CN116769777A - 一种促进脊髓损伤后轴突再生的工程化外泌体、制备方法与应用 - Google Patents
一种促进脊髓损伤后轴突再生的工程化外泌体、制备方法与应用 Download PDFInfo
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Abstract
本发明提供一种促进脊髓损伤后轴突再生的工程化外泌体、制备方法与应用,涉及生物医学技术领域,所述工程化外泌体为人经血干细胞源性外泌体miR‑421,所述miR‑421的前提核苷酸序列如Seq ID No.1所示,其成熟编码序列如Seq ID No.2所示。本申请中基于全基因转录组测序技术,筛选出差异表达的miRNAs,通过生信和在体实验锁定关键分子miR‑421。发现miR‑421能够促进脊髓损伤后大鼠运动感觉功能的恢复,促进轴突再生,降低胶质瘢痕。本发明通过电穿孔技术制备miR‑421过表达工程化外泌体,制备方法效率高,技术可推广程度高且能迅速制备大量不同类型外源物质的外泌体,具有极高的应用价值。本发明为脊髓损伤治疗提供了新方案,为临床转化提供支撑。
Description
技术领域
本发明涉及生物医学技术领域,尤其涉及一种促进脊髓损伤后轴突再生的工程化外泌体、制备方法与应用。
背景技术
全世界每年因车祸、暴力以及跌落等原因导致的脊髓损伤(Spinal cord injury,SCI)患者约25-50万人。这一严重的致残性疾病会导致损伤平面以下感觉和运动功能障碍,并因此产生神经病理性疼痛、痉挛、褥疮以及膀胱和肠道功能障碍等并发症,患者的生活质量严重下降。脊髓损伤过程复杂,早期以轴突、少突胶质细胞和神经元凋亡和缺失为主;继而破坏血脑屏障,诱发大量炎性细胞浸润;后期在损伤部位产生空洞、胶质瘢痕、髓鞘脱落而诱导远端神经元萎缩和凋亡,并且阻断轴突的再生,限制功能恢复。手术治疗、药物治疗、康复治疗等是目前临床上治疗脊髓损伤的主要方案,但皆不能有效逆转受损脊髓的病理程度,影响功能恢复。因此,积极探索SCI治疗新方案具有重要的理论意义与临床转化价值。
经血干细胞(Menstrual blood-derived mesenchymal stem cells,MenSCs)在2007年被首次从女性月经期间脱落的子宫内膜中分离出来,因其来源丰富,采集方式无创、安全、方便,具有较强的多向分化潜能,增殖活性高且遗传稳定性强,自体移植伦理风险低,定向分化为神经元能力强,免疫调节和神经营养因子分泌能力强,逐步成为治疗中枢神经***疾病的“潜力股”。与骨髓干细胞、脐带干细胞、羊膜干细胞外泌体相比,MenSCs外泌体孵育后皮层神经元长度最长,对背根神经元生长刺激效果与骨髓干细胞相近,优于其他干细胞。
外泌体是具有脂质双层膜的细胞外颗粒,直径约30~150nm,可以直接将特定效应物,如蛋白质、mRNA、miRNA和DNA等从分泌细胞传递到受体细胞。与特化的终末分化细胞相比,间充质干细胞的外泌体释放量更高。然而,与干细胞相比,外泌体获取存储便利,伦理限制低,能够穿透血-脊髓屏障,具有更低的免疫原性和非整倍性风险,临床应用更为便利。已有报道表明间充质干细胞分泌的外泌体广泛应用于脊髓损伤后治疗,降低神经细胞凋亡和组织损伤,抑制炎症反应,减少瘢痕面积,促进轴突再生和神经环路重建,改善肢体功能。
外泌体因其良好的生物组织相容性、稳定性、低免疫原性和工程化便利性逐渐成为损伤修复“非细胞治疗(Cell-free therapy)”首选方案。但目前尚无有关经血干细胞源性外泌体用于脊髓损伤治疗领域的相关报道。
发明内容
本发明的目的是为了解决现有技术中针对经血干细胞源型外泌体用于脊髓损伤治疗技术领域并无相关研究的技术问题。
为了实现上述目的,本发明采用了如下技术方案:
本申请提供了一种人经血干细胞源性外泌体miR-421,其特征在于:所述miR-421的前提核苷酸序列如Seq ID No.1所示,其成熟编码序列如Seq ID No.2所示。
本申请还提供了外泌体miR-421在制备用于治疗脊髓损伤轴突再生和功能恢复过程中药物中的应用。
优选的,所述miR-421能够促进脊髓损伤后运动感觉功能的恢复,促进轴突再生,降低胶质瘢痕。
本申请还提供了一种miR-421过表达工程化外泌体的制备方法,所述制备方法为电穿孔技术。
优选的,所述制备方法包含以下步骤:
S1:采用同源重组发构建表达Seq ID No.1的慢病毒颗粒,分装冻存-80℃备用;
S2:于12孔板中按照1*105个细胞/ml接种细胞,待其汇合率达到70-80%时,按照细胞数量:病毒颗粒数量等于0.5、1.0、1.5加入慢病毒,然后混匀;
S3:24h后换液,72h观察感染效率,若感染效率高于40%则按照终浓度1μg/mL剂量加入嘌呤霉素,每2天更换一次,持续至少14天,感染效率达到100%。
S4:冻存细胞并基座P0代,其余细胞继续扩增培养;
S5:用15cm培养皿接种P3-P10代MenSCs,常规培养至汇合率约70%,去除旧培养液,无外泌体PBS洗涤2次,添加DMEM F12于37℃,5%的CO2条件下培养48h,收集上清,冻存于-80℃备用;
S6:上清经过梯度差速离心处理获得外泌体。
优选的,所述S5中DMEM F12含3%胎牛血清,不含有外泌体。
优选的,所述S6的具体步骤为:
首先S5中获得的上清经过4℃,2000rpm离心25min,弃去沉淀,保留上清;随后在4℃,10000rpm离心40min,弃去沉淀,保留上清;接着在4℃,1×105rpm离心150min,弃去上清,保留沉淀,所述沉淀即为外泌体,将外泌体用适量PBS(pH=7.5)重悬并保存在-80℃备用。
本申请中基于全基因转录组测序技术,筛选出差异表达的miRNAs,通过生信和在体实验锁定关键分子miR-421。发现miR-421能够促进脊髓损伤后大鼠运动感觉功能的恢复,促进轴突再生,降低胶质瘢痕。本发明通过电穿孔技术制备miR-421过表达工程化外泌体,制备方法效率高,技术可推广程度高且能迅速制备大量不同类型外源物质的外泌体,具有极高的应用价值。本发明为脊髓损伤治疗提供了新方案,为临床转化提供支撑。
附图说明
图1是实施例1的外泌体粒径分析结果;
图2是实施例1的外泌体颗粒透射电镜结果(Scale bar=100nm);
图3是实施例1中外泌体颗粒标记物的表达;
图4是实施例2中外泌体颗粒在神经元细胞中的内吞情况;
图5是实施例2中HT22神经元细胞中miR-421的表达;
图6是实施例2中原代脊髓神经元细胞中miR-421表达;
图7是实施例2中外泌体调控原代脊髓神经元存活;
图8是实施例3中外泌体颗粒在SCI大鼠脊髓内分布情况;
图9是实施例3中SCI大鼠损伤局部脊髓组织中miR-421的表达;
图10是实施例3中BBB评分体系评估SCI大鼠后肢运动功能;
图11是实施例3中H&E、Nissl、Masson和PAS染色;
图12是实施例3中免疫荧光检测SCI大鼠损伤脊髓处NF200的表达。
具体实施方式
以下结合具体实施例,对本发明作进一步地详细说明。
本申请提供了一种人经血干细胞源性外泌体miR-421,所述miR-421的前提核苷酸序列如Seq ID No.1所示,其成熟编码序列如Seq ID No.2所示,
本申请还提供了外泌体miR-421在制备用于治疗脊髓损伤轴突再生和功能恢复过程中的药物中的应用。
所述miR-421能够促进脊髓损伤后运动感觉功能的恢复,促进轴突再生,降低胶质瘢痕。
本申请还提供了一种miR-421过表达工程化外泌体的制备方法,所述制备方法使用电穿孔技术制备。
在一实施方式中,所述miR-421制备方法如下所示:
S1:采用同源重组发构建表达Seq ID No.1的慢病毒颗粒,分装冻存-80℃备用;
S2:于12孔板中按照1*105个细胞/ml接种细胞,待其汇合率达到70-80%时,按照细胞数量:病毒颗粒数量等于0.5、1.0、1.5加入慢病毒,然后混匀;
S3:24h后换液,72h观察感染效率,若感染效率高于40%则按照终浓度1μg/mL剂量加入嘌呤霉素,每2天更换一次,持续至少14天,感染效率达到100%。
S4:冻存细胞并基座P0代,其余细胞继续扩增培养;
S5:用15cm培养皿接种P3-P10代MenSCs,常规培养至汇合率约70%,去除旧培养液,无外泌体PBS洗涤2次,添加DMEM F12于37℃,5%的CO2条件下培养48h,收集上清,冻存于-80℃备用。
在一实施方式中,所述DMEM F12含3%胎牛血清,不含有外泌体。
S6:上清经过梯度差速离心处理获得外泌体。
具体的,在一实施方中,首先经过4℃,2000rpm离心25min,弃去沉淀,保留上清;随后在4℃,10000rpm离心40min,弃去沉淀,保留上清;接着在4℃,1×105rpm离心150min,弃去上清,保留沉淀(外泌体),用适量PBS(pH=7.5)重悬并保存在-80℃备用。
以下结合具体实施例对本申请进行阐述:
实施例1:工程化外泌体miR-421的构建、提取及鉴定:
采用同源重组法构建表达Seq ID No.1的慢病毒颗粒(镇江维根生物技术有限公司),分装冻存-80℃备用。于12孔板中按照1×105个细胞/mL接种细胞,待其汇合率达到70~80%时,按照细胞数量:病毒颗粒数量等于0.5、1.0和1.5加入慢病毒,混匀。24h后换液,72h观察感染效率,若感染率高于40%则按照终浓度1μg/mL剂量加入嘌呤霉素,每2天更换一次,持续至少14天,感染效率达到100%。冻存细胞并记作P0代,其余细胞继续扩增培养。
用15cm培养皿接种P3-P10代MenSCs,常规培养至汇合率约70%,去除旧培养液,无外泌体PBS洗涤2次,添加DMEM F12(含3%胎牛血清,不含有外泌体)于37℃,5%的CO2条件下培养48h,收集上清,冻存于-80℃备用。
上清经过梯度差速离心处理获得外泌体,具体流程如下:首先经过4℃,2000rpm离心25min,弃去沉淀,保留上清;随后在4℃,10000rpm离心40min,弃去沉淀,保留上清;接着在4℃,1×105rpm离心150min,弃去上清,保留沉淀(外泌体),用适量PBS(pH=7.5)重悬并保存在-80℃备用。用粒径仪(Zetaview)检测所提取外泌体的颗粒直径分布,用BCA试剂盒(碧云天)测定蛋白浓度。
充分混匀后取10μL外泌体溶液滴到铜网上,室温静置10分钟。用纸巾从远端掖干多余溶液,2%的醋酸双氧铀染色1分钟,PBS洗涤1次,重复染色3次,PBS洗涤3遍。室温晾干铜网后置于透射电镜(HT7700)观察外泌体的大小、形貌。
如图1纳米颗粒追踪分析结果表明,外泌体粒径主要分布在100-150nm之间,定量分析结果表明外泌体的浓度为5×1011个/mL。
图2电镜获得外泌体形状呈双凹圆盘状,粒径大小平均处于100nm附近,符合国际通行外泌体标准。
图3蛋白免疫印迹结果显示外泌体表达阳性标记物CD63、CD9,而不表达阴性标记物Calnexin,表明工程化外泌体制备成功。
实施例2:验证工程化外泌体在细胞水平上的神经元保护作用
HT22细胞(购买自中科院细胞库)采用完全培养基DMEM(PM150210)+10%FBS+1%P/S培养于37℃,5% CO2的培养箱中,待其汇合率达到90%时用PBS洗涤3次,0.25%胰酶消化,完全培养基重悬后进行细胞计数。
12孔板铺设细胞爬片后接种1000个细胞/孔,6h后加入105个外泌体/孔,孵育24h后按照常规操作流程行Tuj1、GFP和DAPI染色,封片后镜检。使用常规TRIzol法提取HT22细胞总RNA,qRT-PCR法检测miR-421的表达,结果表明其含量在转染后HT22细胞中极显著高于对照组细胞。常规方法分离大鼠脊髓神经元进行培养,采用终浓度为0.4mM的H2O2孵育构建脊髓神经元损伤模型,48h后收集细胞提取RNA,qRT-PCR法检测miR-421含量,结果表明外泌体孵育组脊髓神经元细胞中miR-421表达量极显著高于对照组。96孔板上加入100μL完全培养基中含有1×104个脊髓神经元细胞,同等培养条件下培养12h,加入终浓度为0.4mM的H2O2,同时每孔加入1×105个外泌体继续培养,空白组加入等量溶剂,对照组加入等量H2O2和溶剂。于12h、24h、48h、72h行CCK-8检测。每个孔中加入10μL CCK-8溶液,培养箱中继续培养4h,随后测定OD450 nm吸光值,按照以下公式计算存活率:细胞存活率=[(实验孔-空白孔)/(对照孔-空白孔)]×100%。结果如图7所示,相对于对照组,外泌体显著促进脊髓神经元细胞的存活。
如图4免疫荧光结果表明携带GFP的工程化外泌体能够被HT22细胞所高效摄取,并在细胞质和细胞核中均有分布。提示工程化外泌体结构完整,能够完整被HT22细胞内吞,且效率较高。
如图5实时荧光定量PCR结果显示miR-421的含量在转然后HT22细胞中显著升高,表明HT22细胞有效内吞了工程化外泌体,并纳入了工程化外泌体所携带的miR-421。
如图6中qRT-PCR结果显示工程化外泌体孵育极显著提升了脊髓神经元中miR-421的表达。
如图7中CCK-8实验结果显示工程化外泌体有效增强了脊髓神经元抵抗H2O2损伤的能力,进而提升了其存活率。
实施例3:验证工程化外泌体在体内水平上的促进SCI后运动功能恢复作用
常规方法构建第10胸椎(T10)脊髓半切损伤模型,术后2h、24h、48h、72h按照200μg/只剂量尾静脉注射外泌体。注射后24h取3只SCI大鼠,麻醉后生理盐水灌注,4%的PFA灌流,随后暴露损伤处脊髓,以切口为中心前后取5mm脊髓组织,OCT包埋后制作20μm切片,行GFP、Synaptophysin和DAPI染色,观察损伤局部外泌体的分布,如图8免疫荧光结果表明外泌体在损伤处大量聚集,形成归巢效应。
注射后48h取对照组、SCI组和外泌体组各3只大鼠,CO2窒息法安乐死后暴露脊髓,同上方法取5mm脊髓组织,置于液氮中冷冻,随后TRIzol法提取总RNA,加A法逆转录(诺唯赞,MR201)获得cDNA,
实时荧光定量PCR检测miR-421表达,程序如下:95℃30Sec;95℃5Sec,60℃34Sec,循环40次;
随后95℃15Sec,60℃1min,95℃15Sec结束反应。采用2-ΔΔCt方法计算相对表达量。
内参采用U6,前引物为TGACACGCAAATTCGTGAAGCGTTC,
后引物为CCAGTCTCAGGGTCCGAGGTATTC;
miR-421前引物为CGGCGGGGCCUCAUUAAAUGUUU,
后引物为通用引物CAGTGCAGGGTCCGAGGTAT。
如图9实时荧光定量PCR结果表明外泌体处理组大鼠脊髓中miR-421的含量显著高于对照组大鼠脊髓组织。结果表明外泌体注射后SCI大鼠损伤处脊髓组织中miR-421含量显著高于对照组。
BBB评分体系(Basso,Beattie,and Bresnahan,BBB)被纳入本研究中用以评估SCI大鼠后肢运动功能恢复情况。两位充分培训后的实验人员各自在双盲的情况下对照BBB评分表对大鼠运动功能进行打分。如图10中BBB评分体系结果表明外泌体注射后显著提高了SCI大鼠后肢运动功能。
脊髓组织用OCT包埋后作20μm切片用于H&E染色。随后切片用苏木素染色2min40sec,双蒸水洗涤2次,1%的HCl分化30sec,双蒸水洗涤2次后伊红染色1min。如H&E,采用Masson试剂盒(索莱宝)、Nissl染色试剂盒(索莱宝)、PAS染色试剂盒(索莱宝)按照说明书进行染色操作。封片后用Olympus DP71采集图像供后续分析。如图11中H&E染色、Masson染色、Nissl染色和PAS染色表明外泌体显著改善了SCI大鼠损伤脊髓局部微环境,即外泌体有效改善了SCI大鼠脊髓损伤局部病理变化,提高了神经元的存活,降低了损伤局部纤维化程度和免疫反应。采用常规免疫荧光法共染NF200和DAPI,如图12免疫荧光结果表明外泌体能够有效促进神经元的存活。
综上所述,本申请中基于全基因转录组测序技术,筛选出差异表达的miRNAs,通过生信和在体实验锁定关键分子miR-421。发现miR-421能够促进脊髓损伤后大鼠运动感觉功能的恢复,促进轴突再生,降低胶质瘢痕。本发明通过电穿孔技术制备miR-421过表达工程化外泌体,制备方法效率高,技术可推广程度高且能迅速制备大量不同类型外源物质的外泌体,具有极高的应用价值。本发明为脊髓损伤治疗提供了新方案,为临床转化提供支撑。
Claims (7)
1.一种人经血干细胞源性外泌体miR-421,其特征在于:所述miR-421的前提核苷酸序列如Seq ID No.1所示,其成熟编码序列如Seq ID No.2所示。
2.外泌体miR-421在制备用于治疗脊髓损伤轴突再生和功能恢复过程中药物中的应用。
3.根据权利要求2所述的应用,其特征在于:所述miR-421能够促进脊髓损伤后运动感觉功能的恢复,促进轴突再生,降低胶质瘢痕。
4.一种miR-421过表达工程化外泌体的制备方法,其特征在于:所述制备方法为电穿孔技术。
5.根据权利要求4所述的miR-421过表达工程化外泌体的制备方法,其特征在于:所述制备方法包含以下步骤:
S1:采用同源重组发构建表达Seq ID No.1的慢病毒颗粒,分装冻存-80℃备用;
S2:于12孔板中按照1*105个细胞/ml接种细胞,待其汇合率达到70-80%时,按照细胞数量:病毒颗粒数量等于0.5、1.0、1.5加入慢病毒,然后混匀;
S3:24h后换液,72h观察感染效率,若感染效率高于40%则按照终浓度1μg/mL剂量加入嘌呤霉素,每2天更换一次,持续至少14天,感染效率达到100%;
S4:冻存细胞并基座P0代,其余细胞继续扩增培养;
S5:用15cm培养皿接种P3-P10代MenSCs,常规培养至汇合率约70%,去除旧培养液,无外泌体PBS洗涤2次,添加DMEM F12于37℃,5%的CO2条件下培养48h,收集上清,冻存于-80℃备用;
S6:上清经过梯度差速离心处理获得外泌体。
6.根据权利要求5所述的miR-421过表达工程化外泌体的制备方法,其特征在于:所述S5中DMEM F12含3%胎牛血清,不含有外泌体。
7.根据权利要求5所述的miR-421过表达工程化外泌体的制备方法,其特征在于:所述S6的具体步骤为:
首先S5中获得的上清经过4℃,2000rpm离心25min,弃去沉淀,保留上清;随后在4℃,10000rpm离心40min,弃去沉淀,保留上清;接着在4℃,1×105rpm离心150min,弃去上清,保留沉淀,所述沉淀即为外泌体,将外泌体用适量PBS(pH=7.5)重悬并保存在-80℃备用。
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