CN115212234A - 纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用 - Google Patents
纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用 Download PDFInfo
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Abstract
本发明提供了纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用。本发明首次提取了FRCs来源的外泌体;并通过实验发现FRCs来源的外泌体能够发挥了肾损伤保护作用,促进损伤的肾小管细胞损伤修复,促进其增殖,减少肾小管坏死,减轻炎症反应和降低氧化应激。FRCs来源的外泌体,应用于小鼠脓毒症急性肾损伤的治疗中,取得了良好的治疗效果。因此提示纤维网状细胞来源的外泌体可以用于制备治疗和/或预防脓毒症急性肾损伤的药物。与传统的细胞治疗相比,外泌体具有稳定性好、保存及应用方便快捷的特点。
Description
技术领域
本发明涉及生物医学技术领域,特别涉及纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用。
背景技术
脓毒症(Sepsis)是由多种病原体、创伤、烧伤及外科大手术等因素造成的机体感染而引起机体对感染反应失调,导致严重的、复杂的、失控的免疫反应并危及生命的多器官功能障碍综合征。肾脏是脓毒症最容易影响的器官,约有50%脓毒症患者发生急性肾损伤(Acute Kidney Injury,AKI)。尽管应用了大量抗生素及器官支持治疗,但由于发病机制不明确,缺乏特异性治疗方法,脓毒症AKI的发病率仍不断增高,消耗的医疗资源也逐年增加。因此深入研究脓毒症AKI的发病机制并不断寻找新的治疗靶点,改善脓毒症AKI预后,具有重要的社会效益和经济效益。
因此,急需开发一种用于治疗和/或预防脓毒症急性肾损伤的药物。
发明内容
本发明目的是提供纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用,本发明经过试验发现FRCs来源的外泌体能够发挥了肾损伤保护作用,促进损伤的肾小管细胞损伤修复,促进其增殖,减少肾小管坏死,减轻炎症反应和降低氧化应激,应用于小鼠脓毒症急性肾损伤的治疗中,取得了良好的治疗效果。。
为实现所述目的,本发明采用如下技术方案:
在本发明的第一方面,提供了纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用。
进一步地,所述纤维网状细胞来源的外泌体的制备方法包括:
获得纤维网状细胞;
将所述纤维网状细胞用含无外泌体的血清的培养基进行培养,获得细胞上清,后进行第一固液分离获得上清液,将所述上清液进行第二固液分离获得固体,后重悬,获得纤维网状细胞来源的外泌体。
进一步地,所述获得纤维网状细胞,包括:
收集脂肪组织,剪碎后消化,过细胞筛后终止消化,通过密度梯度离心法去除红细胞和粒细胞,收集的细胞采用含有FBS的DMEM完全培养基进行培养并传代培养,获得纤维网状细胞。
进一步地,所述消化中,采用0.2±0.02%的胶原酶、2.0±0.5mg/ml中性蛋白酶和0.08 ±0.01mg/ml透明质酸酶的混合酶进行消化。
进一步地,所述第一固液分离采用0.2μM过滤。
进一步地,所述第二固液分离包括:4±2℃下超速离心60±20min,所述超速离心的转速为1.0×105g±1000g。
进一步地,所述含无外泌体的血清的培养基为含0.5-1.5%P/S、0.05-0.15%PB、45-55μM EGF、0.1-0.3mg/mLFGF、50-150nM TGF、8-12%无外泌体血清的DMEM完全培养基。
进一步地,所述纤维网状细胞的标志抗体为CD45-、PDPN+和CD31-。
进一步地,所述纤维网状细胞来源的外泌体特异性标志物为CD9、CD63、HSP70、TSG-101和CD81。
在本发明的第二方面,提供了一种用于治疗和/或预防脓毒症急性肾损伤的药物,所述药物包括纤维网状细胞来源的外泌体。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明提供纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用,本发明首次提取了FRCs来源的外泌体;并通过实验发现FRCs来源的外泌体能够发挥了肾损伤保护作用,促进损伤的肾小管细胞损伤修复,促进其增殖,减少肾小管坏死,减轻炎症反应和降低氧化应激。FRCs来源的外泌体,应用于小鼠脓毒症急性肾损伤的治疗中,取得了良好的治疗效果。与传统的细胞治疗相比,外泌体具有稳定性好、保存及应用方便快捷的特点。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为实施例1中FRCs的原代分离培养及鉴定的结果,其中图1A为小鼠肠系膜脂肪组织中***构;图1B为明场下观察FRCs细胞形态;图1C为CD45、Podoplanin、CD31 抗体标记FRCs,流式细胞仪鉴定FRCs;
图2为实施例1中提取FRCs-外泌体及鉴定的结果;其中,图2A为电镜检测外泌体的大小和形态;图2B为Western blot检测外泌体(2个重复)标志蛋白CD63、CD9、TSG101和HSP70;图2C为纳米颗粒跟踪分析技术(NTA)检测所提取的胞外囊泡的直径;图2D为成脂诱导分化培养基培养FRCs,油红O染色脂肪细胞,红色为FRCs分化为脂肪细胞;
图3为实施例2中FRCs-外泌体能够归巢到损伤的肾脏的结果,其中图3A为活体成像观察FRCs在小鼠体内聚集的部位;图3B流式细胞仪检测腹腔注射的FRCs在腹腔灌洗液、脾脏、肾组织的聚集情况;图3C为Dio(绿色荧光)标记的FRCs与肾小管上皮细胞共培养,使用Transwell小室将FRCs和肾小管上皮共培养24小时,将Dio标记的FRCs-Exos 加入到肾小管上皮细胞,DAPI着色肾小管上皮细胞的细胞核,共聚焦显微镜观察外泌体摄取情况;
图4为实施例2中FRCs来源外泌体对脓毒症AKI的保护作用的结果,其中图4A将FRCs和FRCs-外泌体(Exos)注射到脓毒症小鼠腹腔,观察脓毒症小鼠的120h生存率变化;图4B检测各组小鼠血清肌酐和尿素氮含量;
图5为实施例2中FRCs-外泌体改善LPS诱导的脓毒症的结果。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买获得或者可通过现有方法获得。
本申请实施例的技术方案为解决上述技术问题,总体思路如下:
外泌体由细胞内的多泡小体(Multivesicular Bodies)与细胞膜融合后以外分泌的形式释放到细胞外,直径约为40nm-100nm的脂质膜性微囊泡,通过内吞、直接膜融合或受体配体相互作用进入受体细胞,在细胞间进行信息传递。
1、本申请发明人经分析不同来源的外泌体的成分相差甚大,不同来源的外泌体效果各异,主要有:
(1)内脏脂肪组织来源的外泌体在炎症性肠病中具有促炎作用,第四军医大学张健课题组在ACS Nano杂志上发表文章,研究结果表明内脏脂肪组织衍生的外泌体通过转移促炎性miRNA加剧了结肠炎进程。
(2)循环促炎外泌体在衰老过程中加重中风,心血管领域权威期刊CirculationResearch 杂志,研究发现老年个体的血液外泌体逐渐积累外周促炎介质,并且可以通过C3aR依赖性机制穿过血脑屏障来启动和过度激活小胶质细胞,促进脑梗死的发生发展。
(3)外泌体促进肿瘤相关的炎症反应,南京医科大学刘起展课题组在CancerLetters 上发表文章:在肝癌中,来自亚砷酸盐转化的L-02细胞的外泌体会增加正常L-02和THLE-3 细胞的miR-155表达量和促炎症特性。
(4)肾小管细胞来源的外泌体激活成纤维细胞促进肾纤维化,武汉大学人民医院程帆课题组在Theranostics杂志上发表文章,发现肾小管上皮细胞的外泌体miR-21可能通过靶向miR-21/PTEN/Akt通路,激活输尿管梗阻肾脏中的成纤维细胞,从而加速肾纤维化的发展。
2、本发明通过实验首先成功提取到纤维网状细胞来源的外泌体,所述纤维网状细胞来源的外泌体的制备方法包括:
步骤S1、获得纤维网状细胞;
所述步骤S1具体包括:
收集脂肪组织,剪碎后消化,过细胞筛后终止消化,通过密度梯度离心法去除红细胞和粒细胞,收集的细胞采用含有FBS的DMEM完全培养基进行培养并传代培养,获得纤维网状细胞。所述消化中,采用0.2±0.02%的胶原酶、2.0±0.5mg/ml中性蛋白酶和0.08±0.01mg/ml透明质酸酶的混合酶进行消化。
步骤S2、将所述纤维网状细胞用含无外泌体的血清的培养基进行培养,获得培养基,后进行第一固液分离获得上清液,将所述上清液进行第二固液分离获得固体,后重悬,获得纤维网状细胞来源的外泌体。
所述步骤S2中,
所述第一固液分离采用0.2μM过滤。
所述第二固液分离包括:4±2℃下超速离心60±20min,所述超速离心的转速为1.0×105 g±1000g。
所述含无外泌体的血清的培养基含0.5-1.5%P/S、0.05-0.15%PB、45-55μM EGF(表皮生长因子)、0.1-0.3mg/mLFGF(成纤维细胞生长因子)、50-150nM TGF(转化生长因子TGF-α)、8-12%无外泌体血清的DMEM完全培养基。
优选地,所述含无外泌体的血清的培养基含1%P/S(青霉素-链霉素)、0.1%PB(两性霉素B)、50μM EGF(表皮生长因子)、0.2mg/mLFGF(成纤维细胞生长因子)、100nM TGF (转化生长因子TGF-α)、10%无外泌体血清的DMEM完全培养基。
接着,本发明将提取到的纤维网状细胞来源的外泌体应用于小鼠脓毒症急性肾损伤的治疗中,通过实验发现FRCs来源的外泌体能够发挥了肾损伤保护作用。因此提示纤维网状细胞来源的外泌体可以用于制备用于治疗和/或预防脓毒症急性肾损伤的药物。
可选地,用于治疗和/或预防脓毒症急性肾损伤的药物还可包括药学上可接受的辅料和载体。所述辅料包括填充剂、崩解剂、粘合剂、赋形剂、稀释剂、润滑剂、甜味剂或着色剂中的至少一种。剂型包括颗粒剂、片剂、丸剂、胶囊剂、注射剂或分散剂中的至少一种。
本发明的纤维网状细胞来源的外泌体首次获得,制备提取步骤不同于常规的提取方案,主要有以下几点:
(1)外泌体的来源:来源于小鼠原代纤维网状细胞,不同于传统提取方式该发明使用多种混合酶(采用采用0.2±0.02%的胶原酶、2.0±0.5mg/ml中性蛋白酶和0.08±0.01mg/ml 透明质酸酶的混合酶进行消化),提取方式温和,对细胞损伤小,细胞活力高,细胞纯度高达99.5%,且该方式提取的原代纤维网状细胞冻存后,仍可传至4-6代。
(2)改良培养基成分:本发明采用含无外泌体的血清的培养基为含0.5-1.5%P/S、0.05-0.15%PB、45-55μM EGF(表皮生长因子)、0.1-0.3mg/mLFGF(成纤维细胞生长因子)、50-150nM TGF(转化生长因子TGF-α)、8-12%无外泌体血清的DMEM完全培养基。该改良型培养基选择性极佳,仅适合原代纤维网状细胞的生长。
(3)首次将FRC-EXO应用于脓毒症诱导的急性肾损伤研究中,与***相比,FRCs 的低免疫原性、脂肪组织含量丰富、易于扩增的特性使得FRCs成为脓毒症细胞治疗的理想方法。在本发明中,我们成功分离出脂肪来源的FRCs,并在体外培养FRCs,将体外扩增的FRCs释放的外泌体注射到脓毒症AKI小鼠腹腔,以探讨脂肪中的FRCs-外泌体作为一种新方法对脓毒症AKI的治疗效果。
研究结果表明在盲肠结扎穿孔(CLP)诱导脓毒症AKI小鼠模型中,在感染早期和晚期给予FRCs,以及FRCs释放的外泌体均能降低脓毒症死亡率,FRCs通过调控一氧化氮合酶2(NOS2)降低血清和感染部位的促炎细胞因子水平。我们在FRCs对脓毒症的治疗效果方面也进行了大量研究,证实腹腔注射FRCs能有效改善脓毒症小鼠的死亡率,减少血液和腹腔灌洗液中的炎症因子含量,降低腹腔灌洗液中的细菌负荷。进一步发现腹腔注射FRCs- 外泌体能够降低脓毒症小鼠的血清肌酐和尿素氮含量,改善肾脏损伤程度。
下面将结合实施例及实验数据对本申请的纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用进行详细说明。
实施例1、FRCs来源的外泌体的提取
1、FRCs的原代分离培养及鉴定
(1)提取小鼠FRCs:用小动物麻醉机(异氟烷)吸入的方法麻醉小鼠,消毒后逐层打开腹腔,剪下肠系膜脂肪,注意避开胰腺组织,将剪碎的脂肪组织置于含有0.2%的胶原酶、中性蛋白酶2.0mg/ml、透明质酸酶0.08mg/ml中消化30-60min,过细胞筛后用含有FBS的培养基终止消化,Ficoll密度梯度离心法去除红细胞和粒细胞,所提取细胞用含有FBS的DMEM完全培养基,铺入T-25培养瓶,在37℃、5%CO2培养箱中培养;3天后,弃去未贴壁的细胞与组织,加入新鲜的含有完全培养基;待细胞长至80%融合度时,用0.25%胰酶消化,传代至10cm培养皿中继续扩增培养,并记为第1代,以后每三到四天换液或消化后传代培养,每传代一次记为一代。
(2)流式细胞仪鉴定FRCs:流式细胞术检测第4代FRCs细胞表面CD45、PDPN、 CD31表达情况,CD45抗体用于区分淋巴细胞和非淋巴细胞,CD31是内皮细胞标志物, PDPN是纤维网状细胞特异性标志物,CD45-CD31-PDPN+为FRCs细胞群。
收集小鼠肠系膜脂肪组织,脂肪中***构如图1A所示,图1B为用胶原酶消化脂肪***构并接种到培养皿,明场下观察FRCs细胞形态,可见细胞呈梭形;应用流式细胞仪检测FRCs细胞表面标志物(CD45-Podoplanin+CD31-),Podoplanin+CD31-占CD45-细胞的99.5%(图1C)。
2提取FRCs外泌体及鉴定
(1)提取FRCs外泌体:用无外泌体的血清中培养FRCs,0.2μM过滤上清液,在4℃下超速离心70分钟,弃上清,重悬外泌体。应用透射电镜观察外泌体的形态,纳米粒度分析仪检测外泌体的大小和直径。
(2)透射电镜观察外泌体:将一滴外泌体重悬于PBS中,用镊子将Falvar碳涂层镍网格放置在液滴顶部,确保涂层侧面含有脱落物的外泌体,用透射电镜观察外泌体的形态。
(3)Western blot检测外泌体的标志物:应用蛋白质裂解缓冲液裂解外泌体,加入外泌体跨膜蛋白(CD9、CD63、HSP70、TSG-101和CD81)的抗体,检测目的蛋白表达。
(4)鉴定FRCs的干细胞特性:对FRCs进行成脂、成骨分化诱导,鉴定其干细胞的分化能力。加入成骨分化诱导培养基,qRT-PCR检测成骨分化标志基因Alp、Runx2、Ocn 的表达,茜素红染色检测细胞外钙化基质形成情况;成脂诱导与成骨诱导类似,将培养基换为脂肪细胞诱导培养基,qRT-PCR检测成脂分化标志基因Ppar-γ、Lpl,油红O染色检测细胞内脂滴形成情况。
结果如图2所示,图2A为使用超速离心法提取FRCs释放的外泌体,电镜下观察外泌体的形态,图2B可知,采用Western blot成功检测到了外泌体相关标志蛋白CD63、CD9、TSG101和HSP70。纳米颗粒跟踪分析技术(NTA)检测所提取的胞外囊泡的直径,由图 2C可知,97%颗粒的直径为140.1nm,提示胞外囊泡为外泌体;为了了解FRCs的干细胞特性,用成脂分化培养基诱导FRCs成脂分化,红色为FRCs分化为脂肪细胞(图2D)。
实施例2、纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用
1、盲肠结扎穿孔(CLP)诱导脓毒症AKI模型
用盲肠结扎穿孔(CLP)的方法诱导脓毒症急性肾损伤模型,具体如下:选用8-12周龄的C57BL/6雄性小鼠,麻醉后小鼠置于俯卧位,打开腹腔,结扎1/4长度的盲肠,用22G 针头在盲肠部位穿2个孔,挤出大便,逐层关闭腹腔,20ml/kg的生理盐水皮下注射进行液体复苏。(方法学详见Peng ZY,et al.Crit Care Med.2012Feb;40(2):538-43)。
2、FRCs-外泌体能够归巢到损伤的肾脏
在脓毒症小鼠的腹腔分别注射荧光标记的FRCs-外泌体,小动物活体成像检测FRCs- 外泌体在小鼠多个器官(心脏、肾脏、肝脏等)的分布情况;
结果如图3所示,在盲肠结扎穿孔(CLP)构建脓毒症模型的小鼠腹腔注射荧光标记的 FRCs,活体成像观察FRCs聚集位置,发现FRCs在小鼠腹腔聚集,48h后FRCs的细胞量减少(图3A);收集上述三组小鼠的腹腔灌洗液、脾脏、肾脏,流式细胞仪检测FRCs在不同组织的含量,发现大部分FRCs仍驻留于腹腔,肾脏中FRCs聚集较少,提示FRCs可能通过旁分泌方式影响肾功能(图3B);我们进一步将荧光标记的FRCs、FRCs-外泌体分别和小鼠原代肾小管上皮细胞共培养,发现外泌体能够被肾小管上皮细胞摄取(图3C)。
3、FRCs-外泌体对脓毒症AKI的保护作用
给予FRCs-外泌体对AKI的改善情况:在脓毒症AKI小鼠注射FRCs-外泌体,观察脓毒症AKI小鼠一周生存率和肾功能,以确定FRCs-外泌体对脓毒症AKI生存率改善作用。
FRCs-外泌体对脓毒症AKI的保护作用:给予CLP脓毒症小鼠腹腔注射FRCs或FRCs-外泌体,24h后收集肾组织、血清标本和尿液标本,应用肌酐和尿素氮检测试剂盒测定小鼠血清中血肌酐和尿素氮的水平(肾损伤的标志物主要有肌酐和尿素氮),肾脏组织进行HE染色,按照肾损伤评分量表评估肾损伤严重程度;
结果如图4所示,我们进一步提取了小鼠的FRCs,以及FRCs释放的外泌体,在脓毒症后1小时将FRCs和FRCs-外泌体注射到脓毒症小鼠腹腔,观察脓毒症小鼠120h的生存率。由图4A可知,CLP组小鼠生存中位时间是36小时,CLP+FRCs-外泌体的中位生存时间是72小时,腹腔注射FRCs-外泌体能够明显改善脓毒症小鼠的生存率(P<0.05);由图4B可知,检测小鼠血清肌酐含量,发现注射FRCs和FRCs外泌体24h后,小鼠血清肌酐明显降低。
4、FRCs-外泌体改善LPS诱导的脓毒症AKI
用LPS预处理小鼠肾小管上皮细胞,将FRCs-外泌体和肾小管上皮细胞共培养,检测肾小管上皮细胞焦亡蛋白,检测NOD样受体蛋白3炎症体(NLRP3)、炎性caspases介导的细胞焦亡的关键底物蛋白GSDMD、凋亡蛋白CASPASE-1以及内参GAPDH的表达情况。
结果如图5所示,可知在NC组与LPS组分别加入FRC-外泌体,在LPS诱导下小鼠原代肾小管上皮细胞中,NLRP3与Caspases-1蛋白含量显著增高,说明炎症小体形成,随之GSDMD蛋白在细胞膜打孔,细胞质外流,炎症因子释放,而进一步在LPS刺激AKI的小鼠肾小管上皮细胞中加入FRCs-外泌体,炎症相关NLRP3与Caspases-1含量明显下调, GSDMD蛋白表达水平与NC组相当,细胞恢复到了损伤前水平,说明FRCs-外泌体改善LPS 诱导的脓毒症AKI。
综上可知,FRCs来源的外泌体能够发挥了肾损伤保护作用,促进损伤的肾小管细胞损伤修复,促进其增殖,减少肾小管坏死,减轻炎症反应和降低氧化应激。应用于小鼠脓毒症急性肾损伤的治疗中,取得了良好的治疗效果。与传统的细胞治疗相比,外泌体具有稳定性好、保存及应用方便快捷的特点。外泌体作为一种非细胞生物制剂,克服了细胞治疗的局限性,具有不易致瘤、低抗原性、靶向性较强等优势,有巨大的临床应用潜能。
最后,还需要说明的是,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.纤维网状细胞来源的外泌体在制备用于治疗和/或预防脓毒症急性肾损伤的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述纤维网状细胞来源的外泌体的制备方法包括:
获得纤维网状细胞;
将所述纤维网状细胞用含无外泌体的血清的培养基进行培养,获得细胞上清,后进行第一固液分离获得上清液,将所述上清液进行第二固液分离获得固体,后重悬,获得纤维网状细胞来源的外泌体。
3.根据权利要求2所述的应用,其特征在于,所述获得纤维网状细胞,包括:
收集脂肪组织,剪碎后消化,过细胞筛后终止消化,通过密度梯度离心法去除红细胞和粒细胞,收集的细胞采用含有FBS的DMEM完全培养基进行培养并传代培养,获得纤维网状细胞。
4.根据权利要求3所述的应用,其特征在于,所述消化中,采用0.2±0.02%的胶原酶、2.0±0.5mg/ml中性蛋白酶和0.08±0.01mg/ml透明质酸酶的混合酶进行消化。
5.根据权利要求2所述的应用,其特征在于,所述第一固液分离采用0.2μM过滤。
6.根据权利要求2所述的应用,其特征在于,所述第二固液分离包括:4±2℃下超速离心60±20min,所述超速离心的转速为1.0×105g±1000g。
7.根据权利要求2所述的应用,其特征在于,所述含无外泌体的血清的培养基为含0.5-1.5%P/S、0.05-0.15%PB、45-55μM EGF、0.1-0.3mg/mLFGF、50-150nM TGF、8-12%无外泌体血清的DMEM完全培养基。
8.根据权利要求2所述的应用,其特征在于,所述纤维网状细胞的标志抗体为CD45-、PDPN+和CD31-。
9.根据权利要求2所述的应用,其特征在于,所述纤维网状细胞来源的外泌体特异性标志物为CD9、CD63、HSP70、TSG-101和CD81。
10.一种用于治疗和/或预防脓毒症急性肾损伤的药物,其特征在于,所述药物包括纤维网状细胞来源的外泌体。
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