CN115414360A - 全反式维甲酸联合抗生素在治疗假体周围感染中的应用 - Google Patents
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Abstract
本发明公开了全反式维甲酸联合抗生素在治疗假体周围感染中的应用。本发明通过构建小鼠假体周围感染模型，验证了全反式维甲酸联合抗生素在治疗假体周围感染的应用中具有良好的治疗效果，克服了目前临床上采用抗生素治疗假体周围感染的效果不佳的技术问题。本发明将全反式维甲酸与抗生素联用能够从根本上改善假体周围感染免疫系统低下的状态，从而增强机体免疫细胞的主动杀菌作用，同时增强外源性抗生素杀菌效力；此外，全反式维甲酸与抗生素联用不仅可以减少抗生素的使用疗程和剂量，相应的还避免了细菌的耐药突变，最终提高了小鼠假体周围感染的治愈率，本发明为临床上有效控制假体周围感染提供了新的治疗方案。
Description
技术领域
本发明涉及全反式维甲酸联合抗生素在治疗假体周围感染中的应用，属于生物医药领域。
背景技术
假体周围感染是人工关节置换术后的灾难性并发症，随着人口老龄化的加剧，每年人工关节置换术的需求量日益增长，而与之伴随发生的假体周围感染也居高不下。其中，由金黄色葡萄球菌导致的假体周围感染是发病率最高，骨组织破坏力最大的假体周围感染。目前，假体周围感染临床治疗的金标准仍为二期翻修手术联合大剂量抗生素长期治疗。即使在翻修的围手术期，全身和局部使用了足量敏感抗生素，二期翻修手术的感染控制成功率并不满意，再感染发生率高达25％。
而导致这一失败结果的原因与以下两方面相关，即细菌的逃逸和机体主动免疫微环境的抑制。细菌的逃逸归因于大剂量抗生素长疗程的使用，易迫使细菌产生耐药突变和在假体表面形成生物膜，从而极大的限制抗生素的被动杀伤。而机体免疫微环境的抑制归因于，手术创伤和假体的植入破坏了膝关节组织局部的免疫微环境平衡，使得免疫细胞的杀菌作用降低。此外，细菌及其相关毒素的存在，能进一步地促进骨髓来源的免疫抑制细胞(MDSC)的产生，从而分泌抑炎性细胞因子，使得具有主动杀菌作用的免疫细胞功能耗竭或者凋亡。
目前临床上关于假体周围感染的治疗方案，往往忽略了对于免疫抑制微环境的改善，轻视了机体免疫细胞的主动杀菌作用。而临床上仅依靠抗生素的被动杀菌作用，不仅很容易导致假体周围感染治疗的疗效不佳，容易诱导细菌耐药突变，还无法对假体周围感染进行长期控制，带来感染高复发率。因此，一个具有主动杀菌能力的机体免疫防御系统对于假体周围感染的长期控制显得至关重要。
全反式维甲酸(ATRA)是维生素A的代谢中间产物，在临床上，全反式维甲酸具有广泛的抗增殖及促分化活性，能够抑制多种恶性肿瘤的生长。例如，全反式维甲酸联合三氧化二砷可以有效治疗急性早幼粒细胞白血病，增加患者生存率。机制上，全反式维甲酸被认为既可以通过抑制MDSCs的扩增，又可以诱导MDSCs向成熟免疫细胞分化，来提高机体主动免疫作用。同样，在感染领域，全反式维甲酸也被证明可通过影响脓毒症相关的MDSCs的扩增和功能，降低其对其他免疫细胞功能的抑制，逆转脓毒症后期的免疫抑制状态，提高机体对二次继发性感染的预防效果。然而，其在改善假体周围感染免疫抑制微环境，增强抗生素疗效方面的应用尚无研究报道。
发明内容
本发明的目的在于针对现有技术中的抗生素对于治疗假体周围感染的效果不佳的技术问题，提供一种能够从根本上改善假体周围感染免疫系统低下的状态，从而增强机体免疫细胞的主动杀菌作用，同时增强外源性抗生素杀菌效力的医药方案。
为了实现上述目的，本发明提供了全反式维甲酸联合抗生素在制备预防和/或治疗假体周围感染的药物中的应用。全反式维甲酸联合抗生素作为联用药物可以提高机体免疫细胞的主动杀菌作用，增加抗生素被动杀菌的效力，减轻药物的使用疗程和剂量。所述全反式维甲酸联合抗生素包括：全反式维甲酸与抗生素同时给药治疗；或先单独采用抗生素进行治疗后再采用全反式维甲酸进行单独治疗。
优选地，所述假体周围感染包括膝关节假体周围感染，髋关节假体周围感染以及肩关节假体周围感染中的任一种。
优选地，其特征在于，所述抗生素包括苯唑西林。
本发明还提供了全反式维甲酸联合抗生素在制备假体周围感染后用于抑制免疫抑制细胞的产生和/或免疫抑制性炎症因子的表达的药物中的应用。
本发明还提供了一种用于预防和/或治疗假体周围感染的药物，所述的药物包括药学上可接受的载体和有效量的活性成分，所述活性成分包括全反式维甲酸和抗生素。
与现有技术相比，本发明的有益效果在于：
本发明将全反式维甲酸与抗生素联用能够从根本上改善假体周围感染免疫系统低下的状态，从而增强机体免疫细胞的主动杀菌作用，同时增强外源性抗生素杀菌效力；此外，全反式维甲酸与抗生素联用不仅可以减少抗生素的使用疗程和剂量，相应的还避免了细菌的耐药突变，最终提高了小鼠假体周围感染的治愈率，因此，本发明将全反式维甲酸与抗生素联用为临床上有效控制假体周围感染提供了新的治疗方案。
附图说明
图1显示了全反式维甲酸显著改善了抗生素苯唑西林治疗假体周围感染的疗效；其中，A为各组小鼠膝关节假体周围感染的大体形态照片；B-C为各组小鼠的膝关节假体周围感染X线图，及其影像学评分；D为各组小鼠膝关节假体周围感染，植入物表面的细菌及生物膜形态的扫描电镜图；E-F为各组小鼠膝关节假体周围感染，植入物表面的细菌涂板及定量计数；G为各组小鼠膝关节假体周围感染，植入物周围软组织的细菌涂板定量计数；
图2显示了全反式维甲酸降低了免疫抑制细胞PMN型MDSC的数量，改善了假体周围感染免疫微环境的抑制，从而提高了抗生素治疗假体周围感染的疗效；其中，A为膝关节假体周围感染小鼠的脾脏大体观察结果；B-C为小鼠脾脏中免疫抑制性细胞MDSC比例的流式细胞学分析及其定量结果；D-E为小鼠感染侧骨髓中免疫抑制性细胞MDSC比例的流式细胞学分析及其定量结果；F为小鼠假体周围感染膝关节软组织中的免疫抑制性炎症因子IL-10的水平定量分析；
以上各图中，*表示经过统计学分析，两组间具有显著性差异，P<0.05；**表示经过统计学分析，两组间具有显著性差异，P<0.01；***表示经过统计学分析，两组间具有显著性差异，P<0.001。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
下述实施例中采用的试剂及实验方法如下：
分子生物化学试剂：全反式维甲酸(ATRA)、苯唑西林(oxacillin)、玉米油等为MCE(美国)，CD11b，Ly6c，Ly6G等抗体为Cell Signaling Technology(美国)，金黄色葡萄球菌ATCC 25923为ATCC(美国)，胰酪大豆胨液体培养基(TSB)、胰蛋白胨大豆琼脂(TSA)为海博生物(中国)，2.5％的戊二醛电镜固定液为索莱宝(中国)。
实施例1
小鼠假体周围感染模型的建立与给药处理：
(1)金黄色葡萄球菌悬液的配制和定量：将冻存于-80℃冰箱的金黄色葡萄球菌标准菌株ATCC 25923取出，然后取1毫升的菌液加入到20ml新鲜配制的无菌的TSB溶液中，置于37℃，220转/分钟的摇床上孵育过夜。待液体浑浊，细菌扩增后，再取1mL的细菌悬液加入到无菌的20mL的TSB中，继续置于摇床上孵育4小时，使细菌达到对数生长期。随后，用分光光度计对细菌的浓度进行定量，使稀释后的细菌浓度为106CFU/mL。Aseptic组的小鼠膝关节假体植入术后，仅滴入无菌生理盐水。
(2)小鼠膝关节假体植入术：将8-12周龄的雄性小鼠分别称重后，腹腔注射1％的戊巴比妥钠溶液进行麻醉(剂量50mg/kg)。随后，用小动物剃毛刀将小鼠左膝关节周围的毛发剔除，并用酒精清洁，碘伏消毒膝关节手术区域，铺无菌巾。在左膝关节正中做一长约1.5cm切口，钝性分离皮下组织，并将髌骨向外侧脱位，充分暴露胫骨平台。随后，在切除半月板和交叉韧带后，用直径为0.6mm的克氏针对胫骨平台沿着长轴方向进行钻孔。待小鼠膝关节假体顺利安装完成后，向膝关节腔滴入100uL的处于对数生长期的细菌悬液(浓度为106CFU/mL)，逐层缝合肌肉，韧带，皮肤切口。最后将手术后的小鼠置于恒温加热毯上，直到其麻醉苏醒。整个手术过程约10分钟左右。
在第一部分实验中，将Balb/c背景的小鼠，共随机分为5个实验组：
Aseptic组：行膝关节假体植入，无细菌感染术后，给予每日一次腹腔注射生理盐水溶液，持续4周；
Infected+Vehicle组：行膝关节假体植入，金葡菌感染术后，给予每日一次腹腔注射生理盐水溶液，持续4周；
Infected+Oxacillin组：行膝关节假体植入，金葡菌感染术后，给予每日一次腹腔注射苯唑西林溶液，持续4周；
Infected+ATRA组：行膝关节假体植入，金葡菌感染术后，给予每日一次局部注射全反式维甲酸溶液，持续4周；
Infected+ATRA+Oxacillin组：行膝关节假体植入，金葡菌感染术后，给予每日一次腹腔注射苯唑西林溶液，同时每日一次局部注射全反式维甲酸溶液，持续4周。
术后ATRA和Oxacllin给药：根据生产商的说明，全反式维甲酸按照体积比1:9的比例配制DMSO/玉米油溶剂，苯唑西林钠用生理盐水配制成溶剂。将全反式维甲酸按50mg/kg/d的剂量，苯唑西林按照80mg/kg/d的剂量，每天上午11点注射，持续4周。而对照组注射相应体积的生理盐水溶剂。
小动物膝关节假体X线评价：将小鼠用体积百分比浓度为1％戊巴比妥钠腹腔注射麻醉后，俯卧位放于X线检查台上，并适当调整探测器与小鼠左膝关节的位置。使用Bruker公司X线机器检测小鼠膝关节假体的位置和感染区域骨的炎症反应改变。各组小鼠检查的仪器参数设置保持相同，并由同一人进行图像采集和计算。
小动物膝关节假体细菌学评价：将小鼠置于可封闭的装置，加入二氧化碳安乐死后。解剖分离其左侧膝关节，随后，取出其中3只膝关节假体并放入4℃，2.5％的戊二醛电镜固定液中浸泡过夜，以进行Hitachi公司的扫描电镜检查。此外，为了进行假体周围的细菌定量计数，将其余的小鼠膝关节假体和假体周围的软组织分别取出，放入无菌的生理盐水中，震荡稀释后，涂布TSA平板，以进行假体表面和假体周围软组织的细菌计数。各组小鼠检查的仪器参数设置保持相同，并由同一人进行图像采集和计算。
Balb/c背景的小鼠进行膝关节假体周围感染后，使用小动物X线和细菌学评价分析膝关节假体周围感染区域的骨组织炎症改变，发现Infected+ATRA+Oxacillin组小鼠的骨组织炎症改变程度较Infected+Vehicle组和Infected+Oxacillin组的显著减轻。
进一步的统计分析小鼠膝关节细菌数量的改变，发现Infected+ATRA+Oxacillin组小鼠的细菌数量较Infected+Vehicle组和Infected+Oxacillin组的显著减轻。
图1A为各组小鼠膝关节假体周围感染的大体形态照片。Infected+Vehicle组和Infected+Oxacillin组的小鼠的膝关节周围脓肿较Infected+ATRA+Oxacillin组小鼠的脓肿显著增大。
图1B-C为各组小鼠的膝关节假体周围感染X线图，及其影像学评分，可见Infected+Vehicle组和Infected+Oxacillin组小鼠的感染膝关节炎症反应较Infected+ATRA+Oxacillin组小鼠显著增加。
图1D为各组小鼠膝关节假体周围感染，植入物表面的细菌及生物膜形态的扫描电镜图。可见Infected+Vehicle组和Infected+Oxacillin组小鼠的膝关节假体表面细菌生物膜面积较Infected+ATRA+Oxacillin组小鼠的显著增加。
图1E-F为各组小鼠膝关节假体周围感染，植入物表面的细菌涂板及定量计数。可见Infected+Vehicle组和Infected+Oxacillin组小鼠的膝关节假体表面细菌数量较Infected+ATRA+Oxacillin组小鼠的显著增加。
图1G为各组小鼠膝关节假体周围感染，软组织的细菌定量计数。可见Infected+Vehicle组和Infected+Oxacillin组小鼠的膝关节感染软组织的细菌数量较Infected+ATRA+Oxacillin组小鼠的显著增加。
实施例2
在第二部分实验中，将金葡菌导致的膝关节假体周围感染的Balb/c背景小鼠的脾脏和感染侧胫骨分离出来，并分析各组MDSC的比例与功能。
图2A为膝关节假体周围感染小鼠的脾脏大体观察。可见Infected+ATRA+Oxacillin组小鼠的脾脏较Infected+Vehicle组和Infected+Oxacillin组小鼠的长轴显著减小。
图2B-C为小鼠脾脏中免疫抑制性细胞MDSC比例的流式细胞学分析。将小鼠脾脏取出后，剪碎分离成单细胞悬液，随后，在冰上进行免疫细胞MDSC的表面标志物(CD11b，Ly6c，Ly6G)染色，流式抗体按照1:100的浓度进行稀释。待细胞活死染料孵育完成后，进行CytoFLEX流式细胞仪检测。所产生的原始数据经FlowJo软件圈门分析后，进行统计学计算。可见，Infected+ATRA+Oxacillin组小鼠脾脏的免疫抑制性细胞PMN型的MDSC的数量较Infected+Vehicle组和Infected+Oxacillin组小鼠的显著减少。
图2D-E为小鼠感染侧骨髓中免疫抑制性细胞MDSC比例的流式细胞学分析。将小鼠的感染侧胫骨分离后，用1mL注射器小心冲出其内的骨髓免疫细胞。经70um滤网过滤后，制备成单细胞悬液。随后，对其进行MDSC的表面标志物染色和流式细胞仪检测。所产生的原始数据使用FlowJo软件归一化处理。可见，Infected+ATRA+Oxacillin组小鼠骨髓的免疫抑制性细胞PMN-MDSC的数量较Infected+Vehicle组和Infected+Oxacillin组小鼠的显著减少。
图2F为小鼠假体周围感染膝关节软组织中的免疫抑制性炎症因子IL-10的水平定量分析。可见，Infected+ATRA+Oxacillin组小鼠感染膝关节软组织中，抑炎性细胞因子IL-10水平较Infected+Vehicle组和Infected+Oxacillin组小鼠的显著降低。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (5)
1.全反式维甲酸联合抗生素在制备预防和/或治疗假体周围感染的药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述假体周围感染包括膝关节假体周围感染，髋关节假体周围感染以及肩关节假体周围感染中的任一种。
3.如权利要求1所述的应用，其特征在于，所述抗生素包括苯唑西林。
4.全反式维甲酸联合抗生素在制备假体周围感染后用于抑制免疫抑制细胞的产生和/或免疫抑制性炎症因子的表达的药物中的应用。
5.一种用于预防和/或治疗假体周围感染的药物，其特征在于，所述的药物包括药学上可接受的载体和有效量的活性成分，所述活性成分包括全反式维甲酸和抗生素。
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| 李嵩，等: "饲喂不同水平维生素A对围产期奶牛免疫性能的影响", 饲料工业, vol. 31, no. 17, pages 42 - 45 * |
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