WO2023098101A1 - 一种lncRNA基因修饰细胞分泌的外泌体及应用 - Google Patents
一种lncRNA基因修饰细胞分泌的外泌体及应用 Download PDFInfo
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- the present invention also provides a method for constructing the HEK-293T cell line, comprising the following steps:
- the HEK-293T cell line stably expressing lncENAF was constructed using the construction method, and then cultured, the culture medium was collected, and the exosomes secreted by the HEK-293T cell line were collected by centrifugation; the exosomes were mixed with giant The phagocytes were co-incubated, and the expression levels of the macrophage cytokines were detected.
- Figure 6 shows that exosome lncENAF regulates inflammation
- A exosomes enter cells
- B-C exosomes inhibit LPS-induced IL-6 mRNA synthesis and extracellular IL-6 release
- D-E exosomes inhibit LPS-induced IL-1 ⁇ mRNA synthesis and extracellular IL-1 ⁇ release.
- the lentiviral packaging plasmids are psPAX and pMD2.G respectively, and the extraction steps refer to (OMEGA Endotoxin Removal Plasmid Extraction Kit (D6948-01), as follows:
- step (9) take out DNA Mini Column, add 700 ⁇ L of the liquid in step (8), and centrifuge at room temperature at 20,000 g for 1 min;
- IL-6 kit mouse interleukin 6 ELISA kit (70-EK206/3)
- IL-1 ⁇ kit mouse interleukin 1 ⁇ ELISA kit (70-EK201B/3)
- washing liquid Discard the liquid, add 300 ⁇ L of washing liquid to each well to wash the plate, wash 6 times, wash the plate each time, and pat dry on absorbent paper;
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Abstract
提供了一种lncRNA基因修饰细胞分泌的外泌体及应用,属于细胞生物学领域。所述外泌体是通过基因工程获得的HEK-293T细胞株分泌,所述HEK-293T细胞株稳定表达lncENAF,所述lncENAF的核苷酸序列如SEQ ID NO:1所示。该外泌体可以有效地抑制脂多糖诱导的巨噬细胞细胞因子的产生,是一种细胞因子潜在的抑制剂,为细胞因子风暴和治疗自身免疫性疾病提供了新方向。
Description
本发明涉及细胞生物学领域,特别是涉及一种lncRNA基因修饰细胞分泌的外泌体及应用。
长链非编码RNA(lncRNA)是一类转录长度超过200nt的非编码序列,因其缺少有效开放读框而很少编码或不能编码蛋白质。由于之前对lncRNA的研究尚浅,认为其不具有任何生物学功能,只是作为转录过程的副产物而存在。随着分子生物学测序技术的不断发展,发现它可以从表观遗传调控、转录调控以及转录后调控等不同层面进行基因调控。因此,长链非编码RNA的调控作用正在被越来越多的人关注和研究。
外泌体是指其粒径大小在60-200nm的细胞外囊泡,可以由几乎所有的细胞分泌,能包含一些复杂的物质(如核酸、蛋白质、脂质等),因此外泌体可以作为细胞间通讯的重要介质参与细胞间的信号传递。有研究提出LncRNA HOTAIR能够通过介导外泌体形成相关蛋白的表达,促进外泌体的释放,在一定程度上丰富了我们对长链非编码RNA对外泌体释放调节的认识。但是,目前对长链非编码RNA调控的外泌体对于巨噬细胞因子影响的相关报道很欠缺。
发明内容
本发明的目的是提供一种lncRNA基因修饰细胞分泌的外泌体及应用,以解决上述现有技术存在的问题,该外泌体可以有效地抑制脂多糖诱导的巨噬细胞细胞因子的产生,是一种细胞因子潜在的抑制剂,为细胞因子风 暴和治疗自身免疫性疾病提供了新方向。
为实现上述目的,本发明提供了如下方案:
本发明提供一种抑制巨噬细胞细胞因子的外泌体,所述外泌体是通过基因工程获得的HEK-293T细胞株分泌,所述HEK-293T细胞株稳定表达lncENAF,所述lncENAF的核苷酸序列如SEQ ID NO:1所示。
本发明还提供所述的外泌体在制备抑制脂多糖诱导的细胞因子水平升高的药物中的应用。
本发明还提供所述的外泌体在制备抑制细胞因子风暴或治疗自身免疫性疾病的药物中的应用。
优选的是,所述自身免疫性疾病包括脓毒血症、病毒性肺炎、类风湿性关节炎、脑炎、肺纤维化、脂肪肝炎和多发性硬化症等。
优选的是,所述外泌体通过抑制脂多糖诱导的细胞因子水平的升高,实现对细胞因子风暴抑制或自身免疫性疾病的治疗。
优选的是,所述细胞因子包括IL-6和IL-1β。
本发明还提供一种抑制脂多糖诱导的细胞因子水平升高的药物,包括所述的外泌体,以及药学或者免疫学上可结合的载体或辅料。
本发明还提供一种抑制细胞因子或治疗自身免疫性疾病的药物,包括所述的外泌体,以及药学或者免疫学上可结合的载体或辅料。
本发明还提供一种所述HEK-293T细胞株的构建方法,包括以下步骤:
步骤1:获取lncENAF的基因序列,并构建稳定表达所述lncENAF的慢病毒载体;
步骤2:用HEK-293T细胞与所述慢病毒载体混合进行慢病毒质粒转 染,获取病毒液;
步骤3:将所述病毒液与HEK-293T细胞混合培养,经抗生素筛选,获取稳定表达lncENAF的HEK-293T细胞株。
本申请还公开一种所述的抑制巨噬细胞细胞因子的外泌体的使用方法,包括以下步骤:
利用所述的构建方法构建稳定表达lncENAF的HEK-293T细胞株,然后进行培养,收集培养液,再通过离心收集所述HEK-293T细胞株分泌的外泌体;将所述外泌体与巨噬细胞共孵育,检测所述巨噬细胞细胞因子的表达水平。
本发明公开了以下技术效果:
本发明通过构建的非酒精性脂肪性肝炎小鼠模型,发现一条显著上调的非编码RNA,命名为lncENAF。然后通过基因工程构建了稳定表达lncENAF的HEK-293T细胞株,由该细胞株分泌的外泌体与巨噬细胞孵育,发现该外泌体可以显著抑制由脂多糖诱导的巨噬细胞因子IL-6等的产生。可见,本发明提供的外泌体是一种细胞因子潜在的抑制剂,这为巨噬细胞细胞因子的抑制提供了数据支持,并且为由脂多糖诱导的巨噬细胞因子升高而导致的细胞因子风暴和相关的自身免疫性疾病提供了新的防治策略。
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅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他的附图。
图1为pCDH-GFP-lncENAF质粒菌液PCR后电泳验证结果;
图2为lncEANF全长与重组质粒测序比对结果;
图3为pCDH-GFP-lncENAF载体图谱;
图4为稳定表达lncENAF的HEK-293T细胞株的构建与鉴定;A:HEK-293T-lncENAF细胞株荧光图;B:qPCR检测HEK-293T-lncENAF细胞株内lncENAF的Cq值;
图5为外泌体的分离和鉴定;A:外泌体提取流程;B:外泌体电镜鉴定;C:外泌体粒径分析;D:外泌体特征蛋白(TSG101和CD9)的定性分析;
图6为外泌体lncENAF调控炎症;A:外泌体进入细胞;B-C:外泌体抑制LPS诱导的IL-6的mRNA合成以及细胞外IL-6释放;D-E:外泌体抑制LPS诱导的IL-1β的mRNA合成以及细胞外IL-1β释放。
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
在前期研究中(Chen Q,Xiong C,Jia K,et al.Hepatic transcriptome analysis from HFD-fed mice defines a long noncoding RNA regulating cellular cholesterol levels.J Lipid Res.2019;60(2):341-352),我们在构建的非酒精性脂肪性肝炎小鼠模型中,通过RNA测序发现了一条显著上调的非编码RNA,NONCODE:NONMMUG027912.3,我们命名为lncENAF(Elevated in Non Alcoholic Fatty Liver)。通过转基因技术,构建了稳定表达lncENAF的HEK-293T细胞株,对HEK-293T-lncENAF细胞株进行培养,收集经lncENAF基因修饰的HEK-293T细胞培养基,通过超速离心方法收集分泌的外泌体,将外泌体与巨噬细胞共孵育,发现此种外泌体可以显著抑制由脂多糖诱导的巨噬细胞细胞因子,如IL-6等的产生。
实施例1
一、过表达lncENAF的慢病毒载体构建
课题组保存有pMD-T18-lncENAF质粒和菌株,前期通过设计同源臂引物(酶切位点为:XbaⅠ和SalⅠ),将lncENAF(lncENAF的核苷酸序列如SEQ ID NO:1所示)连接到pCDH-GFP质粒构成pCDH-GFP-lncENAF质粒(质粒图谱见图3,pCDH-GFP-lncENAF的核苷酸序列如SEQ ID NO:2所示),同源臂引物见附图(见表1),具体构建步骤如下:
表1 同源臂引物序列
(1)PCR扩增lncEANF:使用PCR进行lncEANF的扩增,扩增模板为pMD-T18-lncENAF,扩增体系如下:
表2 扩增体系
(2)称取0.5g琼脂糖粉至干净的锥形瓶,加入50mL TAE缓冲液,微波炉加热融化摇匀,流水冲洗锥形瓶降温至大约40℃后,加入0.5μL的Gold ViewⅠ型核酸染料,混匀后倒入胶槽,插入梳子,室温放置30min,待琼脂糖胶凝固后,拔去梳子,将胶转移到含适量1×TAE的电泳槽;
(3)PCR结束后,每个PCR反应体系中加入1μL 10×Loading Buffer, 加样枪混匀后加入到凝胶中的加样孔中,120V电泳25min;
(4)电泳完成后把凝胶放置在凝胶成像系统,在紫外光照射下切下目标条带,称取重量(0.2g)后放至到无菌1.5ml EP管中;
(5)加入200μL Binding Buffer,50℃水浴融化凝胶,每2min震荡一次;
(7)加入300μL Binding Buffer,20000g室温离心1min,弃废液;
(8)加入700μL Washing Buffer,20000g室温离心1min,弃废液,重复此步骤一次;
(10)加入30μL ddH
20,室温静置2min,然后20000g室温离心1min,EP管中收集到的即为lncENAF,采用DeNovix DS-11+Spectrophotometer检测lncENAF浓度和纯度,记录后进行后续操作;
(11)pCDH-GFP质粒双酶切回收,双酶切体系如下:
表3 双酶切体系
混匀后37℃反应2h;
(12)反应结束后进行电泳以及切胶回收,步骤同上;
(13)配制如下无缝克隆体系(使用和元无缝克隆试剂盒):
表4 无缝克隆体系
混匀后37℃反应30min;
(14)-80℃取出DH-5α感受态,冰上融化后取50μL至干净1.5mL EP管,加入10μL无缝克隆体系,冰上放置30min,然后立刻42℃水浴45s,再放回冰上,静置3min,加入940μL LB培养基,37℃震荡培养2h;
(15)培养结束后,3000g室温离心5min,弃去900μL上清液,用加样枪混匀剩余100μL,加到Amp抗性平板中央,用无菌涂布棒均匀涂抹菌液,37℃培养过夜;
(16)培养过夜后,使用接种环挑取单个菌落至400μL含Amp抗性的LB细菌培养基中,37℃震荡培养2h;
(17)培养2h后,取1μL菌液作为模板进行菌液PCR鉴定pCDH-GFP-lncENAF质粒是否成功构建,体系如下:
表5 PCR鉴定体系
混匀后放入PCR仪反应;
(18)PCR反应结束后,进行琼脂糖凝胶电泳,步骤同上,电泳结束后进行曝光鉴定,电泳结果显示第7管和第11管菌液PCR产物片段大小在600bp-800bp,且条带清晰透亮(见图1),随后挑取第7管菌液送至上海桑尼公司进行Sanger测序,测序比对结果显示lncEANF全长与重组质粒100%配对(见图2),表明pCDH-GFP-lncENAF质粒被成功构建(质粒图谱见图3)。
二、慢病毒表达质粒和包装质粒的提取
慢病毒包装质粒分别为psPAX、pMD2.G,提取步骤参考(OMEGA内毒素去除质粒提取试剂盒(D6948-01),具体如下:
(1)打开紫外超净台紫外灯,照射30min;
(2)取50mL EP管,倒入30mL LB培养基,添加30μL氨苄青霉素(Amp+)(1:1000稀释),最后加入50μL菌液,37℃,250rpm培养16h;
(3)取出菌液,10000g离心1min,弃去上清;
(4)加入600μL的SolutionⅠ,吹悬细菌沉淀后转移至2mL EP管,涡旋仪剧烈震荡1min;
(5)加入600μL的SolutionⅡ,轻柔颠倒6次,室温静置2min;
(6)加入300μL提前预冷的N3Buffer,颠倒混匀至白色浑浊沉淀形成,室温静置2min;
(7)20000g室温离心5min,吸取上清至新的2mL EP管,加入1/10体积的ETR Solution,颠倒混匀后,冰上静置10min(期间每过2min颠倒3次),随后42℃放置2min,再20000g室温离心5min;
(8)取上清液至新的2mL EP管,加入1/2体积的无水乙醇,颠倒混匀,室温静置2min;
(10)弃去收集柱液体,重复步骤(9)直至步骤(8)液体全部离心;
(11)弃去收集柱液体,加入500μL HBC Buffer,20000g室温离心1min;
(13)加入50μL DEPC水,室温静置3min后,20000g室温离心1min;
二、慢病毒的包装
(1)取出培养的HEK-293T细胞,接种100×10
4个细胞至6cm培养皿,共计两个皿,采用十字交叉法摇动培养皿使细胞均匀贴壁生长;
(2)观察细胞生长密度至70%时,进行慢病毒质粒转染;
(3)取两个1.5mL EP管,标记为A、B两管,每管加入500μL Opti-MEM培养基;
(4)A管加入10μL lipofectamine3000,吹打混匀,室温静置1min;
(5)B管加入10μL P3000,4μg pCDH-lncENAF质粒(对照组加入pCDH-GFP质粒)、3μg psPAX2质粒和1μg的pMD2.G质粒,吹打混匀,室温静置1min;
(6)将B管全部液体转移至A管,吹打混匀,室温静置15min;
(7)将步骤(6)的混合物均匀滴加至HEK-293T细胞中,培养箱中培养12h,更换DMEM培养基,继续培养24h和48h,收取病毒上清;
(8)使用0.45μm孔径的过滤器过滤收取的病毒液,分装至1.5mL EP管,每管1mL,-80℃保存。
三、HEK-293T细胞致死嘌呤霉素浓度筛选和HEK-293T-lncENAF细胞株构建
(1)把HEK-293T细胞接种至6孔板,每孔25×10
4个细胞;
(2)在细胞密度在50%左右时,培养基更换为含嘌呤霉素的DMEM培养基,分别使用为0μg/mL、0.2μg/mL、0.3μg/mL和0.4μg/mL四个浓度梯度的嘌呤霉素;
(3)继续培养72h后,发现HEK-293T细胞最大致死嘌呤霉素浓度为0.4μg/mL;
(4)确定好最适嘌呤霉素浓度后,把HEK-293T细胞接种到6孔板,每孔25×10
4个细胞,培养12h至细胞贴壁;
(5)每孔加入300μL病毒液,感染细胞12h后更换新鲜的DMEM培养基进行培养72h;
(6)病毒感染72h后,病毒携带的基因将会随机整合进HEK-293T细 胞,此时把培养基更换为含0.4μg/mL的嘌呤霉素培养基,继续培养7天,每天更换一次新鲜含嘌呤霉素的DMEM培养基,筛选7天后,存活下来的细胞即为稳定表达lncENAF的HEK-293T细胞株,命名为HEK-293T-lncENAF,在倒置荧光显微镜下可看到绿色荧光(见图4A)。
四、荧光定量PCR
使用SYBRRPremix Ex TaqTM II试剂盒,参考说明书(Takara:(RR820A))进行加好体系,在BioRad仪器,按照以下程序进行95℃3min,(95℃5s,60℃30s,72℃40s,循环40次),72℃5min,95℃15s,60℃1min,95℃15s。反应结束后观察HEK-293T-lncENAF细胞株与对照细胞株内lncENAF的Cq值差异,结果显示HEK-293T-lncENAF细胞株内lncENAF的Cq值显著低于对照(见图4B),实验涉及到的qPCR引物序列均由擎科生物公司合成,序列见附图(见表6)。
表6 引物序列
五、外泌体的分离纯化及鉴定
5.1外泌体的获取与分离纯化
(1)把HEK-293T-lncENAF细胞株接种至15cm培养皿,每个培养皿接种200×10
4个细胞,放至37℃恒温二氧化碳培养箱培养;
(2)待细胞汇合度至80%时,把培养基更换为不含血清的DMEM培养基,继续培养24h;
(3)收集细胞培养上清至50mL EP管,参考外泌体提取流程进行收集外泌体(见图5A),首先300g 4℃离心10min;
(4)将上清移至新的50mL EP管,弃去沉淀,3000g 4℃离心20min;
(5)收集上清至新的50mL EP管,弃去沉淀,10000g 4℃离心30min;
(6)收集上清至SW 32Ti超离管,100000g 4℃离心90min;
(7)弃去上清,加35mL PBS重悬沉淀,100000g 4℃离心90min;
(8)弃去上清,加200μL PBS重悬外泌体,使用0.22μm孔径过滤器过滤后-80℃保存。
5.2外泌体的鉴定
5.2.1利用TEM观察负染外泌体
用PBS重悬提取的外泌体,滴于孔径2nm的载样铜网上,室温静置2min,用滤纸滤网侧边吸干液体,用2%磷钨酸溶液在室温下负染2min,滤纸吸干负染液,室温晾干,电镜观察拍照,箭头所示大小在100nm左右的囊泡即为外泌体(见图5B)。
5.2.2纳米颗粒跟踪分析(NTA)分析外泌体粒径大小和数量
将分离的外泌体样本用PBS稀释后,取500μL样品稀释10倍后,注入纳米颗粒跟踪分析仪,用激光穿过样本,通过装有摄像头的显微镜收集 散射光,捕捉外泌体的布朗运动,然后Stokes-Einstein方程来测量粒子的平均速度而估算粒子大小和数量,结果显示外泌体平均粒径大约为144nm,其中每1mL含有的外泌体囊泡数为4.39×10
8(见图5C)。
5.2.3 Western Blot检测外泌体特征Marker蛋白
(1)凝胶配制:配制12%的分离胶,充分混匀后加至厚薄板之间,上层用压平,室温放置约30min,弃去上层水,配制5%的浓缩胶,充分混匀后加入厚薄板之间,插入梳子,避免产生气泡,待浓缩胶凝固后可进行电泳;
(2)电泳:将配好的凝胶放置在电泳槽中,加入1×电泳缓冲液,竖直将梳子拔出,每孔上样20μg蛋白样品,浓缩胶70V电泳40min,分离胶110V电泳60min;
(3)转膜:剪取合适大小的PVDF膜,放入甲醇中活化90s后放入转膜缓冲液中平衡,待电泳结束后,根据蛋白Marker的位置切胶,按照黑色转膜夹在下,海绵、滤纸、凝胶、PVDF膜、滤纸、海绵的顺序放置凝胶,固定好转膜夹,放置在转膜槽中,加入转膜缓冲液和冰袋,接通转膜仪,恒流300mA电泳60min;
(4)封闭:转膜结束后,用镊子小心夹取PVDF膜放入5%脱脂牛奶中,室温摇床轻摇封闭2h;
(5)孵育一抗:封闭结束后,弃去封闭液,用1×TBST清洗条带5min,共三次,根据蛋白Marker的位置将目的蛋白分子量大小的条带剪下,用滤纸吸干,放入相应稀释好的抗体中,4℃摇床孵育过夜;
(6)孵育二抗:取出孵育过夜的条带,用1×TBST清洗条带5min, 共三次,根据一抗来源将条带放入相应的二抗中,室温摇床轻摇孵育1h;
(7)显影:从二抗中取出条带,用1×TBST清洗条带15min,共三次,ECL显影液A液和B液按照1:1混合均匀,用滤纸将条带吸干放入曝光夹,均匀加上现配的显影液,设置曝光时间,进行曝光分析,结果显示收集的外泌体中含有特征蛋白TSG101和CD9,GAPDH蛋白含量很低,HEK-293T细胞裂解液作为对照(见图5D)。
六、外泌体进入巨噬细胞激光共聚焦拍摄
6.1 PKH26荧光染料标记外泌体
(1)取出-80℃保存的外泌体,冰上融化;
(2)避光条件下,取出PKH26荧光染料,取1μL至200μL PCR管,加入99μL稀释液稀释100倍;
(3)稀释后的PKH26染料全部加入外泌体中,剧烈涡旋1min混匀,避光孵育20min;
(4)加入35mL PBS,100000g 4℃离心90min;
(5)弃去上清,加100μL PBS重悬外泌体,-20℃避光保存。
6.2外泌体处理枯否细胞、爬片制作以及激光共聚焦拍摄
(1)取出一个24孔板,每个孔放入一个圆形细胞爬片,接种5×10
4个细胞;
(2)培养细胞12h后,每个孔加入20μL荧光标记的外泌体,分别在培养12h和24h时取出爬片,加500μL PBS,轻微晃动几次,弃去PBS,重复三次;
(3)加入200μL 4%多聚甲醛,4℃固定过夜;
(4)取出固定好的爬片,弃去多聚甲醛,加入500μL PBS,轻微晃动几次,弃去PBS,重复三次;
(5)取一块干净的载玻片,滴加一滴抗荧光淬灭剂,用镊子夹出爬片,小心用边缘接触滤纸吸干水分,倒扣在抗荧光淬灭剂上,边缘四周滴上中性树脂,避光室温静置30min,待稳定后4℃保存,第二天进行激光共聚焦观察,发现枯否细胞内出现PKH26红色荧光,表明枯否细胞可以吞噬PKH26标记的外泌体,并且随着孵育时间的延长,吞噬的外泌体数量增加(见图6A)。
七、外泌体与枯否细胞共孵育后,利用脂多糖刺激,检测IL-6、IL-1β变化
(1)把HEK-293T-lncENAF细胞株及对照细胞株接种至24孔板中,每孔接种5×10
4个细胞;
(2)12h后细胞贴壁,每孔加入25μg外泌体,继续培养12h后,每孔加入终浓度为50μg/mL的脂多糖刺激枯否细胞24h,24h后收集细胞上清,使用酶联免疫法检测上清中IL-6、IL-1β浓度(见方法“八、酶联免疫法检测IL-6、IL-1β”),使用qPCR检测枯否细胞内IL-6、IL-1β的mRNA表达变化,结果显示,加入Exo-ENAF之后,可以抑制LPS诱导的IL-6、IL-1β的mRNA表达(见图6B、6D)。
八、酶联免疫法检测IL-6、IL-1β
操作步骤:参考IL-6试剂盒(MuitiSciences:小鼠白介素6ELISA试剂盒(70-EK206/3))和IL-1β试剂盒(MuitiSciences:小鼠白介素1βELISA试剂盒(70-EK201B/3)),具体如下:
(1)细胞培养上清:300g室温离心10min,离心结束后取上清;
(2)稀释标准品:标准品开盖前短暂离心,用蒸馏水溶解小鼠IL-6/IL-1β标准品。轻柔地涡旋震荡,确保充分混匀,标准品的浓度为1000pg/mL,静置20min。
(3)细胞培养上清样本标准曲线的制备:取230μL浓缩标准品,加入230μL细胞培养基,作为标准曲线的最高浓度(500pg/mL)。在每一个试管中加入230μL细胞培养基。使用高浓度标准品做1:1系列稀释。每次移液时,确保充分混匀,以细胞培养基作为标准曲线的零浓度。
(4)检测之前请将所有的试剂、样本平衡至室温,准备好所有需要的试剂及工作浓度标准品;
(5)将不需要的板条拆卸下来,放回装有干燥剂的铝箔袋,重新封好封口;
(6)浸泡酶标板:加入300μL 1×洗液静置浸泡30s,弃掉洗液,在吸水纸上将微孔板拍干(洗板完成之后,立即使用微孔板,不要让微孔板干燥);
(7)加样:标准品孔加入100μL 2倍倍比稀释的标准品,空白孔加入100μL培养基,样本孔加入100μL细胞培养上清;
(8)加检测抗体:每孔加入50μL稀释的检测抗体(1:100稀释);
(步骤(6)、(7)、(8)连续加样,不要间断,加样过程在15min内完成)。
(9)孵育:使用封板膜封板,300rpm/min振荡,室温孵育1.5h;
(10)洗涤:弃掉液体,每孔加入300μL洗液洗板,洗涤6次,每次 洗板,在吸水纸上拍干;
(11)加酶孵育:每孔加入100μL稀释的辣根过氧化物酶标记的链霉亲和素(1:100稀释);
(12)孵育:使用新的封板膜封板,300rpm/min振荡,室温孵育0.5h;
(13)洗涤:重复步骤(10);
(14)加底物显色:每孔加入100μL显色底物TMB,室温避光孵育20min;
(15)加终止液:每孔加入10μL终止液,颜色由蓝色变为黄色(如果颜色呈现绿色或者颜色的变化明显不均匀,轻轻叩击板框,充分混匀);
(16)检测读数:在30min之内,使用酶标仪进行双波长检测,测定450nm最大吸收波长和570nm参考波长下的OD值,校准后的OD值为450nm的测定值减去570nm的测定值,仅使用450nm测定会导致OD值偏高,并且准确度降低。结果显示,加入稳定表达lncENAF细胞分泌的外泌体后,可以抑制LPS诱导的IL-6、IL-1β细胞因子的释放(结果见图6C、6E)。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (10)
- 一种抑制巨噬细胞细胞因子的外泌体,其特征在于,所述外泌体是通过基因工程获得的HEK-293T细胞株分泌,所述HEK-293T细胞株稳定表达lncENAF,所述lncENAF的核苷酸序列如SEQ ID NO:1所示。
- 权利要求1所述的外泌体在制备抑制脂多糖诱导的细胞因子水平升高的药物中的应用。
- 权利要求1所述的外泌体在制备抑制细胞因子风暴或治疗自身免疫性疾病的药物中的应用。
- 根据权利要求3所述的应用,其特征在于,所述自身免疫性疾病包括脓毒血症、病毒性肺炎、类风湿性关节炎、脑炎、肺纤维化、脂肪肝炎和多发性硬化症。
- 根据权利要求3所述的应用,其特征在于,所述外泌体通过抑制脂多糖诱导的细胞因子水平的升高,实现对细胞因子风暴抑制或自身免疫性疾病的治疗。
- 根据权利要求5所述的应用,其特征在于,所述细胞因子包括IL-6和IL-1β。
- 一种抑制脂多糖诱导的细胞因子水平升高的药物,其特征在于,包括权利要求1所述的外泌体,以及药学或者免疫学上可结合的载体或辅料。
- 一种抑制细胞因子或治疗自身免疫性疾病的药物,其特征在于,包括权利要求1所述的外泌体,以及药学或者免疫学上可结合的载体或辅料。
- 一种权利要求1中所述HEK-293T细胞株的构建方法,其特征在于,包括以下步骤:步骤1:获取lncENAF的基因序列,并构建稳定表达所述lncENAF的 慢病毒载体;步骤2:用HEK-293T细胞与所述慢病毒载体混合进行慢病毒质粒转染,获取病毒液;步骤3:将所述病毒液与HEK-293T细胞混合培养,经抗生素筛选,获取稳定表达lncENAF的HEK-293T细胞株。
- 一种如权利要求1所述的抑制巨噬细胞细胞因子的外泌体的使用方法,其特征在于,包括以下步骤:利用权利要求9所述的构建方法构建稳定表达lncENAF的HEK-293T细胞株,然后进行培养,收集培养液,再通过离心收集所述HEK-293T细胞株分泌的外泌体;将所述外泌体与巨噬细胞共孵育,检测所述巨噬细胞细胞因子的表达水平。
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