%，而在75岁及以上人群中，膝OA的患病率则达到80.0%，将面对巨大的OA患者的社会经济压力。然而目前并没有针对OA的特效药物，晚期OA患者只能接受手术治疗。
OA被认为是一种“全关节疾病”，其病变包括软骨退变磨损、半月板变形、韧带撕裂、软骨下骨硬化以及滑膜组织的炎症等。滑膜组织炎症是OA的典型表现，包括关节周围红、肿、热、痛。关节软骨出现形态学改变之前，滑膜组织已存在明显的炎症反应，巨噬细胞是滑膜组织中的主要炎症反应细胞，滑膜组织炎症刺激巨噬细胞产生MMP13、ADAMTS5降解软骨加剧OA进展；另外，巨噬细胞分泌大量可溶性促炎因子，包括IL-1β，TNF-α等，作用于滑膜细胞、免疫细胞等，进一步加重关节内的炎症反应，招募更多的巨噬细胞，从而形成关节内炎症反应的恶性循环。因此，靶向OA早期滑膜巨噬细胞，是延缓OA进展的有效途径。
单细胞转录组测序结果显示，在OA的进展过程中，滑膜中巨噬细胞大量增加，并特异性高表达CXCL16，而CXCL16可以表达于细胞膜表面，并与CXCR6特异性结合，因此，本发明通过构建过表达CXCR6质粒，并将此质粒转染至293T细胞中，使293T细胞膜表面高表达CXCR6，提取该细胞膜作为药物载体（MembraneCXCR6），可达到靶向CXCL16+巨噬细胞的目的，MembraneCXCR6可进一步负载抗炎药物，从而制备靶向巨噬细胞抗炎药物，减轻膝关节内炎症反应及OA进展。
发明内容
本发明的目的是提出一种靶向CXCL16+巨噬细胞的药物载体及其制备方法和应用，以解决现有技术中的膝骨关节炎缺乏特异性靶向抗炎药物的问题，同时提供了一种新的膝关节滑膜组织炎症的治疗靶点。
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一种靶向CXCL16+巨噬细胞的药物载体的制备方法，包括以下步骤：
步骤1、构建过表达CXCR6的重组质粒；
步骤2、将步骤1得到的重组质粒转染至293T细胞中，使293T细胞膜高表达CXCR6；
步骤3、提取293T细胞膜作为药物载体，得到靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6。
进一步的，所述步骤1具体包括：
以初始骨架载体pKD-EF1a-IRES2-Puro为基础，将标签基因HA和绿色荧光基团EGFP以PCR方法扩增，构建pKD-EF1a-HA-EGFP-IRES2-Puro空载质粒；
pKD-EF1a-HA-EGFP-IRES2-Puro经限制性内切酶BamH I和EcoR I 双酶切后回收，DNA重组连接酶将小鼠CXCR6基因插入到线性化的空载质粒上，构建pKD-EF1a-HA-EGFP-mCXCR6-IRES2-Puro重组质粒。
进一步的，所述步骤2具体包括：
进行慢病毒包装，将步骤1得到的重组质粒转染入293T细胞中，使293T细胞膜表面过表达CXCR6。
进一步的，所述pKD-EF1a-HA-EGFP-IRES2-Puro空载质粒的上游引物如 SEQ IDNo .1所示，所述pKD-EF1a-HA-EGFP-IRES2-Puro的下游引物如SEQ ID No .2所示。
进一步的，所述CXCR6的引物如SEQ ID No .1、SEQ ID No .3、SEQ ID No .4和SEQID No .5所示。
本发明另一方面提供了上述的制备方法得到的靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6。
本发明另一方面提供了上述的制备方法得到的靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6在制备治疗膝关节骨性关节炎的药物或药物组合物中的应用。
进一步的，在所述靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6上偶联抗炎药物。
本发明的突出效果为：
本发明首先构建了过表达CXCR6的质粒并将其转染至293T细胞中，使293T细胞膜表面过表达CXCR6，最后提取293T细胞膜作为药物载体，从而构建通过CXCR6靶向CXCL16+巨噬细胞的药物载体（MembraneCXCR6）。MembraneCXCR6可进一步负载抗炎药物。本发明有助于推动相关靶向抗炎药物的制备，提高骨性关节炎相关药物的疗效，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中正常及OA小鼠滑膜炎HE染色及评分；
图1B为本发明实施例2中合并分析正常及OA小鼠滑膜组织单细胞转录组测序后整合分析UMAP分群图；
图1C为本发明实施例2中各群细胞的标志基因表达；
图2A为本发明实施例3中正常及OA小鼠滑膜组织单细胞转录组测序后分开分析UMAP图；
图2B为本发明实施例3中正常及OA小鼠UMAP图整合分析，显示各细胞群的差异情况；
图2C为本发明实施例3中正常及OA小鼠小提琴图，显示相比于Normal组，OA组巨噬细胞中CXCL16表达增加；
图2D为本发明实施例3中正常及OA小鼠UMAP图分析，显示相比于Normal组，OA组巨噬细胞中CXCL16表达增加；
图3为本发明实施例3中Control及OA小鼠滑膜组织中CXCL16的免疫荧光染色结果图；
图4为本发明实施例3中Control及OA患者滑膜组织中CXCL16的免疫荧光染色结果图；
图5为本发明实施例4空载质粒图谱；
图6为本发明实施例4 pKD-EF1a-HA-EGFP-mCXCR6-IRES2-Puro重组质粒图谱；
图7为本发明实施例5中靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6的结构示意图；
图8为本发明实施例6的EGFP（MembraneCXCR6）荧光信号在Raw 264.7细胞分布图；
图9为本发明实施例7的OA小鼠关节腔内注射MembraneCXCR6后，CXCL16免疫荧光染色结果图；
图10为本发明实施例8中MembraneCXCR6-PLGA-Capsaicin合成及结构示意图；
图11A为本发明实施例9中OA关节腔注射PBS，Capsaicin以及MembraneCXCR6-PLGA-Capsaicin后，软骨组织番红O/快绿染色染色结果图；
图11B为本发明实施例9中OA严重程度的Mankin score，分值越高，OA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的试剂，均通过市售获得。本发明CXCR6基因根据NCBI网站查找小鼠CXCR6(NM_030712.4)的CDS区序列，由小鼠cDNA序列经PCR扩增而得；pKD-EF1a-IRES2-Puro质粒由中国科学院广州生物医药与健康研究院赠。本发明所使用的实验方法，如分子克隆、细胞实验及动物实验等均为本领域的常规方法和技术。对于动物实验，相关程序及方法符合医学伦理要求。本发明所使用的实验方法均为本领域的常规方法和技术。
实施例1：DMM术后滑膜组织出现明显的炎症反应
此项研究中进行的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。为了研究巨噬细胞在不同程度滑膜炎中的特点，申请人利用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。收集正常小鼠以及术后7天小鼠膝关节滑膜组织样品进行HE染色，发现DMM手术引起滑膜组织的急性炎症反应，滑膜炎评分明显增加（如图1A所示）。
实施例2：不同炎症程度滑膜炎组织单细胞转录组学测序。
收集正常小鼠以及DMM术后7天小鼠膝关节滑膜组织样品，提取细胞进行单细胞转录组测序。对单细胞测序结果进行分析。将2批样品进行整合分析、预处理和质控，根据标志基因的表达，对巨噬细胞及间充质系细胞进行分析，得到肌成纤维细胞（Myofibroblast，cluster 0），成脂前体细胞（Pre-adipocyte，cluster 1），巨噬细胞1（Macrophage 1,cluster 2），滑膜衬里层细胞（Synovial lining layer cell，cluster 3），巨噬细胞1（Macrophage 1, cluster 4），早期间充质祖细胞（Early mesenchymal progenitor，EMP，cluster 5），滑膜衬里下层细胞（Synovial sublining layer cell，cluster 6）（如图1B和图1C所示）。
实施例3：OA滑膜组织中，CXCL16+巨噬细胞数量大量增加
将2批单细胞测序结果分开分析，发现相比于正常组，OA组巨噬细胞数量明显增加（图2A和B, cluster 2 and 4），并且特异性高表达CXCL16（图2C和D），故称为CXCL16+巨噬细胞。免疫荧光染色显示，在OA小鼠滑膜组织中，F4/80+CXCL16+的巨噬细胞数量也明显增加（图3）。同样，免疫荧光也显示，相比于对照组，滑膜炎患者的滑膜组织中F4/80+CXCL16+的巨噬细胞也明显增加（图4）。
实施例4：CXCR6稳定细胞系筛选体系建立
1、引物设计：
设计pKD-EF1a-HA-EGFP-IRES2-Puro空载质粒的引物对pKD ( BamH I)-F和pKD( EcoR I )-R的序列如SEQ ID No .1和SEQ ID No .2所示；设计CXCR6基因的多片段克隆扩增引物pKD ( BamH I)-F、pKD( HA-GFP+)-R1、pKD( HA-GFP+)-F2和pKD( EcoR I )-R2如SEQ ID No .1、SEQ ID No .3、SEQ ID No .4和SEQ ID No .5所示。
pKD ( BamH I)-F，SEQ ID No .1：
taaactacgggatccatgtacccatacgacgtcccagactacgctggcggccgcatggtgagcaagggcgag；
pKD ( EcoR I )-R，SEQ ID No .2：
gtcgtccttgtagtcgaattcttacttgtacagctcgtccatgcc；
pKD( HA-GFP+)-R1，SEQ ID No .3:
gcctccgctgcctcccttgtacagctcgtccatgcc；
pKD( HA-GFP+)-F2，SEQ ID No .4:
acaagggaggcagcggaggcaggcgcgccgatgatgggcatcaagagtcagc；
pKD( EcoR I )-R2, SEQ ID No .5:
gtcgtccttgtagtcgaattcctacaattggaacatactggtggtct。
2、构建重组质粒：
以初始骨架载体pKD-EF1a -IRES2-Puro为基础，将HA和EGFP以PCR方法扩增的。pKD-EF1a -IRES2-Puro经限制性内切酶BamH I和EcoR I 双酶切后回收，DNA重组连接酶将HA标签和EGFP荧光基团的基因插入到线性化的初始骨架载体pKD-EF1a -IRES2-Puro上，构建pKD-EF1a-HA-EGFP-IRES2-Puro空载质粒，质粒图谱如图5所示。
以小鼠鼠尾提取cDNA为模板，PCR技术扩增小鼠CXCR6基因。pKD-EF1a-HA-EGFP-IRES2 - Puro经限制性内切酶BamH I和EcoR I 双酶切后回收，DNA重组连接酶将小鼠CXCR6基因插入到线性化的空载载体上，构建pKD-EF1a-HA-EGFP-mCXCR6-IRES2-Puro重组质粒，质粒图谱如图6所示。
实施例5：构建靶向CXCL16+的巨噬细胞的药物载体MembraneCXCR6
1、稳定细胞系的构建方式是将CXCR6重组质粒（或者空载质粒）、包装质粒psPAX2和pMD2.G以4:3:1的比例，通过lipo2000转染试剂转染293T细胞，转染48 h后，收集病毒上清，用 0.45 μm 滤器过滤，将病毒液加入293T细胞中48h后换液，并加入含嘌呤霉（3μg /mL）的培养基继续进行筛选；
2、通过细胞膜和细胞浆蛋白提取试剂盒试剂和1x 蛋白酶抑制剂充分裂解过表达有CXCR6的细胞；
3、把样品在液氮和室温依次反复冻融3次，4℃，14000 g离心30分钟，以得到膜碎片MembraneCXCR6。
实施例6：Raw 264.7巨噬细胞体外摄取MembraneCXCR6
将10万个Raw 264.7巨噬细胞种于12 孔板中，6 h后加入MembraneCXCR6，处理24 h后，用PBS清洗3次，每次5 min，之后用4%的多聚甲醛室温固定10 min，PBS再次清洗后，加入1 ml的PBS，在共聚焦显微镜下观察，发现Raw 264.7巨噬细胞可以很好地摄取MembraneCXCR6， 并且MembraneCXCR6主要位于细胞膜表面。（如图8所示）。
实施例7：MembraneCXCR6靶向小鼠滑膜炎症组织中CXCL16+巨噬细胞
利用DMM手术构建OA模型后，通过关节腔注射技术将MembraneCXCR6输送到小鼠关节腔内，使其作用于膝关节局部组织，24 h后收集小鼠膝关节标本并制作冰冻切片，进行CXCL16免疫荧光染色，结果如图9显示，MembraneCXCR6与CXCL16存在明显的共定位现象，表明MembraneCXCR6可以很好地靶向CXCL16+巨噬细胞。
实施例8：构建靶向CXCL16+巨噬细胞的靶向抗炎药物MembraneCXCR6-Capsaicin
首先构建载有Capsaicin 的PLGA纳米载体，具体步骤如下：1) 取10mg PLGA颗粒溶于700 μL二氯甲烷及300 μL乙酸乙酯混合有机溶剂中； 2) 在混有PLGA的有机溶剂中加入溶解在DMSO溶剂中的Capsaicin药物；3) 混合物于水浴超声5分钟后，将声探头置于配置好的3%聚乙烯醇（PVA）溶液中，功率设置为50w, 开3s, 关2s。在超声刺激下，将PLGA-Capsaicin有机溶剂混合物逐滴PVA溶液中充分乳化5分钟，形成“水包油”混合体系；4) 将充分乳化后的有机体系置于0.3%的PVA溶液中，磁力搅拌4小时，从而充分挥发有机溶剂；5)待充分搅拌后，将锥形瓶中的溶液倒入50 mL离心管中，配平离心12000 rpm, 40分钟，第一次离心结束后，倒出溶液，PBS重悬沉淀，再次离心12000 rpm, 40分钟，此步骤重复2遍，待第二次PBS重悬离心后，倒出管中液体，用1mL PBS重悬沉淀后，置于4度冰箱保存；6) 将MembraneCXCR6与规定重量比的PLGA-Capsaicin混合，连续涡旋5 min，超声5 min；7) 使用Miniextruder (Avanti)，将混合物与400 nm、200 nm、100 nm聚碳酸酯薄膜前后共挤15次，得到MembraneCXCR6膜包覆的PLGA NPs (MembraneCXCR6-PLGA-Capsaicin)（其合成及结构示意图如图10所示）。
实施例9：MembraneCXCR6-PLGA-Capsaicin有效延缓小鼠OA的进程
在体内水平验证MembraneCXCR6-Capsaicin对小鼠OA进程的影响。通过DMM手术后，立即开始关节腔注射PBS，Capsaicin（浓度：50 μM，体积：15 μl），MembraneCXCR6-PLGA-Capsaicin（浓度：50 μM，体积：15 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。结果如图11A和11B所示，番红O染色显示，PBS以及单纯Capsaicin组呈现出明显的软骨组织破坏，而MembraneCXCR6-PLGA-Capsaicin组的软骨组织较完整，OA严重程度的Mankin score，数值越高代表OA越严重。以上结果提示MembraneCXCR6-PLGA-Capsaicin能够有效延缓OA的进展。
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 (8)
1.一种靶向CXCL16+巨噬细胞的药物载体的制备方法，其特征在于包括以下步骤：
步骤1、构建过表达CXCR6的重组质粒；
步骤2、将步骤1得到的重组质粒转染至293T细胞中，使293T细胞膜高表达CXCR6；
步骤3、提取293T细胞膜作为药物载体，得到靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6。
2.根据权利要求1所述的制备方法，其特征在于：
所述步骤1具体包括：
以初始骨架载体pKD-EF1a-IRES2-Puro为基础，将标签基因HA和绿色荧光基团EGFP以PCR方法扩增，构建pKD-EF1a-HA-EGFP-IRES2-Puro空载质粒；
pKD-EF1a-HA-EGFP-IRES2-Puro经限制性内切酶BamH I和EcoR I 双酶切后回收，DNA重组连接酶将小鼠CXCR6基因插入到线性化的空载质粒上，构建pKD-EF1a-HA-EGFP-mCXCR6-IRES2-Puro重组质粒。
3.如权利要求1所述的制备方法，其特征在于：
所述步骤2具体包括：
进行慢病毒包装，将步骤1得到的重组质粒转染入293T细胞中，使293T细胞膜表面过表达CXCR6。
4. 如权利要求2所述的制备方法，其特征在于：所述pKD-EF1a-HA-EGFP-IRES2-Puro空载质粒的上游引物如 SEQ ID No .1所示，所述pKD-EF1a-HA-EGFP-IRES2-Puro的下游引物如SEQ ID No .2所示。
5. 如权利要求2所述的制备方法，其特征在于：所述CXCR6的引物如SEQ ID No .1、SEQID No .3、SEQ ID No .4和SEQ ID No .5所示。
6.根据权利要求1-5任一项所述的制备方法得到的靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6。
7.根据权利要求1-5任一项所述的制备方法得到的靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6在制备治疗膝关节骨性关节炎的药物或药物组合物中的应用。
8.根据权利要求7所述的应用，其特征在于：在所述靶向CXCL16+巨噬细胞的药物载体MembraneCXCR6上偶联抗炎药物。
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| CN117180443A (zh) * | 2023-10-23 | 2023-12-08 | 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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| CN117018229A (zh) * | 2023-08-14 | 2023-11-10 | 暨南大学附属第一医院（广州华侨医院） | 过表达Clec1b蛋白质细胞膜在制备膝骨关节炎药物的用途 |
| CN117018229B (zh) * | 2023-08-14 | 2024-05-24 | 暨南大学附属第一医院（广州华侨医院） | 过表达Clec1b蛋白质细胞膜在制备膝骨关节炎药物的用途 |
| CN117180443A (zh) * | 2023-10-23 | 2023-12-08 | 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 |
| 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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