CN116421613A - 一种过表达miR-146a-5p的间充质干细胞外泌体药物及应用 - Google Patents
一种过表达miR-146a-5p的间充质干细胞外泌体药物及应用 Download PDFInfo
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Abstract
本发明提供了一种过表达miR‑146a‑5p的间充质干细胞外泌体药物及应用,属于生物药物技术领域。本发明将制备的过表达miR‑146a‑5p的间充质干细胞外泌体作用于脂多糖诱导BV2小胶质细胞和RAW246.7巨噬细胞炎症损伤海马神经元HT22共培养体系中,结果表明过表达miR‑146a‑5p的间充质干细胞外泌体较对照间充质干细胞外泌体在抗炎作用方面提高了1~2倍,能够对小胶质细胞、巨噬细胞以及神经细胞中下调炎症因子的表达,同时抑制炎症相关通路NF‑κB信号通路的激活,有效抑制炎症反应。因此,本发明为神经炎症相关的疾病的治疗提供了新手段。
Description
技术领域
本发明属于生物药物技术领域,具体涉及一种过表达miR-146a-5p的间充质干细胞外泌体药物及应用。
背景技术
随着人口老龄化的加剧,神经退行性疾病的发病率日益升高。目前认为,神经退行性疾病的发生与年龄的增加呈正相关。很多神经退行性疾病包括阿尔茨海默病(Alzheimer’s Disease,AD)、视网膜色素变性(Retinitis pigmentosa,RP)等在中老年人群中高发。随着我国人口老龄化的加剧,神经退行性疾病的预防和治疗将成为一项新的困难和挑战。然而,神经退行性疾病导致神经元死亡、脑功能或者视觉功能逐渐丧失,常规药物治疗难以修复神经元的损伤和促进功能的恢复。因此,寻找新的治疗方式成为目前亟待解决的问题之一。世界卫生组织在2008年发布的一项报告显示,全球约5000万痴呆患者,AD患者占60%~70%。流行病学数据预测,2050年全球AD患者可能超过1.52亿[1]。大多数的AD患者在65岁发现患病,发现此病后患者的平均寿命为7年[2-3]。RP是一种不可逆转的致盲性眼科疾病,RP的患病率为1/4000,目前有超过150万的患者[4-5]。因此,迫切需要探索临床有效治疗如AD、RP等神经退行性疾病的的方式。
神经退行性疾病以神经元继发性死亡为特征,并且导致神经功能丧失。如阿尔茨海默症(Alzheimer,s Disease,AD),患者主要表现为认知功能障碍、记忆力衰退和其他精神障碍;视网膜色素变性(Retinitis Pigmentosa,RP),患者主要表现为夜盲、视野变窄和逐渐失明。此外,神经元继发性的死亡与神经炎症高度相关[5]。神经炎症的发生表现为神经胶质细胞的反应性形态改变和功能异常,如小胶质细胞和星形胶质细胞[6],并且激活态的小胶质细胞能够损伤或者杀死神经元[7],提示抑制神经炎症是治疗神经退行性疾病的一个切入口。研究表明,AD小鼠海马区域小胶质细胞大量增殖[8],并且形态明显发生改变(小胶质细胞突起回缩、胞体增大)[9],同时伴随炎症因子的表达异常升高[10-11]。在视网膜退行性疾病小鼠视网膜中同样也发现小胶质细胞具有同样的反应性激活[12-13]。除此之外,本团队成员前期的研究也明确了小胶质细胞在RP小鼠中反应性激活的表现[14-15]。越来越多的临床和动物研究数据表明,视网膜可能是AD早期诊断的窗口。AD早期发生时,视网膜结构与功能开始发生异常,具体表现为:视网膜神经纤维层变薄和视网膜血管***缺陷、视力下降、对比敏感度、颜色分辨力、图形视网膜电图(pERG)反应和视野缺陷[16]。最近的研究也表明AD小鼠视网膜中小胶质细胞反应性激活[17]。由此强烈提示,治疗RP对抗神经炎症的治疗机制可能也适用于AD的治疗。
干细胞或者前体细胞的移植治疗成为目前临床上最为可行的新型治疗方法,大量研究也报道了其在神经营养、免疫调节、抑制炎症中的作用[18]。但是,干细胞移植治疗效果有限,不能长久维持,而且涉及细胞的移植相关的各种风险及综合征,比如临床标准级细胞的生产、移植手术、免疫排斥及潜在的致癌风险等等[18]。虽然目前有研究报导间充质干细胞的治疗在临床上是安全有效的[19],但报导仍指出间充质干细胞的移植治疗仍需要深入的分子研究和仔细的安全性评估[18]。甚至,有研究报告了玻璃体或视网膜下腔注射间充质干细胞治疗引起视网膜损伤和眼部并发症,表明仍应谨慎应用干细胞移植治疗[20-21]。因此,寻求临床上治疗神经退行性疾病,对抗神经炎症的新型安全的有效的方法仍是一个亟待解决的问题。
干细胞来源的外泌体是是干细胞分泌的直径为30~200纳米大小囊泡(extracellμlarvesicles,EVs),其成分主要包括脂质、蛋白质、核酸等成分[22]。与间充质干细胞移植治疗相比,其外泌体(mesenchymal stem cells derived exosomes,MSCs-Exo)免疫原性低,易于储藏和给药,降低了干细胞移植相关风险。相较MSCs细胞移植,外泌体具有明显的优点:(1)MSCs在体内的分化、迁徙需要一定的时间;而外泌体体积小,易于穿过微血管到达靶器官,既能在较短时间内启动组织修复,又可减少因微循环阻塞所致的组织损伤;(2)外泌体具有良好的稳定性,其具有脂质双分子膜结构,能防止内容物的降解;(3)外泌体表面存在众多的粘附分子和锚定受体,具有高度的靶向性;(4)移植入体的MSCs具有成瘤癌变、病毒感染、基因突变等风险,而外泌体操作简单、花费低廉、安全有效。本团队前期发现MSCs-Exo有效促进RP小鼠视网膜神经元的存活,抑制神经炎症激活,且无明显副作用[15]。鉴于此,推测MSCs-Exo对于AD小鼠海马神经元也能起到很好的神经保护作用。然而MSCs-Exo中抗炎活性成分并不明确,并且神经细胞的抗炎能力也有待进一步提高。
参考文献
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发明内容
有鉴于此,本发明的目的在于提供一种过表达miR-146a-5p的间充质干细胞外泌体药物,通过将miR-146a-5p在间充质干细胞中过表达,收集的外泌体具有较高的神经抗炎效果。
本发明还提供了过表达miR-146a-5p的间充质干细胞外泌体在制备治疗神经炎症的药物中的应用,外泌体通过携带miR-146a-5p较间充质干细胞外泌体,在神经抗炎效果方面有极大提高。
本发明提供了一种过表达miR-146a-5p的间充质干细胞外泌体在制备治疗神经炎症的药物中的应用。
优选的,所述miR-146a-5p的核苷酸序列如SEQ ID NO:1所示。
优选的,所述过表达miR-146a-5p的间充质干细胞外泌体的制备方法,包括以下步骤:
制备过表达miR-146a-5p的重组慢病毒;
将所述过表达miR-146a-5p的重组慢病毒转染间充质干细胞,得到过表达miR-146a-5p的间充质干细胞;
从所述过表达miR-146a-5p的间充质干细胞的培养液中分离提取外泌体,得到过表达miR-146a-5p的间充质干细胞外泌体。
优选的,所述制备过表达miR-146a-5p的重组慢病毒的方法,包括将过表达miR-146a-5p的重组人源载体和PXPAX2、PMD2G在转染试剂的作用下共同转染哺乳细胞,培养,收集过表达miR-146a-5p的重组慢病毒颗粒。
优选的,所述分离提取外泌体的方法为将所述培养液依次在2000g、10000g的离心力条件下离心收集上清,再在100000g的离心力条件下离心,弃去上清,重悬沉淀。
优选的,神经炎症相关的疾病包括神经退行性疾病;
所述神经退行性疾病包括阿尔茨海默病和/或视网膜色素变性。
优选的,所述治疗神经炎症是通过保护小胶质细胞和/或巨噬细胞炎症损伤海马神经元实现。
优选的,所述神经炎症包括脂多糖诱导形成。
优选的,所述药物具有下调炎症因子表达水平和/或抑制NF-κB炎症信号通路的药效。
本发明还提供了一种过表达miR-146a-5p的间充质干细胞外泌体药物,其特征在于,以过表达miR-146a-5p的间充质干细胞外泌体为活性成分,还包括药学上可接受的辅料。
本发明提供了一种过表达miR-146a-5p的间充质干细胞外泌体在制备治疗神经炎症的药物中的应用。本发明首次证明间充质干细胞中微小RNA(miRNA)-146a-5p具有良好的抗炎作用,通过构建过表达miR-146a-5p的重组慢病毒,得到稳定过表达的间充质干细胞,通过超高速梯度离心法收集过表达miR-146a-5p的间充质干细胞外泌体。本发明再以LPS诱导BV2细胞和RAW246.7细胞炎症损伤HT22细胞模型为研究对象,给药过表达miR-146a-5p的间充质干细胞外泌体处理后,能够有效下调BV2细胞和RAW246.7细胞中炎症因子IL-1β、TNF-α和IL-6mRNA表达水平,同时抑制炎症信号通路NF-κB信号激活。并且过表达miR-146a-5p的间充质干细胞外泌体明显逆转LPS诱导导致的HT22神经元细胞活力下降、细胞凋亡率升高和细胞凋亡信号通路激活等问题,并且过表达miR-146a-5p的间充质干细胞外泌体的抗炎效果是普通MSCs-Exo的2倍左右。可见本发明提供的方案为神经炎症相关的疾病的治疗提供了新手段。
附图说明
图1为本发明技术方案的技术路线图;其中A为过表达miR-146a-5p的MSCs-Exo的收集与验证,B为LPS诱导BV2细胞和RAW246.7细胞炎症损伤HT22细胞模型的建立;C为过表达miR-146a-5p的MSCs-Exo的抗炎效果与对HT22细胞的神经保护作用;
图2为在间充质干细胞和外泌体中过表达miR-146a-5p结果图;其中A为普通显微镜下P3代间充质干细胞形态(scale bar=200μm),B为荧光显微镜下观察慢病毒感染间充质干细胞荧光效率(scale bar=200μm),C为间充质干细胞与过表达间充质干细胞中miR-146a-5p的mRNA的表达情况,D为间充质干细胞外泌体与过表达间充质干细胞外泌体中miR-146a-5p的mRNA的表达情况;
图3为使用LPS诱导小胶质细胞和巨噬细胞炎症模型结果图;利用0-10.0μg/mLLPS处理RAW264.7和BV2细胞,其中A为刺激RAW264.7细胞活力变化,B为刺激RAW264.7细胞形态变化,C为刺激RAW264.7细胞炎症因子IL-1β、TNF-α和IL-6变化,D为1μg/mL LPS刺激后RAW264.7细胞Nr4a3与miR-146a-5p的mRNA的表达情况,F为刺激BV2细胞活力变化,E为刺激BV2细胞形态变化,G为刺激BV2细胞活力变化,H为1μg/mL LPS刺激后BV2细胞Nr4a3与miR-146a-5p的mRNA的表达情况;
图4为LPS诱导小胶质细胞和巨噬细胞对神经元HT22共培养后的细胞活力、细胞凋亡、炎症因子、信号通路表达结果图;其中A为神经炎症共培养体系示意图,B为两种神经炎症共培养体系中HT22细胞活力的变化,C为HT22-RAW264.7共培养体系中LPS处理后HT22细胞凋亡情况,D为HT22-BV2共培养体系中LPS处理后HT22细胞凋亡情况,E为HT22-RAW264.7共培养体系中LPS处理后HT22细胞炎症因子及NF-κB信号通路的表达情况,F为HT22-BV2共培养体系中LPS处理后HT22细胞炎症因子及NF-κB信号通路的表达情况;
图5为采用过表达miR-146a-5p的MSCs-Exo和普通MSCs-Exo作用到共培养体系后的抗炎效果以及对HT22细胞的治疗效果图;其中A为HT22-RAW264.7共培养体系中两种外泌体治疗后HT22细胞活力变化,B为HT22-BV2共培养体系中两种外泌体治疗后HT22细胞活力变化,C为两种炎症共培养体系中两种外泌体治疗后凋亡蛋白的表达情况,D为HT22-RAW264.7共培养体系中两种外泌体治疗后HT22细胞炎症因子变化,E为HT22-BV2共培养体系中两种外泌体治疗后HT22细胞炎症因子变化,F为HT22-RAW264.7共培养体系中两种外泌体治疗后HT22细胞NF-κB信号通路的表达情况,G为HT22-BV2共培养体系中两种外泌体治疗后HT22细胞NF-κB信号通路的表达情况;
图6为本发明探索过表达miR-146a-5p抗炎疗效通过Nr4a3-NF-κB信号轴的机制结果图,其中A为RAW264.7瞬时转染mimic miR-146a-5p和inhibitor miR-146a-5p后Nr4a3和miR-146a-5p的表达,B为BV2瞬时转染mimic miR-146a-5p和inhibitormiR-146a-5p后Nr4a3和miR-146a-5p的表达,C为过表达miR-146a-5p慢病毒感染RAW264.7细胞后Nr4a3蛋白、mRNA的表达以及炎症因子的变化,D为过表达miR-146a-5p慢病毒感染BV2细胞后Nr4a3蛋白、mRNA的表达以及炎症因子的变化。
具体实施方式
本发明提供了一种过表达miR-146a-5p的间充质干细胞外泌体在制备治疗神经炎症的药物中的应用。
在本发明中,所述miR-146a-5p的核苷酸序列优选如SEQ ID NO:1(ACAGGGCUGGGACAGGCCUGGACUGCAAGGAGGGGUCUUUGCAC CAUCUCUGAAAAGCCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGUGGGCUUGAGGACCUGGAGAGAGUAGAUCCUGAAGAACUUUUUCAGUCUGCUGAAGAGCUUGGAAGACUGGA)所示。
在本发明中,所述过表达miR-146a-5p的间充质干细胞外泌体的制备方法,优选包括以下步骤:
制备过表达miR-146a-5p的重组慢病毒;
将所述过表达miR-146a-5p的重组慢病毒转染间充质干细胞,得到过表达miR-146a-5p的间充质干细胞;
从所述过表达miR-146a-5p的间充质干细胞的培养液中分离提取外泌体,得到过表达miR-146a-5p的间充质干细胞外泌体。
在本发明中,所述制备过表达miR-146a-5p的重组慢病毒的方法,优选包括将过表达miR-146a-5p的重组人源载体和PXPAX2、PMD2G在转染试剂的作用下共同转染哺乳细胞,培养,收集过表达miR-146a-5p的重组慢病毒颗粒。所述miR-146a-5p在人源载体中的多克隆位点优选为AgeI/EcoRI。所述过表达miR-146a-5p的重组人源载体购自吉凯基因,产品型号为hsa-mir-146a(81952-2)。所述PXPAX2、PMD2G和过表达miR-146a-5p的重组人源载体的质量比为2:1:2。所述PXPAX2、PMD2G和过表达miR-146a-5p的重组人源载体的总质量和转染试剂的体积比优选为1μg:2μl。本发明对所述转染试剂的种类没有特殊限制,采用本领域所熟知的转染试剂即可,例如lipofectamine 3000。本发明对所述哺乳细胞的种类没有特殊限制,采用本领域所熟知的哺乳动物种类即可,例如293T细胞。所述培养的条件优选为在5%CO2、37℃下培养。
在本发明中,所述转染的方法优选为将所述过表达miR-146a-5p的重组慢病毒和细胞培养基加入到铺板的间充质干细胞中,培养48h传代,用嘌呤霉素筛选后经鉴定,得到过表达miR-146a-5p的间充质干细胞。所述鉴定方法优选包括蛋白免疫印迹或qPCR检测。所述qPCR检测的方法优选为提取细胞中RNA,经逆转录,得到cDNA,以所述cDNA为模板配制qPCR反应体系,经qPCR扩增反应,经过2-ΔΔCt方法计算mRNA和miRNA表达水平,确定间充质干细胞过表达miR-146a-5p。所述qPCR反应体系中扩增引物包括Hsa-U6和Hsa-miR-146a-5p引物;Hsa-U6优选包括核苷酸序列如SEQ ID NO:2所示的正向引物和核苷酸序列如SEQ IDNO:3所示的反向引物。所述Hsa-miR-146a-5p引物优选包括核苷酸序列如SEQ ID NO:4所示的正向引物和核苷酸序列如SEQ ID NO:5所示的反向引物。所述qPCR反应程序优选为95℃10min;95℃10s,65℃20s,72℃10s,共40个循环。
在本发明中,所述分离提取外泌体的方法优选为将所述培养液依次在2000g、10000g的离心力条件下离心收集上清,再在100000g的离心力条件下离心,弃去上清,重悬沉淀。在本发明中,所述离心的时间优选为25~35min,更优选为30min。所述离心的温度优选为4℃。所述重悬沉淀用溶液优选为PBS溶液。所述100000g的离心力条件下离心,弃去上清,重悬沉淀的操作步骤优选重复2次。
在本发明中,神经炎症相关的疾病优选包括神经退行性疾病。所述神经退行性疾病优选包括阿尔茨海默病和/或视网膜色素变性。所述神经炎症优选包括脂多糖(LPS)诱导形成BV2细胞和RAW246.7细胞炎症损伤HT22细胞引起的。
在本发明中,所述治疗神经炎症优选是通过保护小胶质细胞和/或巨噬细胞炎症损伤海马神经元实现。所述药物优选具有下调炎症因子表达水平和/或抑制NF-κB炎症信号通路的药效。所述炎症因子优选包括IL-1β,TNF-α和IL-6。所述抑制NF-κB炎症信号通路优选为下调Nr4a3的表达。
在本发明实施例中,首先通过建立LPS诱导BV2细胞和RAW246.7细胞炎症模型,结果表明,RAW264.7和BV2细胞中IL-1β,TNF-α和IL-6mRNA表达水平上调,同时促进了NF-κB炎症信号通路的激活。为了模拟体内炎症环境下细胞之间的相互作用,建立了LPS诱导BV2细胞和RAW246.7细胞共培养HT22细胞的体系来模拟细胞炎症模型,结果表明HT22细胞活力下降,细胞凋亡信号通路激活。而当向共培养体系中给药过表达miR-146a-5p的间充质干细胞外泌体后,使炎症因子IL-1β,TNF-α和IL-6mRNA表达水平显著下调,炎症信号通路NF-κB信号通路明显抑制。miR-146a-5p-OE-MSCs-Exo与普通MSCs-Exo相比,其抗炎作用提升1-2倍。并且海马神经元细胞HT22的细胞活力显著提升,细胞凋亡信号通路明显受到抑制。为了进一步明确过表达miR-146a-5p的间充质干细胞外泌体的下游作用机制,检测了BV2小胶质细胞或者RAW246.7巨噬细胞中转录因子Nr4a3的表达,结果发现miR-146a-5p能够调控转录因子Nr4a3的表达,过表达miR-146a-5p能够明显下调Nr4a3的表达。通过在BV2小胶质细胞或者RAW246.7巨噬细胞中过表达Nr4a3,同样发现细胞中IL-1β,TNF-α和IL-6mRNA表达水平显著上调,炎症信号通路NF-κB信号通路激活,证实miR-146a-5p-OE-MSCs-Exo通过下游抑制Nr4a3来实现抗炎作用。
鉴于过表达miR-146a-5p的间充质干细胞外泌体具有良好的抗神经炎性的作用,本发明还提供了一种过表达miR-146a-5p的间充质干细胞外泌体药物,以过表达miR-146a-5p的间充质干细胞外泌体为活性成分,还包括药学上可接受的辅料。
本发明对所述药物的制备方法没有特殊限制,采用本领域所熟知的外泌体药物的制备方法即可。
下面结合实施例对本发明提供的一种过表达miR-146a-5p的间充质干细胞外泌体药物及应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
一种稳定过表达miR-146a-5p的间充质干细胞的构建方法
(1)过表达miR-146a-5p重组质粒的复制和提取
A.LB培养基的配制
先用ddH2O润洗所需的试剂瓶,高温灭菌后,配制培养基,如下表1所示液体培基定容至250ml,固体平板定容至200ml,再次进行高温灭菌。
表1培养基配方
B.过表达miR-146a-5p重组质粒转化
取50μl大肠杆菌感受态细胞置于冰浴中,待感受态细胞融化后,向感受态细胞悬液中加入500μg过表达miR-146a-5p重组质粒(购自于吉凯基因),用移液枪吹打混合2-3次,冰上放置30min。随后将离心管置于42℃水浴中,热击90秒,迅速将离心管置于冰上,放置2~3min。
然后在生物安全柜中向每个离心管中加入500μl无菌不含抗生素的LB培养基,37℃摇床,150rpm振荡培养60min,使质粒上相关抗性基因表达,菌体复苏。
在4000rpm下离心4min,留取200μl已转化的感受态细胞,在含有氨苄青霉素抗性的LB固体平板上,用无菌涂布棒将细胞均匀涂开,将平板置于37℃培养箱里直至液体完全吸收,倒置培养过夜。隔天挑取单克隆菌落在含有氨苄青霉素的LB培基中扩增。
C.摇菌
在配制好的LB培养基中加入氨苄青霉素,以上操作在生物安全柜中进行操作,避免杂菌污染。取15ml离心管加入5~8ml LB培养基,加入100μl甘油菌,37℃摇菌6~8h左右,随后将15ml离心管中菌液转移至含有100ml LB(加入氨苄青霉素)锥形瓶中,37℃摇菌12h左右。
D.菌液保存
提前准备50%的甘油高压分装,吸取400μl扩增好的菌液加入600μl的50%甘油中进行菌液保存,置于-80℃。
E.质粒抽提
根据天根大提试剂盒按照说明书进行操作提取重组质粒。利用紫外分光光度计对提取的重组质粒浓度进行测定。
(2)慢病毒质粒的包被
第一天将汇合度80%~90%左右的293T细胞,消化传代,接种于6cm培养皿中,密度为60%~70%。第二天选取三质粒***,PSPAX2、PMD2G(购自于汉恒生物)和目的质粒,按照汉恒生物包装病毒说明书进行操作。按照说明书参考比例T75中,PXPAX2:PMD2G:目的质粒=10μg:5μg:10μg,在6cm培养皿中等比缩减。将质粒质量与转染试剂(lipofectamine3000)体积按照1μg:2μl混合,在转染293T细胞前,去除细胞培养基,PBS清洗2~3次,去除血清干扰,随后加入10ml含有10%灭活血清培养基,将转染复合物均匀滴加至细胞板在5%CO2、37℃培养24h,在荧光显微镜下观察荧光,第三天加入1ml新鲜培养基在5%CO2、37℃培养24h,准备收获慢病毒颗粒。
(3)获取慢病毒感染
将制备的慢病毒病毒上清收集,2000g离心10min获取含有慢病毒颗粒的上清,随后利用工具293T细胞进行慢病毒滴度检测,筛选合适慢病毒上清体积。
(4)慢病毒感染目的细胞
在慢病毒感染前24h,在3cm的培养皿中铺板间充质干细胞或RAW264.7细胞或BV2细胞,每孔密度为40%~50%,加入2ml培养基,加入含有重组慢病毒颗粒的上清,48h后传代,加入终浓度为2μg/mL的嘌呤霉素进行筛选,得到筛选的细胞。将收集的细胞通过qPCR检测目的基因的表达,具体方法如下:
A.提取RNA以及逆转录
将200μl外泌体加入500μlTrizol裂解液,冰上静置5-10min,加入100μl氯仿冰上静置5min,离心机4℃预冷,13,000g离心15min,将离心后的上清尽可能多的移到新的EP管中,加入等体积预冷异丙醇溶液,冰上静置30min,13,000g离心10min,弃掉上清加入500μl75%酒精冰上静置5min,最后7,500g离心5min,弃掉上清,室温干燥,加入适量DEPC水,测定RNA浓度。
B.根据All-in-OneTMmiRNAFirst-Strand cDNA Synthesis Kit 2.0说明书进行配制,所有过程均在冰上进行,最后加入RNA。
表2逆转录反应体系
动作轻柔地混匀已准备好的反应混合物,短暂离心后置于37℃孵育60min,接着置于85℃温育5min,4℃保存。
C.实时荧光定量PCR扩增,按照表3所示配制反应体系,miRNA反应体系配置每个样本目的基因和内参均设3个复孔。
表4实时荧光定量PCR扩增反应体系
反应程序为下表5所示。
表5反应程序
采用BIO-RAD荧光定量PCR进行实验,结果采用相对定量使用2-ΔΔCt方法计算mRNA和miRNA表达水平,并以内参对数据进行标准化处理。
(5)检测结果
a)基因信息
基因名称:Hsa-miR-146a
物种:Human载体信息;
克隆位点:AgeI/EcoRI。
b)阳性重组克隆测序结果及结果分析
GATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGTACAGGGCTGGGACAGGCCTGGACTGCAAGGAGGGGTCTTTGCACCATCTCTGAAAAGCCGATGTGTATCCTCAGCTTTGAGAACTGAATTCCATGGGTTGTGTCAGTGTCAGACCTCTGAAATTCAGTTCTTCAGCTGGGATATCTCTGTCATCGTGGGCTTGAGGACCTGGAGAGAGTAGATCCTGAAGAACTTTTTCAGTCTGCTGAAGAGCTTGGAAGACTGGATTTTTTGAA TTCTCGACCTCGAGACAAATGGCAGTATTCATCCACGGATCCTAACCCGTGTCGGCTCCAACATAACTTACGGTAAATGGCCCGCCTG(SEQ ID NO:6)。其中下划线部分序列表示酶切位点,加粗部分序列为miR-146a-5p。
结果见图2中A和B。构建了过表达miR-146a-5p的重组质粒并在脐带间充质干细胞中稳定过表达。
实施例2
一种过表达miR-146a-5p的间充质干细胞外泌体(miR-146a-5p-MSCs-Exo)的分离提取方法
将实施例1制备的稳定过表达miR-146a-5p的间充质干细胞在10%不含有外泌体的血清配制的完全培养基中,培养24~48h。用梯度离心法获得外泌体,首先将培养上清在2,000g、4℃条件下离心30min保留上清,随后在10,000g、4℃离心30min留取上清,最后在100,000g、4℃条件下离心90min弃掉上清,PBS重悬后100,000g、4℃离心90min,最后200μlPBS收集外泌体,得到过表达miR-146a-5p的间充质干细胞外泌体。
实施例3
对过表达miR-146a-5p的间充质干细胞外泌体(miR-146a-5p-OE-MSCs-Exo)的检测方法
采用实施例1中记载的实时荧光定量PCR检测实施例2中提取的外泌体中目标基因的表达情况。结果见图2中C和D。结果表明本发明提取的miR-146a-5p-MSCs-Exo中过表达miR-146a-5p。
对比例1
一种人源脐带间充质干细胞外泌体(MSCs-Exo)的分离提取方法
首先对血清进行超高速120,000g 4℃离心18小时获取无外泌体血清。在间充质干细胞密度长到80%~90%左右,弃掉上清,加入PBS清洗三次,加入10%不含有外泌体的血清配制的完全培养基,培养24~48h。选用梯度离心法获得外泌体,首先将培养上清在2,000g 4℃条件下离心30min保留上清,随后在10,000g4℃离心30min留取上清,最后在100,000g 4℃条件下离心90min弃掉上清,PBS重悬后100,000g 4℃离心90min,最后200μl PBS收集外泌体。
实施例4
LPS诱导BV2细胞和RAW246.7细胞炎症损伤HT22细胞模型的建立
(1)通过查阅相关文献发现LPS诱导RAW264.7和BV2细胞浓度不同,随后设置0~10ug/ml不同浓度梯度的LPS刺激RAW264.7和BV2细胞构建炎症模型,然后利用CCK8检测LPS对RAW264.7和BV2细胞两种细胞活力的影响。
(2)为了确定是否LPS刺激会引起细胞炎症因子升高以及对LPS刺激细胞浓度的选择,我们检测了不同浓度下LPS诱导作用下,RAW264.7和BV2细胞中IL-1β,TNF-α和IL-6mRNA表达水平的变化,其中炎症因子及其引物序列见表6。
表6炎症因子扩增用引物
综合以上表型及炎症因子变化情况,最终选取合适浓度的LPS刺激细胞建立炎症模型。然后利用WB实验检测RAW264.7和BV2细胞中NF-κB信号通路NF-κB、IKBα和P-IKBα的变化。WB具体实验方法如下:具体方法如下:
A.提取总蛋白
PBS清洗目的细胞,利用细胞刮将目的细胞收集在1.5mL离心管中,每个样品中加入适量RIPA裂解液以及相应蛋白酶抑制剂和磷酸酶抑制剂,放在冰上裂解30min左右,之后用超声细胞破碎仪进行超声2次,每次5s间隔10s,将样品移入至预冷4℃离心机,12000g离心10min后取上层澄清液体,标记样本名称以及时间。
B.蛋白浓度测定
根据BCA试剂盒说明书(货号:PC0020)进行实验操作,将样品进行统一定量,2按照总体积计算加入5×loading buffer,在95-100℃条件下变性5-10min,随后-40℃保存。
C.蛋白电泳
将预制胶(货号:F11010LGel)固定在电泳槽上,拔出梳子,确定蛋白上样量为25~50μg,在上样孔中加入蛋白条带指示剂和样品,电泳时间30min,160恒压电泳,根据蛋白分子量的位置,适当终止电泳。
D.转膜
PVDF膜裁剪适当大小,用甲醇浸泡5-10min激活,利用“三明治夹心法”放置在转印夹中,随后移入到转膜槽,向转膜槽中添加预冷转膜液,300mA恒流条件下转膜90min。
E.封闭及孵育抗体
用TBST配制5%的脱脂奶粉,封闭PVDF膜1-2h。随后TBST清洗三次,每次5min。随后,根据蛋白位置裁剪PVDF,在加入一抗的孵育盒中4℃过夜。抗体明细见表7。第二天,用TBST缓冲液洗膜3次,每次5min,加入相应的羊抗兔二抗(1∶2000)和羊抗小鼠二抗(1∶2000),室温摇床孵育1-2h,配制超敏发光液,在BIO-RAD显影仪进行显影。
表7抗体明细表
(3)为了模拟体内的炎症环境下细胞之间相互作用,构建了一种共培养体系。将RAW264.7和BV2细胞分别放在0.4μm的transwell上室,将HT22接种在下室。在LPS作用RAW264.7和BV2细胞24小时后检测共培养体系中HT22细胞的变化。用CCK8法检测共培养后HT22细胞的活力、HT22细胞的凋亡情况、HT22细胞凋亡信号的改变。
结果见图3和图4。结果表明,脂多糖(LPS)诱导BV2小胶质细胞或者RAW246.7巨噬细胞炎症与海马神经元TH22的共培养体系中炎症因子IL-1β,TNF-α和IL-6mRNA表达水平显著升高,炎症信号通路NF-κB信号通路明显激活。
实施例5
不同外泌体的抗炎效果的检测
(1)使用PBS、普通MSCs-Exo、miR-146a-5p-OE-MSCs-Exo作用到LPS诱导RAW264.7和BV2细胞炎症与HT22共培养的体系中,分别观察PBS、普通MSCs-Exo、miR-146a-OE-MSCs-Exo作用后RAW264.7和BV2细胞中炎症因子IL-1β,TNF-α和IL-6mRNA表达水平的变化,其中炎症因子引物序列见表6。
(2)使用PBS、普通MSCs-Exo、miR-146a-OE-MSCs-Exo作用到LPS诱导RAW264.7和BV2细胞炎症与HT22共培养的体系中的体系中,分别检测PBS、普通MSCs-Exo、miR-146a-OE-MSCs-Exo作用后RAW264.7和BV2细胞中炎症信号通路NF-κB信号通路的变化。
(3)HT22细胞CCK8细胞活力检测,将细胞铺板于96孔板中,每孔约1×104个细胞,在LPS和加入外泌体24h后,加入CCK8试剂(MCE)按照试剂说明书进行操作,每200μl溶液加入10μl试剂,37℃避光孵育1-2h。在450nm处测定吸光度,根据公式I得到细胞存活百分率。通过此实验观察PBS、普通MSCs-Exo、miR-146a-OE-MSCs-Exo作用后HT22细胞活力的变化。
细胞存活百分率(%)=(实验组-空白组)/(对照组-空白组)_×100%公式I
(4)使用PBS、普通MSCs-Exo、miR-146a-OE-MSCs-Exo作用到LPS诱导RAW264.7和BV2细胞炎症与HT22共培养的体系中的体系中,利用荧光显微镜和WB分别检测PBS、普通MSCs-Exo、miR-146a-OE-MSCs-Exo作用后HT22细胞的凋亡率和凋亡信号通路的变化。
结果见图5。使用了miR-146a-5p-OE-MSCs-Exo作用到共培养体系后炎症因子IL-1β,TNF-α和IL-6mRNA表达水平显著下调,炎症信号通路NF-κB信号通路明显抑制。miR-146a-5p-OE-MSCs-Exo与普通MSCs-Exo相比,其抗炎作用提升1-2倍。使用了miR-146a-5p-OE-MSCs-Exo作用到共培养体系后海马神经元细胞HT22的细胞活力显著提升,细胞凋亡信号通路明显受到抑制。
实施例6
miR-146a-5p-OE-MSCs-Exo下游的作用机制
(1)对RAW264.7和BV2细胞分别瞬时转染mimic miR-146a-5p和inhibitormiR-146a-5p后观察RAW264.7和BV2细胞中Nr4a3和miR-146a-5p的表达变化。
(2)使用过表达Nr4a3慢病毒感染RAW264.7和BV2细胞,通过荧光显微镜观察细胞感染效率,利用WB和qPCR检测Nr4a3的表达情况以及过表达Nr4a3后炎症因子IL-1β,TNF-α和IL-6mRNA表达水平变化,其中炎症因子引物序列见表6,Nr4a3引物序列见表8。
表8相关引物序列
结果见图5。结果表明,在BV2小胶质细胞或者RAW246.7巨噬细胞中发现miR-146a-5p能够调控转录因子Nr4a3的表达,过表达miR-146a能够明显下调Nr4a3的表达。随后过表达Nr4a3,同样发现细胞中IL-1β,TNF-α和IL-6mRNA表达水平显著上调,炎症信号通路NF-κB信号通路激活,证实miR-146a-5p-OE-MSCs-Exo通过下游抑制Nr4a3来实现抗炎作用(如图6所示)。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种过表达miR-146a-5p的间充质干细胞外泌体在制备治疗神经炎症的药物中的应用。
2.根据权利要求1所述应用,其特征在于,所述miR-146a-5p的核苷酸序列如SEQ IDNO:1所示。
3.根据权利要求1所述应用,其特征在于,所述过表达miR-146a-5p的间充质干细胞外泌体的制备方法,包括以下步骤:
制备过表达miR-146a-5p的重组慢病毒;
将所述过表达miR-146a-5p的重组慢病毒转染间充质干细胞,得到过表达miR-146a-5p的间充质干细胞;
从所述过表达miR-146a-5p的间充质干细胞的培养液中分离提取外泌体,得到过表达miR-146a-5p的间充质干细胞外泌体。
4.根据权利要求3所述应用,其特征在于,所述制备过表达miR-146a-5p的重组慢病毒的方法,包括将过表达miR-146a-5p的重组人源载体和PXPAX2、PMD2G在转染试剂的作用下共同转染哺乳细胞,培养,收集过表达miR-146a-5p的重组慢病毒颗粒。
5.根据权利要求4所述应用,其特征在于,所述分离提取外泌体的方法为将所述培养液依次在2000g、10000g的离心力条件下离心收集上清,再在100000g的离心力条件下离心,弃去上清,重悬沉淀。
6.根据权利要求1所述应用,其特征在于,神经炎症相关的疾病包括神经退行性疾病;
所述神经退行性疾病包括阿尔茨海默病和/或视网膜色素变性。
7.根据权利要求1所述应用,其特征在于,所述治疗神经炎症是通过保护小胶质细胞和/或巨噬细胞炎症损伤海马神经元实现。
8.根据权利要求7所述应用,其特征在于,所述神经炎症包括脂多糖诱导形成。
9.根据权利要求1~8中任意一项所述应用,其特征在于,所述药物具有下调炎症因子表达水平和/或抑制NF-κB炎症信号通路的药效。
10.一种过表达miR-146a-5p的间充质干细胞外泌体药物,其特征在于,以过表达miR-146a-5p的间充质干细胞外泌体为活性成分,还包括药学上可接受的辅料。
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