CN110038032A - 人脐带msc外泌体的新型抗肾纤维化的生物制剂及制备方法 - Google Patents
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Abstract
本发明属于生物制剂技术领域,公开了一种人脐带MSC外泌体的新型抗肾纤维化的生物制剂及制备方法。人脐带MSC外泌体的新型抗肾纤维化的生物制剂主要成分是由胞内多泡体与细胞膜融合后分泌到细胞外的大小100nm左右的杯状膜形囊泡。人脐带MSC外泌体的新型抗肾纤维化的生物制剂的制备方法包括:超速离心法、化学试剂沉淀法、超滤离心法、磁珠免疫法。本发明将MSC细胞的外泌体提取后用于糖尿病肾病动物模型的治疗,并发现MSC‑Ex具有抑制YAP活性减轻肾间质纤维化延缓糖尿病肾病的治疗作用。
Description
技术领域
本发明属于生物制剂技术领域,尤其涉及一种人脐带MSC外泌体的新型抗纤维化的生物制剂及制备方法。
背景技术
目前,业内常用的现有技术是这样的:糖尿病(diabetes mellitus,DM)是一类由于胰岛素分泌缺陷或胰岛素作用障碍所致的以高血糖为特征的代谢性疾病。国际糖尿病联合会(IDF)最新数据显示,2017年全球20-79岁的糖尿病患者达到4.25亿;随着我国老龄化加剧,中国糖尿病全国流行率上升到10.96%,患者人数1.14亿人,是全球DM增长最快且患病人数最多的国家。糖尿病肾病(diabetic kidney diseases,DKD)是糖尿病最主要的微血管并发症之一,也是引起终末期肾病(ESRD)的首要原因。DKD在糖尿病患者中发病率为30%-50%,已成为糖尿病患者的主要致死、致残原因之一。DKD起病隐匿,一旦进入大量蛋白尿期后,进展至ESRD的速度大约为其他肾脏病变的14倍。因此,亟需寻找降低糖尿病患者血糖,减轻治疗副作用并延缓糖尿病病情发展的新方法。预防与延缓糖尿病肾病的发生发展对提高糖尿病患者存活率,改善其生活质量具有重要意义。人脐带间充质干细胞来源外泌体(hucMSC-Ex)降低糖尿病肾病大鼠的血糖并延缓糖尿病肾病进程,作用机制可能与调控YAP蛋白磷酸化抑制YAP有关。国内外诸多研究表明,间质干细胞(mesenchymal stemcell,MSC)在血糖调节和减轻高糖引起的肾小管损伤保护肾功能方面具有重要的作用。MSC在肾脏疾病的干预方面表现出了一定的效果,但MSC的应用标准、免疫反应性、潜在致瘤性及转基因技术的安全性等因素使MSC的在糖尿病肾病治疗中的应用受到限制。MSC旁分泌外泌体(MSC-Ex)作为发挥其损伤修复的主要作用成分,因此MSC-exosomes在保护肾功能具有生物特性的优势,在糖尿病肾病的治疗将发挥更有效的作用。
目前外泌体与糖尿病肾病的研究主要集中在疾病诊断方面,而外泌体用于治疗糖尿病肾病的实验研究比较少:Nagaishi K等发现MSC-上清纯化的外泌体减轻肾小管内皮细胞凋亡保护肾组织紧密连接结构,抑制肾小球系膜基质增加来预防糖尿病肾病。NesrineEbrahim等发现间充质干细胞的外泌体通过mTOR信号通路诱导自噬改善糖尿病肾病。最近研究发现间充质干细胞外泌体在糖尿病肾病中具有潜在的治疗价值。
综上所述,现有技术存在的问题是:明确间充质干细胞来源外泌体(hucMSC-exosomes)抑制肾间质纤维化延缓糖尿病肾病的作用方式,剂量疗效,及作用机制。现有技术存在较多毒副作用,仅用于控制病情对于病因没有实质性的改善;或存在伦理,剂量不可控及安全性问题。
解决上述技术问题的难度和意义:糖尿病肾病病程长预后差,间充质干细胞外泌体在通过促进YAP蛋白泛素化降解抑制肾间质纤维化延缓糖尿病肾病的作用机制。在干细胞治疗糖尿病肾病具有十分重要的临床应用价值。
发明内容
针对现有技术存在的问题,本发明提供了一种人脐带MSC外泌体的新型抗肾纤维化的生物制剂及制备方法。
本发明是这样实现的,一种人脐带MSC外泌体的新型抗纤维化的生物制剂,所述人脐带MSC外泌体的新型抗纤维化的生物制剂的有效成分是蛋白激酶。
进一步,所述蛋白激酶由对人脐带间充质干细胞进行培养,细胞数目比例为1:1,细胞瓶培养收集MSC上清;蛋白激酶为胞内多泡体与细胞膜融合后分泌到细胞外的大小100nm的杯状膜形囊泡。
本发明的另一目的在于提供一种所述人脐带MSC外泌体的新型抗纤维化的生物制剂的制备方法,所述人脐带MSC外泌体的新型抗纤维化的生物制剂的制备方法包括以下步骤:
第一步,通过超速离心法300g离心10min,2000g离心10min,10000g离心30min,收上清离心100000g离心70min;
第二步,通过化学试剂沉淀法去上清,收集沉淀PBS溶解;
第三步,通过超滤离心法提取干细胞培养上清中的外泌体。
本发明的另一目的在于提供一种所述人脐带MSC外泌体的新型抗纤维化的生物制剂在制备抗肾间质纤维化的药物中的用途。
综上所述,本发明的优点及积极效果为:间充质干细胞外泌体在通过促进YAP蛋白泛素化降解抑制肾间质纤维化延缓糖尿病肾病的作用机制。对于干细胞治疗糖尿病肾病,抑制肾脏组织间质纤维化,保护肾功能具有十分重要的临床意义。
附图说明
图1是本发明实施例提供的人脐带MSC外泌体的新型抗纤维化的生物制剂的制备方法流程图。
图2是本发明实施例提供的建立hucMSC分离培养鉴定方法示意图;
图中:A.透射电镜观察hucMSC-Ex、HFL1-Ex的形态;B.纳米颗粒分析仪检测hucMSC-Ex、HFL1-Ex的粒径及浓度;C.Western blot检测exosomes标记蛋白CD9、CD63和CD81。
图3是本发明实施例提供的hucMSC-Ex干预能延缓DKD大鼠病程示意图;
图中:A.大鼠体重测定;B.血糖值检测;C.血清尿素氮水平检测;D.24h尿白蛋白排泄率;E.肾组织HE染色;F.肾组织天狼星红染色。
图4是本发明实施例提供的hucMSC-Ex抑制DKD模型大鼠肾组织中YAP蛋白的表达示意图;
图中:A.Western blot检测DKD肾组织不同时间YAP蛋白表达水平;B.大鼠血清尿素氮检测;C.Western blot检测第24周肾组织YAP蛋白表达;D.免疫荧光检测第24周肾组织YAP和α-SMA的表达。
图5是本发明实施例提供的hucMSC-Ex促进YAP蛋白胞质内滞留和泛素化降解示意图;
图中:A.免疫荧光检测不同处理HBZY-1细胞YAP的表达与定位;B.Western blot检测YAP细胞核、胞质及全细胞中YAP蛋白表达;C.Western blot检测磷酸化YAP的表达;D.蛋白定量统计分析pYAP(S127)/YAP比值;E.蛋白定量统计分析pYAP(S381)/YAP比值。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
本发明将MSC细胞的外泌体提取后用于糖尿病肾病动物模型的治疗,并发现MSC-ex具有抑制YAP活性减轻肾间质纤维化延缓糖尿病肾病的治疗作用。
下面结合附图对本发明的应用原理作详细的描述。
本发明实施例提供的人脐带MSC外泌体的新型抗纤维化的生物制剂主要成分是由胞内多泡体与细胞膜融合后分泌到细胞外的大小100nm左右的杯状膜形囊泡;含有络蛋白激酶(ck1δ)有效成分。对人脐带间充质干细胞进行培养,细胞数目比例为(1:1),细胞瓶培养收集MSC上清。
如图1所示,本发明实施例提供的人脐带MSC外泌体的新型抗纤维化的生物制剂的制备方法包括以下步骤:
S101:通过超速离心法300g离心10min,2000g离心10min,10000g离心30min,收上清离心100000g离心70min;
S102:通过化学试剂沉淀法去上清,收集沉淀PBS溶解;
S103:通过超滤离心法提取干细胞培养上清中的外泌体。
进一步,所述外泌体的成分就是一种纳米级生物膜囊泡,称为外泌体。在细胞间的物质和信息传递有重要作用,通过递送特异性蛋白质、信使RNA(mRNA)各种生物活性分子到靶细胞,干预多种疾病的发生和发展。
T2DM(Type 2 diabetes mellitus;二型糖尿病);DKD(Diabetic kidneydisease;糖尿病肾病);MSC(Mesenchymal stem cells;间质干细胞);exosomes(外泌体);PBS(磷酸盐缓冲液);YAP(Yes associated protein);Interstitial fibrosis(间质纤维化)。
下面结合具体实施例对本发明的应用原理作进一步的描述。
实施例1MSC外泌体的提取与鉴定
实例1所使用的主要材料和来源分别如下:
MSC培养试剂:α-MEM、胎牛血清(Gibco公司产品)、胰蛋白酶(Sigma公司产品)、二氧化碳培养箱(Forma公司)、无血清培养基(上海依科赛公司;倒置显微镜,荧光显微镜,生物显微镜,电子显微镜,超净工作台,台式离心机,超速离心机(美国贝克曼公司)。
Exosome提取试剂:重水(D2O,上海创赛公司),分析纯蔗糖(广州化学试剂厂),兔抗人CD9抗体(美国Bioworld Technology公司),兔抗人CD63抗体(美国Epitomics公司),兔抗人CD81抗体(美国Epitomics公司),辣根过氧化物酶(HRP)标记的山羊抗兔IgG二抗(北京康为世纪公司),HRP化学发光底物、100-kDa MWCO超滤离心管、0.22μm无菌滤膜(美国Millipore公司);透射电子显微镜(FEI Tecnai 12,Philips公司)。
对本发明实例1具体实施步骤描述如下:
(1)脐带MSC的分离培养:本发明先前构建人HucMSC分离培养及鉴定方法体外培养扩增MSC(Qiao Chun et al.Human mesenchymal stem cells isolated from theumbilical cord.Cell Biol Int.2008 Jan;32(1):8-15),选择增殖能力强,培养MSC,生长80%用无血清培养基培养48h收集上清,2000g/10min离心除去细胞碎片后即为MSC-CM。
(2)脐带间质干细胞外泌体的分离纯化(本发明应用蔗糖密度梯度离心法,此方法不作为本发明保护的重点):差速离心去细胞碎片及细胞器,后15ml规格100000Da MWCO超滤管浓缩MSC-CM,浓缩液移至5ml浓度为30%的蔗糖/D2O密度垫上,4℃条件下,100000g离心120min,收集底部5ml的缓冲垫(含exosome)用PBS稀释洗涤后,置入100000Da MWCO超滤离心管中洗涤,最后将收集的exosome浓缩液定量,0.22μm滤膜过滤除菌,分装,-70℃冷藏备用。
(3)透射电镜观察exosomes形态:MSC-exosomes 20μL,混匀后滴加于直径2mm的载样铜网上,静置5min后,用滤纸吸去残余液体,将铜网倒扣于30g/L磷钨酸(pH 6.8)液滴上,室温下负染5min,白炽灯下烘干,透射电镜下观察拍照,图2A所示exosome直径约为120nm左右囊泡状结构;
(4)western blot检测exosomes表面标记蛋白:配制15%SDS-PAGE电泳胶,exosomes充分裂解加入1/4体积的5×SDS上样缓冲液,煮沸10min,按200μg蛋白质总量上样电泳,电转移(350mA,120min)将蛋白质转至PVDF膜上,50g/L脱脂牛奶室温封闭1h,与兔抗人CD9抗体和兔抗人CD63抗体兔抗、人CD81抗体(1:500)4℃反应过夜,次日TBS/0.5%Tween20洗膜3次,与HRP标记的山羊抗兔IgG二抗37℃温育1h),TBS/0.5%Tween 20洗膜3次,加入预混HRP化学发光底物,化学发光凝胶成像系统曝光检测,如附图2C所示脐带来源的exosome的标记。
实施例2糖尿病肾病大鼠模型构建及MSC-exosomes的治疗干预效果
本实施例所使用的主要材料和来源分别如下:
动物模型所需材料:8周龄雄性SD大鼠,普通饲料(舒克贝塔公司),45%高脂饲料(江苏美迪森公司),链脲佐菌素(Sigma公司),血糖仪及试纸(罗氏金锐);其他器材:一次性胰岛素注射针筒,胰岛素(Sigma公司),葡萄糖(食用),YAP检测组化试剂盒(Excell公司)。
对本发明实施例2具体实施步骤描述如下:
(1)构建MSC-exosomes干预DKD大鼠模型
选择8周龄雄性SD大鼠作为T2DM造模对象,45%高脂饲料喂养5周后尾静脉注射STZ(35mg/kg,链脲佐菌素,Sigma),第7天用罗氏便携式血糖仪(活力型)检测血糖有无≥16.7mmol/L,继续普通饲料喂养至12周,收集大鼠血液尿液检查肾功能相关指标,HE染色检测肾组织结构病理变化,辅助判断模型构建成功,STZ造模DKD成功后,尾静脉注射hucMSC-Ex(10mg/k溶于200μl PBS)干预,每3日注射一次共注射10次,不同时间点观察大鼠体重、血糖值和血清尿素氮以及尿白蛋白水平升高,显示DKD模型构建成功,模型大鼠随着病程进展,体重逐渐下降,但经hucMSC-Ex处理后,其体重可维持在一定水平hucMSC-Ex处理可延缓糖尿病肾病进程(图3)。
(2)MSC-exosomes抑制YAP减轻间质纤维化延缓糖尿病肾病的治疗;
(3)通过Western blot检测不同时间点模型鼠YAP表达,结果发现,造模24周随着造模时间延长,YAP表达明显上升,α-SMA表达增强同时血清尿素氮水平也明显增加,提示肾间质纤维化肾功能损伤加重。hucMSC-exosomes干预组肾组织内YAP明显降低,肾纤维化改善。高糖刺激肾小球系膜细胞,YAP核内显著增加,hucMSC-Exosomes干预组YAP入核则减少,核质分离分析显示,hucMSC-Exosomes处理后YAP蛋白整体下调,胞核减少,胞质内Ser381、Ser127磷酸化的YAP明显增加,提示hucMSC-Exosomes诱导YAP胞质内滞留。因此,MSC-exosomes通过抑制YAP减轻间质纤维化延缓糖尿病肾病发挥作用(图4和图5)。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种人脐带MSC外泌体的新型抗纤维化的生物制剂,其特征在于,所述人脐带MSC外泌体的新型抗纤维化的生物制剂的有效成分是蛋白激酶。
2.如权利要求1所述的人脐带MSC外泌体的新型抗纤维化的生物制剂,其特征在于,所述蛋白激酶由对人脐带间充质干细胞进行培养,细胞数目比例为1:1,细胞瓶培养收集MSC上清;蛋白激酶为胞内多泡体与细胞膜融合后分泌到细胞外的大小100nm的杯状膜形囊泡。
3.一种如权利要求1所述人脐带MSC外泌体的新型抗纤维化的生物制剂的制备方法,其特征在于,所述人脐带MSC外泌体的新型抗纤维化的生物制剂的制备方法包括以下步骤:
第一步,通过超速离心法300g离心10min,2000g离心10min,10000g离心30min,收上清离心100000g离心70min;
第二步,通过化学试剂沉淀法去上清,收集沉淀PBS溶解;
第三步,通过超滤离心法提取干细胞培养上清中的外泌体。
4.一种如权利要求1~2任意一项所述人脐带MSC外泌体的新型抗纤维化的生物制剂在制备抗肾间质纤维化的药物中的用途。
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