CN105395571A - 间充质干细胞分泌的外泌小体治疗心肌梗死的新用途 - Google Patents
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Abstract
本发明公开了一种间充质干细胞分泌的外泌小体治疗心肌梗死的新用途,建立分离足够量外泌小体的方法及全面分析外泌小体的体系,规范外泌小体治疗心肌梗死的疗程、疗效及评估体系。利用干细胞分泌的外泌小体治疗心脏病的创新方案,不但解决了干细胞治疗带来的致癌及免疫排斥的危害,而且与干细胞治疗相比具有便于保存、定量以及移植简单的优点。外泌小体的治疗方案将为社会提供治疗心血管疾病的新途径,并节省大量基金。
Description
技术领域
本发明涉及间充质干细胞分泌的外泌小体治疗心肌梗死的新用途,属于医药技术领域。
背景技术
心肌梗死是一种严重危害人类健康的心血管疾病,随着我国人民生活水平的不断提高,生活节奏的不断加快,缺血性心肌梗死的发病率也在不断上升。缺血性心肌梗死会导致心肌细胞坏死和瘢痕形成,进而影响心脏的功能。虽然目前通过冠状动脉介入术和冠状动脉搭桥术等方法,能够改善患者症状,但却不能替换已坏死的心肌细胞。心脏移植虽能彻底改善心脏状态,但因供体来源稀缺、免疫排斥以及昂贵的治疗费用等因素,在临床上很难广泛应用。
移植干细胞治疗心肌梗死,因其各方面的优势有望成为治疗心肌梗死的一个治疗手段。干细胞是一种具有多分化潜能和自我复制功能的早期未分化细胞,在特定条件下它可以分化成不同的功能细胞,形成多种组织和器官。特别是,骨髓间充质干细胞(BMSCs),它存在骨髓基质系统中,对造血系统起支持诱导作用,具有高度增殖和自我更新能力,在一定条件下也可以诱导分化成特异的组织细胞,修复受损的组织或器官。
可研究表明,基于干细胞的心血管疾病治疗方案虽能够在一定程度上缓解病情,但治疗效果并不理想。这主要是由于人们对干细胞的作用机制仍缺乏认识。另外干细胞移植有很多副作用包括导致心律失常,并具有致癌性及免疫排斥。以往认为干细胞的治疗效果依赖于其分化潜能,但最新研究成果表明移植的干细胞主要是通过旁分泌作用而发挥其治疗作用,比如干细胞分泌物能刺激心肌祖细胞的细胞周期再进入(cellcyclereentry)。最近,本申请提出了无细胞的干细胞治疗(stemcelltherapywithoutstemcells),也就是移植的不是干细胞,而是利用干细胞分泌的外泌小体(exosome)治疗心肌梗死的设想。本发明就是用间充质干细胞分泌的exosome治疗心肌梗死的方案。
外泌小体是细胞内来源的一群直径在30–100nm之间的纳米级颗粒,通过细胞内的多泡体(MVB)与细胞膜融合而释放到细胞外。通常B细胞、T细胞、树突细胞、内皮细胞、成纤维细胞、间充质干细胞和肿瘤细胞等多种不同细胞类型的细胞都可以分泌外泌小体。外泌小体成分除了脂质、蛋白质外,还包括microRNA,lncRNA,circRNA等一些非编码RNA。通过蛋白组学及表达谱分析发现外泌小体中的蛋白组成及所含的RNA分子较为复杂,不同细胞来源的外泌小体其组成成分也不尽相同,这说明了外泌小体的功能的特异性。研究表明血液中外泌小体的特异性蛋白的表达可以做胃癌、胰腺癌等多种疾病的诊断标志。研究还发现,相对传统的干细胞治疗,干细胞来源的外泌小体,可以利用携带的干细胞特异的miRNAs分子和蛋白分子,刺激靶细胞向心脏保护和再生方向发生转化。除此之外,外泌小体还具有稳定性高、应用方便、定量精准等优点,因此本发明将BMSC来源的外泌小体应用于心肌梗死的治疗之中。
发明内容
针对现有干细胞移植的副作用,本发明采用了无细胞的干细胞治疗,也就是移植的不是干细胞,而是利用干细胞分泌的外泌小体治疗心脏病。本发明的目的是为临床提供更具特异性、更安全和更易操作的治疗心肌梗死方案。
本发明提供了间充质干细胞分泌的外泌小体治疗心肌梗死的新用途。具体生物学实验研究结论包括以下方面:
一种骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用。
所述的骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用,其特征在于:所用的外泌小体是来源于健康的骨髓间充质干细胞分泌的。
所述的骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用,其特征在于:所述心肌梗死为因缺血导致的心肌细胞的坏死。
所述的骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用,其特征在于:将骨髓间充质干细胞分泌的外泌小体溶解在磷酸盐缓冲液中制成浓度为1mg/mL的针剂使用。
有益效果:本发明提供了间充质干细胞分泌的外泌小体治疗心肌梗死的新用途,建立分离足够量外泌小体的方法及全面分析外泌小体的体系,规范外泌小体治疗心肌梗死的疗程、疗效及评估体系。利用干细胞分泌的外泌小体治疗心脏病的创新方案,不但解决了干细胞治疗带来的致癌及免疫排斥的危害,而且与干细胞治疗相比具有便于保存、定量以及移植简单的优点。外泌小体的治疗方案将为社会提供治疗心血管疾病的新途径,并节省大量基金。
具体实施方式
以下所列实施例,仅为帮助本领域技术人员更全面理解本发明,但不以任何方式限制本发明。
实施例1大鼠BMSCexosome的分离鉴定
实例1所使用的主要材料和来源分别如下:
BMSC培养试剂 DMEM:F12培养基(hyclone公司,美国)、胎牛血清(BI公司,以色列)、胰蛋白酶(Sigma公司,美国)、二氧化碳培养箱(Thermol公司,美国)、倒置显微镜(ZEISS公司,德国),超净工作台(安泰公司,苏州),台式离心机(Thermol公司,美国),超速离心机、超速离心管(Beckman公司,美国);BCA蛋白定量试剂盒(Takara公司,日本)、辣根过氧化物酶(HRP)标记的IgG二抗(杭州联科生物公司,杭州),ECL化学发光底物(BI公司,以色列)、0.22um无菌滤膜(Millipore公司,美国);透射电子显微镜(FEITecnai12,Philips公司)、FACS检测的微球(Lifetechnology公司,美国),PE标记的CD63抗体(biolegend公司,美国)。
1.大鼠骨髓间充质干细胞(BMSC)的获得
a)选择2-3周龄的雄性SD大鼠,颈椎脱臼法处死大鼠,将大鼠浸泡在75%酒精中5min-10min消毒;
b)在无菌条件下取大鼠的双下肢浸泡于DMEM/F12无血清基础培养基中,用剪刀和镊子去除股骨及胫骨上的肌肉;
c)用1ml注射器吸取添加青、链霉素的DMEM/F12无血清基础培养基,慢慢冲出骨髓,每个骨头冲洗2遍,(不可冲洗次数过多,冲的次数过多可能会冲出来更多的杂细胞);
d)待骨髓全部冲出,1000rpm,离心5min,弃上清,加入新鲜的DMEM/F12+10%FBS的培养基,反复吹打,将冲出的骨髓制成单细胞悬液,按照1.5×105/cm2的密度将细胞悬液接种于培养皿中;
e)48h后换液并去除未贴壁的细胞,以后每两天换液一次,待培养皿中的集落之间互相融合细胞密度达80%左右时进行传代,用0.125%胰酶37℃消化1min15s,收集细胞1:2传代,此时标记为P1代;
f)待细胞传代至P2代或者P3代时,取一部分细胞,用FACS鉴定MSCs表面标志分子(CD34-CD45-CD90+CD29+CD105+)。
2.BMSC外泌小体的获取
a)将P3代的BMSC传至P4代时,换用去除外泌小体的血清进行培养,培养48h后收集培养上清;
b)将收集的细胞上清2,000g,离心10min,以去除死细胞,并将离心后的上清转移到另一新的离心管,再次10,000g离心30min,以去除细胞碎片;
c)将离心后的培养上清转移入超速离心管中,100,000g离心70min,弃上清,用PBS重悬底部沉淀后,再次100,000g离心70min;
d)再次用PBS重悬后,用0.22um无菌滤膜过滤除菌,用BCA蛋白质定量试剂盒进行蛋白质定量检测后,分装后于-80℃保存。
注:本步骤中的离心均在4℃条件下进行。
外泌小体的鉴定
3.1电镜鉴定
a)将获得exosome调整浓度至1000ug/mL,加入等体积的浓度为5%的戊二醛(exosome终浓度为500ug/mL,戊二醛终浓度为2.5%)固定exosome;
b)吸取5ul悬液滴到碳膜铜网上,红外灯下5min至干燥,然后滴加1%磷钨酸1滴,红外灯下5min至干燥,120KV下电镜观察。
检测
提取exosome后,利用BCA试剂盒测定exosome蛋白浓度。取10ug的exosome于EP管中用于流式测定,步骤如下:
a)加入10ul的乳胶免疫微球,室温孵15min;再加PBS至1ml,继续在室温下孵育2h,间断混匀;
b)加110ul1M甘氨酸,轻柔混匀,室温孵育30min;
c)室温4000rpm离心3min,弃上清。1ml0.5%BSA重悬免疫微球沉淀,室温4000rpm离心3min,弃上清,重复洗涤2次;
d)100ul0.5%BSA重悬,加入5ul抗大鼠CD63-PE抗体,4℃孵育30min;1ml的0.5%BSA离心洗涤3次;
e)200ul0.5%的BSA重悬后,用流式进一步检测。
鉴定
a)用PBS将exosome蛋白浓度调至1.25mg/ml,吸取80ul加入20ul的5XSDS上样缓冲液,此时蛋白浓度即为1mg/ml,100℃,5min煮样;
b)吸取蛋白量20ug的exosome上样,行分离胶浓度为15%的SDS-PAGE。100v恒压,2h将蛋白转移至PVDF膜,用5%牛奶室温封闭2h;
c)将封闭好的膜与兔抗大鼠抗体(1:500稀释)进行孵育,4℃,过夜。PBST洗膜,3次,每次5min,然后与辣根过氧化物酶标记的羊抗兔二抗(1:10000稀释)室温孵育45min;
d)PBST洗膜,3次,每次5min,加上ECL发光底物,化学发光凝胶成像系统下进行检测。
实施例2大鼠BMSC来源的外泌小体对心肌梗死的治疗作用
实例2所使用的主要材料和来源分别如下:
SD大鼠(昭衍(苏州)新药研究中心提供,此实验由苏州大学伦理委员会批准);小动物呼吸机(上海奥尔科特生物科技有限公司,上海),心电监护仪(日本光电公司,日本),硫酸阿托品(浙江金力制药有限公司,海宁)水合氯醛(苏州大学附属第一医院自制,苏州),手术器械(六六视觉公司,苏州),缝合针线(上海浦东金环医疗用品有限公司,上海),组织化学染色试剂(索莱宝公司,北京);
1、大鼠心肌梗死模型的建立
a)选用280g左右的雄性SD大鼠为实验对象,采用LAD结扎法制作心梗模型。模型组大鼠在麻醉前5-10min注射0.1ml的硫酸阿托品,抑制气管分泌物,然后10%的水合氯醛(30mg/kg)腹腔注射麻醉,切开气管行气管插管,接空气呼吸机,呼吸频率80次/min,潮气量8ml,吸呼比1∶1。左胸纵切口切开外层皮肤,剥离胸大肌,第三、四肋间横切口开胸,暴露心脏,用镊子撕开心包膜。在左心耳下缘与肺动脉圆锥间可以看见左冠状静脉主干走行,LAD与之伴行。在左心耳与肺动脉圆锥之间的连线距左心耳下缘约3mm处,将LAD连同少量心肌组织一起缝扎,进针深度约1mm,宽度控制在4-6mm。逐层关胸,缝合气管。结扎后30min记录心电图。假手术组仅缝线而不打结,其余同模型组;
b)结扎后肉眼可见心尖变白,心电图监测有ST段的抬高,2周后取左心室组织进行心脏组织染色,可看到明显的纤维化,证明心梗模型建立成功。
2.BMSC来源的外泌小体在心梗模型治疗中的应用
a)按照上述方法LAD结扎后,选择结扎位点附近的左下、右下两个位点注射PBS重悬外泌小体悬液进行治疗,每只大鼠exosome用量为20ug/20ul,每个位点10ug,以PBS作为阴性对照;
b)分别在术后的1周和2周处死大鼠,取左心室组织进行心脏组织染色,观察治疗效果。
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 (4)
1.一种骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用。
2.根据权利要求1所述的骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用,其特征在于:所用的外泌小体是来源于健康的骨髓间充质干细胞分泌的。
3.根据权利要求1所述的骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用,其特征在于:所述心肌梗死为因缺血导致的心肌细胞的坏死。
4.根据权利要求1所述的骨髓间充质干细胞分泌的外泌小体在制备治疗心肌梗死药物中的应用,其特征在于:将骨髓间充质干细胞分泌的外泌小体溶解在磷酸盐缓冲液中制成浓度为1mg/mL的针剂使用。
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