CN112921003A - 表达pd-l1分子的间充质干细胞来源外泌体及其制备方法与应用 - Google Patents
表达pd-l1分子的间充质干细胞来源外泌体及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开一种表达PD‑L1分子的间充质干细胞来源外泌体及其制备方法与应用。本发明利用间充质干细胞对机体的免疫调控功能及PD‑1/PD‑L1信号通路在免疫应答中的抑制作用,通过病毒转染技术介导PD‑L1分子在间充质干细胞内持续过表达,以使分泌的外泌体携带PD‑L1分子,这种基因工程化的外泌体不仅保持间充质干细胞的免疫调控功能,而且能够通过与免疫细胞表面的PD‑1分子作用负调控免疫应答,从而有效治疗免疫炎性疾病。本发明的PD‑L1分子表达的间充质干细胞来源外泌体制备工艺简单成熟,生物相容性好,免疫抑制效果显著,可作为免疫疾病新型治疗制剂,具有重要的研究价值和广阔的应用前景。
Description
技术领域
本发明涉及一种表达PD-L1分子的间充质干细胞来源外泌体及其制备方法与应用,属于生物材料技术领域。
背景技术
炎症是人体非常重要的防御反应,它可以限制炎症扩散、清除坏死组织、恢复器官功能;但它还对人体有一定的危害性,当炎症引起细胞发生严重的变性和坏死时,会影响受累组织和器官的功能,炎症作为一把双刃剑,它既能保护我们的身体免受病原体感染,又是诸多疾病的罪魁祸首。炎症疾病发病率高,病情迁延,严重影响患者的生活质量,自身免疫病的治疗主要包括两个目标,第一是症状缓解和功能维持,第二是延缓组织损害进程。现有的药物多是抑制多种细胞因子分泌、阻止炎症细胞向炎症部位聚集,抑制T、B淋巴细胞增殖和分泌,需长时间连续用药方可获得比较稳定的疗效。炎症细胞因子、细胞表面分子及其介导的信号通路参与免疫细胞功能紊乱和自身免疫病的病理进程,以细胞因子、受体和信号分子为治疗靶点的生物制剂近年来获得较快发展,但是很多药物只能治标,不能治本,不能控制疾病的活动及进展,而且具有很强的不良反应,如中枢神经系统症状、心血管损害、胃肠道症状、造血系统改变、肝肾功能不全等。因此,亟需开辟新的有效且毒副作用小的免疫疾病治疗方法。
间充质干细胞(MSCs)作为一类多能干细胞,不仅具有强大的增殖能力和多向分化潜能,还具有免疫调节功能,通过细胞间的相互作用及产生细胞因子发挥免疫重建作用;而且间充质干细胞来源方便,易于分离、培养、扩增和纯化,不存在免疫排斥的特性,由于间充质干细胞具备的这些优点使其具有广阔的临床应用前景,临床上一些难治性疾病,如系统性红斑狼疮、系统性硬化症、糖尿病、肝硬化、类风湿关节炎等,根据初步的临床报告数据显示,间充质干细胞对这些疾病的治疗取得显著性疗效。间充质干细胞还是非常理想的基因治疗细胞载体,易被临床上普遍应用的病毒载体系统所转染,间充质干细胞特殊的生物学特性和基因治疗二者协同作用扩大疾病治疗范围。然而间充质干细胞在应用过程中逐渐暴露了很多缺点,如间充质干细胞移植到宿主体内后不可控的分化或生长,产生的肺栓塞危害以及移植率、存活率低等问题,而且很多研究表明间充质干细胞发挥疾病治疗的作用可能是通过分泌外泌体并携带间充质干细胞的活性物质实现的。外泌体作为无细胞治疗的一种工具,不但具备间充质干细胞移植疗效,而且减低了移植风险,在现代医学领域具有不可估量的应用价值。
为避免过度活跃的免疫应答导致过度的炎症反应和自身免疫性疾病,人体进化出免疫检查点机制来控制免疫反应的强度和持续时间,最大限度地减少免疫应答对健康组织的损害。其中PD-1/PD-L1通路在抑制免疫应答的初始与效应阶段、维持机体的免疫自稳中扮演重要角色;而且,通过阻断PD-1/PD-L1信号通路增强肿瘤免疫应答的过程中引起机体其他的炎症副作用,可累及各器官。利用PD-1/PD-L1通路负面调节免疫反应的能力治疗免疫炎性疾病具有潜在的临床意义。
目前关于间充质干细胞来源外泌体与PD-L1基因治疗相结合在免疫炎性疾病的研究还未见报道。
发明内容
为解决上述技术问题,本发明提供一种表达PD-L1分子的间充质干细胞来源外泌体,具有显著的免疫抑制作用,生物相容性好,制备工艺简单成熟,可用作炎症治疗制剂,该制剂可有效作用在炎症部位,通过抑制炎症部位炎性细胞浸润和细胞因子分泌,在炎症治疗中呈现显著的疗效。
本发明的第一个目的是提供一种表达PD-L1分子的间充质干细胞来源外泌体,采用病毒介导间充质干细胞表达PD-L1分子,表达PD-L1分子的间充质干细胞分泌得到所述的外泌体。
进一步地,所述的外泌体粒径为80~120nm。
进一步地,所述的外泌体通过静脉注射方式进行给药。
本发明的第二个目的是提供所述的表达PD-L1分子的间充质干细胞来源外泌体的制备方法,包括如下步骤:
间充质干细胞培养;采用病毒介导间充质干细胞表达PD-L1分子;提取表达PD-L1分子的间充质干细胞分泌的外泌体。
进一步地,所述的方法具体包括如下步骤:
(1)分离纯化得到间充质干细胞;
(2)利用载有PD-L1质粒的慢病毒转染间充质干细胞,并筛选获得表达PD-L1分子的间充质干细胞;
(3)将表达PD-L1分子的间充质干细胞置于无血清的低糖DMEM中培养20~30h,收集培养液上清,通过差速和高速离心获得表达PD-L1的间充质干细胞来源外泌体。
进一步地,所述的差速和高速离心是依次进行如下离心:250~350g离心5~15min,1500~2500g离心5~15min,8000~12000g离心20~40min,80000~120000g离心60~80min。
进一步地,在步骤(2)中,筛选是通过6~8μg/ml的嘌呤霉素进行筛选。
本发明的第三个目的是提供一种炎症治疗制剂,包括所述的表达PD-L1分子的间充质干细胞来源外泌体。
进一步地,所述的炎症为慢性炎症和/或急性炎症。
优选地,慢性炎症为结肠炎,急性炎症为肺炎或银屑病。
进一步地,炎症治疗制剂的给药方式为静脉注射。
本发明的表达PD-L1分子的间充质干细胞来源外泌体在间充质干细胞本身具有免疫抑制能力的基础上,表达PD-L1分子,显著性增强间充质干细胞来源外泌体的免疫抑制效果,通过抑制炎症部位免疫细胞浸润和细胞因子分泌,在炎症治疗中展现出优越的疗效。
借由上述方案,本发明至少具有以下优点:
(1)间充质干细胞来源广泛,易获取、分离,可大规模扩增,慢病毒转染效率高,细胞膜表面高表达PD-L1分子。
(2)本发明中的基因工程化的外泌体制备工艺简单成熟,生物安全性高,外泌体治疗属于无细胞疗法,排斥几率很低。
(3)基因工程化的外泌体一方面具备间充质干细胞的免疫抑制特性,另一方面通过携带PD-L1分子,靶向炎症部位活化的免疫细胞,通过PD-1/PD-L1信号通路抑制炎症信号激活,显著地抑制炎症部位过度激活的免疫反应,从而有效缓解组织的炎性损伤。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1为本发明中间充质干细胞在GFP-PD-L1慢病毒转染后,PD-L1表达量的流式结果分析图;
图2为本发明中间充质干细胞在GFP-PD-L1慢病毒转染后,细胞膜上表达PD-L1的免疫荧光分析图;
图3为本发明中透射电镜显示表达PD-L1分子的间充质干细胞来源的外泌体(Exo-PD-L1)粒径大小及形态;
图4为本发明中Exo-PD-L1的粒径分布图;
图5为本发明中免疫印迹分析GFP-PD-L1慢病毒转染后,间充质干细胞及其来源外泌体PD-L1分子的表达;
图6为本发明中结肠炎小鼠静脉注射Exo-PD-L1后结肠组织内免疫细胞表型分析结果;
图7为本发明中结肠炎小鼠静脉注射Exo-PD-L1后结肠组织内免疫细胞因子表达;
图8为本发明中结肠炎小鼠静脉注射Exo-PD-L1后,Elisa检测血清中炎症因子IFN-γ、IL-6、TNF-α的表达水平;
图9为本发明中结肠炎小鼠静脉注射Exo-PD-L1后,小鼠体重、疾病活动指数、结肠长度的变化;
图10为本发明中结肠炎小鼠静脉注射Exo-PD-L1后,小鼠结肠组织病理切片HE分析;
图11为本发明中结肠炎小鼠静脉注射Exo-PD-L1后,小鼠结肠组织紧密连接蛋白表达及通透性分析;
图12为本发明中肺炎小鼠静脉注射Exo-PD-L1后肺组织内免疫细胞表型分析结果;
图13为本发明中肺炎小鼠静脉注射Exo-PD-L1后肺组织内免疫细胞因子表达;
图14为本发明中肺炎小鼠静脉注射Exo-PD-L1后,Elisa检测血清及肺泡灌洗液中炎症因子IL-1β、IL-6、TNF-α的表达水平;
图15为本发明中肺炎小鼠静脉注射Exo-PD-L1后,小鼠肺组织病理切片HE分析;
图16为本发明中肺炎小鼠注射Exo-PD-L1后,masson染色分析小鼠肺组织的纤维化程度;
图17为本发明中银屑病小鼠静脉注射Exo-PD-L1后,对小鼠的临床学评分、体重、皮肤厚度及皮肤鳞屑程度的影响;
图18为本发明中银屑病小鼠静脉注射Exo-PD-L1后,小鼠皮肤内免疫细胞表型分析结果;
图19为本发明中银屑病小鼠静脉注射Exo-PD-L1后,小鼠皮肤内免疫细胞因子表达;
图20为本发明中银屑病小鼠静脉注射Exo-PD-L1后,Elisa检测皮肤中炎症因子IL-1β、IL-6、TNF-α及IL-17A的表达水平;
图21为本发明中银屑病小鼠注射Exo-PD-L1后,小鼠皮肤组织的病理切片分析;
图22为本发明中Exo-PD-L1与LPS刺激的RAW264.7细胞共孵育后,流式细胞仪检测巨噬细胞的极化表型;
图23为本发明中Exo-PD-L1与LPS刺激的RAW264.7细胞共孵育后,ELisa检测培养基上清细胞因子IL-1β、TNF-α、IL-10的水平;
图24为本发明中Exo-PD-L1与LPS刺激的DCs共孵育后,流式细胞仪检测CD80、CD86的表达;
图25为本发明中Exo-PD-L1与anti-CD3/CD28刺激的淋巴结T细胞共孵育后,流式细胞仪检测CD4、Foxp3、IFN-γ的表达;
图26为本发明中Exo-PD-L1与anti-CD3/CD28刺激的T淋巴结细胞共孵育后,Elisa检测培养基上清IFN-γ、IL-4的水平。
具体实施方式
下面结合具体实施方式对本发明做进一步说明,以使相关技术人员更好的理解本发明并予以实施,但所述实例并不限定本发明的范围。
本发明各实施例中的材料来源为:
GFP-PD-L1慢病毒购自汉恒生物科技有限公司。小鼠粒细胞-巨噬细胞集落刺激因子GM-CSF(315-03-50)购自PeproTech公司。脂多糖LPS购自sigma公司。PD-L1抗体购自Proteintech公司。
间充质干细胞由小鼠骨髓中提取并利用其贴壁特性在低糖DMEM培养基中进行纯化和扩增。
6-8周龄雌性C57BL/6小鼠购自常州卡文斯实验动物有限公司。小鼠按照中国科学院生化与细胞所实验动物管理委员会(IACUC)的指导操作方法进行处理。
实施例1:GFP-PD-L1慢病毒转染间充质干细胞使其过表达PD-L1分子
(1)从C57BL/6小鼠的胫骨和股骨骨髓提取骨髓细胞,然后根据间充质干细胞的贴壁特性,利用低糖DMEM培养基进行纯化和扩增。
(2)小鼠骨髓间充质干细胞分离培养后,利用慢病毒介导的基因转染方式使间充质干细胞高表达PD-L1,免疫荧光(图1)和流式细胞术(图2)结果显示,PD-L1在间充质干细胞上高表达,说明成功建立高表达PD-L1的间充质干细胞。
实施例2:高表达PD-L1的间充质干细胞来源外泌体提取及表征
(1)高表达PD-L1的间充质干细胞更换为无血清低糖DMEM培养基培养24h,收集上清;
(2)将上清在4℃下依次经过300g离心10min,去沉淀,2000g离心10min,去沉淀,10000g离心30min,去沉淀,100000g离心70min,弃上清,加PBS悬浮沉淀,即为获得的间充质干细胞来源外泌体;
(3)并对步骤(2)获得的外泌体进行透射电镜分析其粒径大小及分布(图3,图4),免疫印迹分析外泌体上PD-L1的表达(图5)。
实施例3:高表达PD-L1的间充质干细胞来源外泌体在结肠炎治疗效果的分析
(1)6-8周龄雌性C57BL/6小鼠称重,随机分为4组,分别为阴性对照组、模型组、外泌体组和表达PD-L1外泌体组,除阴性对照组,其他组小鼠均自由引用5%葡聚糖硫酸钠(DSS)溶液,建立结肠炎模型;
(3)给与小鼠DSS水的第二天开始静脉注射治疗,其中,阴性对照组和模型组给与等量PBS(体积约100μl),治疗组分别给与50μg间充质干细胞来源外泌体和表达PD-L1的外泌体,并进行体重监测、疾病活动指数(DAI)评估;
(4)8天造模结束后,收集各组小鼠血清及结肠组织,取结肠测量长度;
(5)取出适量步骤(4)的结肠组织样本进行破碎,使其成为单细胞悬液,随后采用流式细胞术对组织内T细胞、巨噬细胞、DC细胞等进行免疫表型分析,其中,用流式抗体对巨噬细胞的CD45、F4/80、CD80、CD206进行染色,对T细胞的CD45、CD3、CD4、CD25、Foxp3、IFN-γ、IL17A及IL-4进行染色,对DC的CD45、CD11c、CD80、CD86、CD103进行染色。结果显示,与阴性对照组相比,模型组小鼠结肠组织内浸润大量活化的巨噬细胞、T细胞、DC细胞,给与表达PD-L1分子的间充质干细胞来源外泌体后,显著性减少结肠组织内免疫细胞浸润和活化以及免疫细胞因子分泌,并且免疫调节细胞Treg和M2型巨噬细胞增多,表明外泌体表达PD-L1可以地有效缓解结肠炎进展(图6,图7);
(6)取步骤(4)获得的血清利用Elisa试剂盒检测细胞因子水平,结果如图8所示,结肠炎小鼠血清中IFN-γ、IL-6、IL-1β的水平明显增高,当给与表达PD-L1的外泌体后,这些细胞因子均显著减少,而未转染PD-L1慢病毒的间充质干细胞来源外泌体对结肠炎小鼠血清炎症因子的水平没有产生显著地抑制作用,表明外泌体表达PD-L1后有效减少肠炎小鼠血清炎症因子的水平;
(7)通过对各组小鼠体重、疾病活动指数及结肠长度的统计,结果如图9所示,表达PD-L1分子的间充质干细胞来源外泌体显著缓解DSS导致的小鼠体重减轻、疾病活动指数增高以及结肠变短;
(8)造模结束后取出的各组小鼠结肠组织进行H&E染色,做病理切边分析,根据对样本病理切片的观察,结果显示,DSS诱导结肠组织淋巴细胞、单核细胞、中性粒细胞浸润增多,并出现肠黏膜脱落及溃疡病变;给与PD-L1表达的的间充质干细胞来源外泌体后,这些炎症病变部位均得到有效缓解(图10);另外,恢复了DSS导致的结肠组织紧密连接蛋白ZO-1的减少以及结肠组织的通透性的增加,但是未经过病毒转染MSC来源外泌体对肠炎的病变未产生明显影响(图11)。
实施例4:表达PD-L1的间充质干细胞来源外泌体在肺炎治疗效果的分析
(1)6-8周龄雌性C57BL/6小鼠称重,随机分为4组,分别为阴性对照组、模型组、外泌体组和表达PD-L1外泌体组,除阴性对照组,其他组小鼠均咽部给与10mg/kg的LPS,持续24h,建立肺炎模型;其中,造模0h时阴性对照组和模型组给与等量PBS(体积约100μl),治疗组分别给与50μg外泌体和表达PD-L1的外泌体;
(2)24h造模结束后,收集各组小鼠血清、肺泡灌洗液及肺组织;
(3)取适量步骤(2)取出的肺组织样本进行破碎,使其成为单细胞悬液,随后采用流式细胞术对组织内T细胞、巨噬细胞、DC细胞等进行免疫表型分析。其中,用流式抗体对巨噬细胞的CD45、F4/80、CD80、CD206进行染色,对T细胞的CD45、CD3、IFN-γ、IL17A进行染色,对DC的CD45、CD11c、CD80、CD86、CD103进行染色。结果显示,与阴性对照组相比,模型组小鼠肺组织内浸润大量活化的巨噬细胞、T细胞、DC细胞,表达PD-L1的外泌体显著性减少肺组织内免疫细胞浸润和活化以及免疫细胞因子IFN-γ、IL17A表达(图12,图13),表明外泌体表达PD-L1可以地有效缓解LPS诱导的肺炎;
(4)利用Elisa试剂盒检测各组小鼠血清及肺泡灌洗液中的细胞因子水平,结果如图14所示,LPS诱导的肺炎小鼠血清及肺泡中IL-1β,IL-6及TNF-α的水平明显增高,当给与表达PDL1的外泌体后,这些细胞因子均显著减少,而未转染PD-L1慢病毒的间充质干细胞来源外泌体对肺炎小鼠血清及肺泡炎症因子的水平没有产生显著地抑制作用,表明表达PD-L1的外泌体有效缓解肺炎进展;
(5)造模结束后取出的各组小鼠肺组织进行H&E染色,做病理切边分析,并且进行了masson染色,根据对样本病理切片及masson染色的观察,结果显示,LPS诱导肺泡内淋巴细胞、单核细胞浸润增多,肺泡破裂塌陷(图15),并且出现明显的纤维化现象(图16),这些肺炎小鼠给与表达PD-L1间充质干细胞来源外泌体后,这些炎症病变部位及纤维化程度得到有效缓解。
实施例5:表达PD-L1的间充质干细胞来源外泌体在银屑病治疗效果的分析
(1)6-8周龄雌性C57BL/6小鼠称重,随机分为4组,分别为阴性对照组、模型组、外泌体组和PD-L1表达外泌体组,各组小鼠的背部2*2cm2脱毛处理,除阴性对照组,其他组小鼠均在脱毛区涂抹62.5mg的咪喹莫特乳膏(艾达乐),连续4天,建立银屑病模型;
(2)造模同时给与小鼠静脉注射治疗,其中,阴性对照组和模型组给与等量PBS(体积约100μl),治疗组分别给与50μg间充质干细胞来源外泌体和表达PD-L1的外泌体,并进行体重监测、临床评分;造模组小鼠体重减轻,临床病理学评分增加,抹药处逐渐出现红斑、鳞屑、皮肤增厚(图17),表达PD-L1的外泌体治疗后,这些病例特征明显得到缓解。
(3)并于末次涂药24h后处死,收集各组小鼠血清及皮肤组织,并且用游标卡尺量取皮肤厚度。
(4)取出适量皮肤组织样本进行破碎,使其成为单细胞悬液,随后采用流式细胞术对组织内T细胞、巨噬细胞、DC细胞等进行免疫表型分析,其中,用流式抗体对巨噬细胞的CD45、F4/80、CD80进行染色,对T细胞的CD45、CD3、IFN-γ、IL17A进行染色,对DC的CD45、CD11c、CD103进行染色。结果显示,与阴性对照组相比,咪喹莫特诱导小鼠皮肤组织内浸润的巨噬细胞、T细胞、DC细胞增多,间充质干细胞分泌的外泌体在表达PD-L1后,显著性减少皮肤组织内免疫细胞浸润以及免疫细胞因子分泌(图18,图19),表明表达PD-L1分子的外泌体可以地有效缓解银屑病炎症进展。
(5)将皮肤组织进行匀浆破碎后,利用Elisa试剂盒检测皮肤组织内细胞因子水平,结果如图20所示,银屑病小鼠皮肤组织内IL-1β、IL-6,TNF-α、IL17A的水平明显增高,当给与表达PD-L1的外泌体后,这些细胞因子均显著减少,而未转染PD-L1慢病毒的间充质干细胞来源外泌体对银屑病小鼠皮肤组织内炎症因子的水平没有产生显著地抑制作用。
(6)造模结束后取出的各组小鼠皮肤组织进行H&E染色,做病理切片分析,根据对切片的观察,结果显示,咪喹莫特诱导皮肤组织炎症细胞浸润,并且表皮厚度明显增加;给与表达PD-L1分子的间充质干细胞来源外泌体后,银屑病样病理特征得到有效缓解(图21)。
实施例6:验证Exosome-PD-L1体外的免疫抑制效果
(1)巨噬细胞株RAW264.7细胞均铺于6孔板进行培养,设置4组,阴性对照组、LPS组、外泌体组、表达PD-L1外泌体组,除阴性对照组外,其余三组均给与20ng/ml LPS刺激,同时,LPS组加入10μl PBS,给药组各加入1μg的外泌体和表达PD-L1外泌体,24h后收集培养液上清及细胞;
(2)流式分析巨噬细胞的极化表型,对RAW 264.7细胞进行F4/80、CD80、CD206染色,结果显示,LPS诱导巨噬细胞CD80的表达升高,表明向促炎的M1型极化,给与表达PD-L1的外泌体后,CD80表达有所减少,CD206表达增加,表明表达PD-L1的外泌体促进M1型巨噬细胞向抑炎的M2型转化(图22);
(3)利用Elisa试剂盒检测各组巨噬细胞培养上清的细胞因子表达,结果显示,表达PD-L1的外泌体有效减少LPS诱导的IL-1β、TNF-α的水平,并且促进IL-10的表达(图23);
(4)取小鼠骨髓细胞,在GM-CSF的诱导下分化为DC细胞,并且在50ng/ml LPS的刺激下进行活化,流式细胞术分析DC的CD80、CD86的表达,结果显示LPS刺激24h后,CD80、CD86的表达显著增加,当给与表达PD-L1的外泌体治疗后,显著性抑制CD80、CD86的表达,说明表达PD-L1的外泌体有效抑制DC细胞活化(图24);
(5)取小鼠淋巴结细胞种于12孔板,除了阴性对照组,均加入2μg的CD3和CD28抗体活化T细胞,并给与表达PD-L1的间充质干细胞来源外泌体进行治疗,24h后收集细胞及细胞培养上清;利用流式细胞术对淋巴细胞进行CD3、CD4、FOXP3、IFN-γ染色,结果显示,表达PD-L1的外泌体有效抑制CD4 T细胞活化及IFN-γ的表达,并且促进免疫调节性T细胞Treg数量的增加(图25);Elisa试剂盒检测细胞培养上清细胞因子表达水平,结果显示表达PD-L1的外泌体抑制活化T细胞分泌的IFN-γ,促进IL-4的表达(图26);上述结果表明,表达PD-L1的间充质干细胞来源外泌体显著性抑制T细胞活化。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
1.一种表达PD-L1分子的间充质干细胞来源外泌体,其特征在于,采用病毒介导间充质干细胞表达PD-L1分子,表达PD-L1分子的间充质干细胞分泌得到所述的外泌体。
2.根据权利要求1所述的表达PD-L1分子的间充质干细胞来源外泌体,其特征在于,所述的外泌体粒径为80~120nm。
3.根据权利要求1所述的表达PD-L1分子的间充质干细胞来源外泌体,其特征在于,所述的外泌体通过静脉注射方式进行给药。
4.一种权利要求1~3任一项所述的表达PD-L1分子的间充质干细胞来源外泌体的制备方法,其特征在于,包括如下步骤:
间充质干细胞培养;采用病毒介导间充质干细胞表达PD-L1分子;提取表达PD-L1分子的间充质干细胞分泌的外泌体。
5.根据权利要求4所述的方法,其特征在于,所述的方法具体包括如下步骤:
(1)分离纯化得到间充质干细胞;
(2)利用载有PD-L1质粒的慢病毒转染间充质干细胞,并筛选获得表达PD-L1分子的间充质干细胞;
(3)将表达PD-L1分子的间充质干细胞置于无血清的低糖DMEM中培养20~30h,收集培养液上清,通过差速和高速离心获得表达PD-L1的间充质干细胞来源外泌体。
6.根据权利要求5所述的方法,其特征在于,所述的差速和高速离心是依次进行如下离心:250~350g离心5~15min,1500~2500g离心5~15min,8000~12000g离心20~40min,80000~120000g离心60~80min。
7.根据权利要求5所述的方法,其特征在于,在步骤(2)中,筛选是通过6~8μg/ml的嘌呤霉素进行筛选。
8.一种炎症治疗制剂,其特征在于,包括权利要求1~3任一项所述的表达PD-L1分子的间充质干细胞来源外泌体。
9.根据权利要求8所述的炎症治疗制剂,其特征在于,所述的炎症为慢性炎症和/或急性炎症。
10.根据权利要求8所述的炎症治疗制剂,其特征在于,炎症治疗制剂的给药方式为静脉注射。
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